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1. 7 410 Time s 20 Time s D E 1 0 0 12 1 0 0 12 10 0 12 0 8 Toa earl Sos 010 Zoos D Zo 08t y 0 081 d 0 6 E d 0 6 0 6 z ie S W 0 06 S 0 06 S 2 0 06 0 41 8 9 4 8 0 4 8 o oO o o 0 gt 0 04 gt g 0 04 g 0 04 w w amp D ow a O oc 2 2 0 02 202 0 02 er 0 2 0 02 0 0 0 00 0 0 0 001211 0 0 0 00 91 ctrl PIBPAb ctriPI3PAb ctrl PI4PAb ctrl PI4PAb ctrl PISPAb ctrl PISPAb PI4PAb PI4PAb Figure 3 6 Non SG fusion is not blocked by antibodies against PI 3 P and PI 4 P In whole cell recording antibodies 1 100 dilution were added to the cytoplasmic pipette solution which was dialyzed into cells for 5 min before Ca was infused to trigger fusion A A typical record from an RBL cell The Ca activated conductance rise was used to define the amp point B The rise of capacitance was well described by the delayed mono exponential function given in the figure The variable k2 is the rate constant used for statistical analysis The number of the data points was reduced to highlight the fitted curve Antibodies against PI 3 P C PI 4 P D and both E fail to block non SG fusion 112 A B RBL ctrl BHK 120 r Pr Acap 119 12 e n 5 2 lt 80 T 26 3 1s 2 200 uM Ca2 40 SS i EEE EEE EE Ee 60 Time s 100 T RBL treated 2 80 5uM wortmannin 0 5 mM adenosine 2 80 5 60 PF Acap 114 5 2 S m 60 n 6 S 1 3
2. et ict y The codes here assign data for offline phase shift If square waves were applied X and Y are assigned from handles PSDofSQA else they are assigned from handles aidata directly PS str2double get handles Phase_Shift String Rc 3R is the correlation coefficient Rg 3R is the correlation coefficient M tmethod of correlation if handles menuindex 1 1 amp amp isnan handles shiftswitch amp amp abs PS handles shiftvalue lt 0 0001 PS Cap Cond R_c R_ g PhaseMatcher2 handles shiftswitch handles aidata indexl index2 1 handles aidata indexl index2 2 Y X 0 1 handles shiftvalue PS set handles Phase Shift String num2str PS if handles shiftswitch 1 M G gt elseif handles shiftswitch 0 M Igt else M C G end disp CorrMethod M Pi num2str PS R_e num2str R_c R_g num2str R_g The function calls PhaseMatcher2 to scan the phase only when all three criteria are met 1 SQA were used 2 handles shiftswitch is not a NaN and 3 the input value in the Shift edit box is not modified Variable handles shiftswitch is a NaN when the User defined option in the Shift context menu is selected If the Shift edit box is modified 150 the program will use the modified value to adjust the angle rather than calling PhaseMatcher2 to scan the phase The correlation method phase angle and the c
3. dependent manner at physiological Ca concentrations Brose et al 1992 and the Ca binding affinities are well correlated with the Ca sensitivities of neurotransmitter release Fernandez Chacon et al 2002 Rhee et al 2005 In Sytl knockout mice the fast synchronous neurotransmitter release was selectively attenuated Geppert et al 1994 indicating that Syt1 is crucial for this type of membrane fusion Together with the evidence mentioned above it is now widely accepted that Sytl is the Ca sensor for fast synchronous neurotransmitter release in neurons It is still not totally clear how Sytl triggers membrane fusion in a Ca dependent manner As mentioned previously the Ca binding affinities and the Ca sensitivities of neurotransmitter release are well correlated Fernandez Chacon et al 2001 Rhee et al 2005 Apparently the Ca dependent phospholipid binding must play certain roles in triggering fusion and it is proposed recently that Sytl together with SNAREs may bring two opposing lipid bilayers very close to each other and accelerate membrane fusion Rizo et al 2006 In addition to Ca dependent phospholipid binding Sytl also interacts with SNAREs in both Ca dependent and independent manners Siidhof and Rizo 1996 As suggested recently the Ca induced displacement of complexin a protein that binds SNARE complex from SNARE complex might account for the triggering of fast neurotransm
4. PFLO CTRO 58 6 EXT Command front switched The connection is compatible with that of Capmeters Things you need to know before using 1 There is no paper for this program yet as of March 2008 Please cite the link 2 This program is still under development and there is only limited error check and control 3 Please remember to adjust the command sensitivity in line 144 default 20 175 mV V Current in the I V plot is estimated using Eq 3 2 which is derived from an exponential function so please do not compensate membrane capacitance completely There are two methods for charge integration and you may switch from one to the other during the experiment Please make a note to specify the method used Unlike charge integration in Capmeter6v3 IQplot2 does not correct the integrated charges using Eq 4 34 It is because time constant from a single curve is not accurate and introduces excessive noise if Eq 4 34 is used Variable datasample in the Workspace does not represent accurate time signal relationship 176 Running the program zini 1 0 8 0 6 0 4 0 2 0 0 2 0 4 0 6 0 8 1 1 1 eo 1 n 0 500 1000 1500 80 60 40 20 o 20 40 amp H o8 axis2 15 axis5 Jost sampled data Q Veurve oat F ge ost A P Y scale of axis2 J F 0 pia A ye loll f7 tet y T 0 5 Fa all at Integration method Programst
5. ajqnop OL E zt0 6 1S T ajqnop lt alqnop x OE gt paysauoa si AA ajqnop lt a qnop LXL0E gt Yso HH p 22 31109 10114 aiqnop lt a qnop x pE gt iso A ajqnop lt alqnop x LOE gt vo HH ajqnop lt ajqnop x 0E gt OH eesgo s ajqnop lt alqnop LXL0E gt ajqnop ZLO 3621F 1 10114 ajqnop nuaw dja y ayy wos Souad 40 TSH AW IDEN Deas payes 326 01 ED E aseg pres fj ie eaga D e mopu pueunuoy X 2 O aoedsyi0my MS Sy 212 Saturation To Wo tkepace Pulse Generator Terminato d QW Sine Wave Terminator I 1V 50e6 Sa Repeating Terminatori Sequencel B x10 T T T T T T T i i 1 o ny o z N T T T T T L L 1 L i j Figure 4 9 Generation of the model current using Simulink Model diagram of the whole cell patch clamping configuration is shown in A Cell parameters can be adjusted in the model red circles The wave protocol square pulses in this case a in A drives the circuit and the total current b in A black in B current charging the membrane c in A red in B and current leaking through the membrane d in A blue in B are displayed The sampled data are exported e in A to the Workspace of MATLAB for analysis Saturation To Wo tkepace Pulse Generator Terminato og Sine Wave Terminator Repeating Terminatori Sequencel Figure 4 10 Some more about the diagram in Simulink To explain a little bit more about how
6. amplitude giving pulses 5 Inthe presence of noise I SQA gives better closer to the actual value estimation of membrane capacitance compared with Q SQA However unlike Q SQA which is almost unaffected by the filter function the estimation of I SQA is greatly influenced by the filter 6 Q SQA is the preferred SQA method because the estimation of Cm is not affected by Ra fluctuation according to my experience Running the program Current Directory CapEngine4 mexw32 MEY fila Capmeter6v3 fig aA Right click Capmeter6y3 m M fila E CapmeterSettin open Capmodule4 fig Capmoduled m View Help CapmoduleAO1 Open as Text CapmoduleAO1 Open Outside MATLAB 4 Dfilter2 mexw3z 8 DispCtrl mexw3 Import Data In MATLAB change the Current Directory to the one containing Capmeter components and then right click on Capmeter6v3 m file double click on the file will open the M file editor Click Run in the context menu and MATLAB will launch the program as shown below Capmeter6v3 08326 p O xj i T T T T T T T T 1 axis 05i bs v chity c 0 HE Right click on it to show the chat context menu and change Ch4 ACH2 Er chs Ra the displayed channel 4 1 l l 1 0 8 0 6 0 4 0 2 0 0 2 04 0 6 0 8 T T T T T T T 5 axis 2 Digital filter panel gt A slider1 a A F Label panel Y axes contr
7. dataBtime lastmax i lny SXXSX SX SxX fenmu double fladd1 sx2 sxxsx means denominator in Chinese if fenmu 0 amp amp fladdl sxy sx sy 0 get peak and tau peak exp sy sx2 sx sxy fenmu asymp tau 1 fladd1 sxy sx sy fenmu else quality 0 Variable firstmin was the first minimal point in the curve Now it represents the end of the region that is used for linear regression solid section in Fig 4 5B It is important to adjust firstmin to ensure that all data points in the natural logarithm are positive within the region Negative points might exist if the trace is noisy i e current crosses the middle line occasionally or there are unexpected glitches in the trace The peak current and the steady state current are obtained after linear regression All valid data sets are added and averaged to generate one digitized data set Ra Rm and Cm in MATLAB using Eq 3 5 and Eqs 3 9 11 Square wave perturbation based on fitting the transferred charges To increase the signal to noise ratio of I SQA my mentor suggested me to develop an algorithm using integrated charges Q SQA Since noise tends to cancel each other after summation the outputs of curve fitting procedures in Q SQA are of better quality compared with I SQA Considering a decaying exponential curve with peak amplitude and asymptote of a and b respectively I subtract b from the net current and only i
8. of the original signal amplitude gt wo a 16 I b a b e T12 b E 0 z8 to b Q 34 0 4 0 200 400 600 800 1000 C time us Vc Vss fVss Figure 4 5 Square wave perturbation based on fitting the current transient Model current for whole cell recording is shown in A Peak current a and the projected steady state current b were determined as described in the text B Half of the current from a real recording dots with the fitted exponential function is shown The asymptote of current was determined using averages of dashed sections according to Eq 3 2 Data range from the peak to a point located at 3t was used to determine the exponential constants solid section C Membrane potential during fast voltage oscillation Vc is shown Because the oscillation is too fast membrane potential is not able to reach its theoretical steady state value Vss and oscillating between fVss where fis the fraction of Vss across the membrane at the end of the voltage step of duration ta Please refer to the text for some more details and equations 208 I b a b e Os a b 1 e 0 Figure 4 6 Square wave perturbation based on fitting the transferred charges Half of the model current for whole cell recording is shown in A with peak current a and the projected steady state current b which is obtained using Eq 3 2 The gray area is integrated and the resulting charge Qs time r
9. 92 DMEM 15 FBS Plate 2 ml of cells on each 35 mm uncoated Petri dish and culture the cells at 37 C in a humidified CO incubator The diameter of mast cells is about 10 um The reason for using a Petri dish rather than a cell culture dish is to prevent attachment of cells onto the dish thus reducing the mechanical force needed for isolating the cells In my experience too much mechanical force triggers fusion of SGs prior to experiments which reduces the releasable pool as a consequence For isolating rat peritoneal mast cells I use a total amount of about 40 ml mast cell buffer and follow the same protocol as described above Rat peritoneal mast cells are bigger than those from mouse Tips for reducing noise in patch amperometric recordings The recording chamber and solution lines are filled up with electrolytes which behave like a big antenna and collect a lot of electrical noise In regular patch clamp configuration the ground is connected to the recording chamber thus electrical noise from outside of the system is sequestered As mentioned in section 3 3 for patch amperometric recordings the ground has to be in the pipette and the headstage for recording capacitance etc is connected to the bath In this case noise introduced by the recording chamber and solution lines is not neutralized and will be detected by the headstage used for capacitance recording Here I will describe some tips that were found to be useful
10. P pi 2 2 pi F T L handles PSD90 sin linspace P pi P pi 1 2 F T L guidata FH handles Every time when you change the oscillating frequency and or adjust the phase angle the program will call the function Refcalc to adjust the reference waves Ref and Ref2 in Fig 4 4 Since the program uses simply averaging to extract the DC component it is important to make sure that the time span of the manipulated data points is the multiple of the period of a single sine wave Variables PPS and L above ensure that it is the case Notice that the last time point represented by the reference wave is L handles aiSR but not L handles aiSR where handles aiSR is the acquisition speed of the analog input points s Time point L handles aiSR is the first time point of the next processing cycle The phase shift P or handles PSDphase above is added into the calculation of the reference waves handles PSDref and handles PSD90 You might have noticed that the first point of handles PSDref is sin P 7 2 but not sin P as introduced in Eq 4 2 in earlier section It is because the acquisition of the signals is initiated by a trigger signal added on top of the peak of the command sine waves the first acquired point is resulted from command potential Vsin 7 2 but not Vsin 0 which is zero The multiplication of the current with the reference waves is carried out in one of the subfunctions in CapEngi
11. ee eeceeeeeseeeteeeeeceneeesneeeeaees 78 Non SG fusion is probably phosphatydialinositide independent ceeeseeeeteeeees 79 Wortmannin adenosine insensitivity of non SG fusion in whole cell recording 80 Non SG fusion in whole cell recording is blocked by cell swelling eee 81 Synaptotagmin VII and PLC s are not required for non SG fusion cceeeeeees 83 Ca dependence of non SG fusion in excised RBL patches c ccccccseseseseseeeneeees 84 3 5 DISCUSSION yh 20dssisuncerivosa EE A E SA EEE EE anes uence EER tA 86 Membrane fusion in excised giant membrane patches ss ssssssseseessersseeseesseessreeseee 86 Non secretory fusion in excised patches ccccecccccsccssseceseceeeeeeeceeeeeeseeeteeeeeenteeeeseaes 87 AT PoSensitivity of non SG TIS Of coccreisers toes cradincccaitta stuerediaw stint nce UA Mawetaes aes labs 88 Wound repair and non SG tusiOti es 3 2c ssebensancehae sees ais osczaacevaescancosans yas eacknins Goss elsinuwaeass 89 3 6 Some more methodological detaills cccccesssceceeeceeeeeeeeeeeeeneees 92 Isolation of rodent peritoneal mast CEUs Uiscccs cionssecicomdien sadsencdoccoulewssnavetent eollebiiitadvees 92 Tips for reducing noise in patch amperometric recordingS ceceesceseeeeeeneeeeeeseeees 93 Tips for making carbon electrodes sessseseseeseesesseseesetseesessestestssessesrstesessresssesseesse 97 Efforts for preserving secretory vesicles in excised
12. equation can be expressed as 9 arctan 4 6 90 arctan Y Y 4 6 It is important to keep in mind however that this method is valid only when the time constant of capacitance compensation is adjusted properly else the changes will not be reflected at the actual phase angle For excised patches because Ra is almost non existing T is superfast as mentioned in the Introduction In this case the knob of fast component compensation should be used and the shortest time constant setting is 125 selected For whole cell recording the series resistance knob on the capacitance compensation panel of the patch clamp has to be adjusted properly To do so the only way I know is to play with the knobs and see at which setting the cell capacitance is compensated properly Phase sensitive detection offline adjustment of the phase angle As introduced previously when the reference phase angle 6 is not set properly X and Y may not reflect the actual G and C If it is the case an offline adjustment of the reference phase angle is desired To do so one can rotate the PSD1 PSD2 coordinate with a desired phase shift to reconstruct a new set of X and Y Considering that what the lock in amplifier sees are two orthogonal vectors X and Y that compose a vector with R and 0 Figs 4 3A By rotating the PSD1 PSD2 coordinate Fig 4 3B C the new PSD1 readout Xag is the sum of the projections of the orig
13. experiments Chen et al 2006 there are some more creative reconstitution systems made during these years One of them is the flipped SNAREs system Hu et al 2003 The v and t SNAREs were inverted and expressed on the plasma membrane of two different cell populations one with red fluorescent cytoplasm and one with blue fluorescent nucleus The two populations were mixed and cell cell fusion was counted as cells with both fluorescence Hu et al 2003 Without commenting on it too much personally I think the method is cool and the only advantage is that purification of recombinant proteins is not required in this system Another two reconstitution systems monitored fusion by using total internal reflection fluorescence microscopy TIR FM The first one labeled v SNARE containing vesicles with fluorescent lipids and then put them onto supporting lipid bilayer containing t SNAREs Approaching of the vesicles can be observed as increasing fluorescence intensity and membrane fusion result in dissipation of the fluorescence due to diffusion of fluorescent lipids in the supporting lipid bilayer Fix et al 2004 The other approach prepared the supporting lipid bilayer differently Instead of making a sheet of lipids on the quartz slide they coated the slide with NeutrAvidin and the v SNARE vesicles containing biotinylated lipids were tethered on the slide without forming a lipid lawn Yoon et al 2006 Since t and v SNARE vesicles were
14. vi is not required for non SG fusion in excised patches Although non SG fusion is ATP dependent and blocked by PI kinase inhibitors wortmannin and adenosine the dependency is not neutralized by the PI 3 kinase inhibitor LY294002 PI 4 5 P gt ligands such as neomycin a Pl transfer protein that can remove PI from membranes and PI 4 5 P2 PI 3 P and PI 4 P antibodies etc In whole cell recording non SG fusion is strongly reduced by osmotically induced cell swelling and subsequent recovery after shrinkage is inhibited by wortmannin indicating that membrane stretch occurring during patch formation may be a major cause of the ATP dependency in excised patches Syt7 and several PLCs are not required for non SG fusion because fusion remains robust in mouse embryonic fibroblasts deficient of Syt7 PLCd1 PLC61 64 or PLCy1 Furthermore the Ca dependence of non SG fusion reflects a lower Ca affinity Kp 71 uM than expected for these C2 domain containing proteins I also developed a program for measuring and analyzing membrane capacitance The program uses either sine waves or square waves to estimate cell parameters Phase sensitive detection is utilized in both cases For square wave perturbation either integrated charges or direct current trace is used for calculating cell parameters Other functions like digital filtering pulse stimulation offline phase angle adjustment baseline subtraction and data normalization are also im
15. 2 meaning step 1 of 2 and the program takes the averages of 20 data points from channels 1 red and 2 blue After changing the compensated capacitance red click on PAdj button again and Capmeter get the averages and then adjust the phase angle automatically using Eq 4 6 When the phase angle is properly adjusted changing capacitance compensation does not affect the reading in conductance channel blue You may also set the angle by putting specific value in the edit box and then press Enter or play with the slider bar in the panel Occasionally you might get an angle that is 2 m or m or so on away from the actual angle when using PAdj In this case it is also useful to use the 90 button to correct the angle To adjust the phase angle offline simply put a value in the edit box next to the Shift button and then click Shift For PSD analysis of SQA data you can right click on the Shift button and select a method for cross correlation from the context menu and then click Shift to start phase scanning Detail about PSD analysis of SQA data can be found in the last paragraph of section 4 2 Besides adjusting phase angle online you can also change oscillating frequency and the peak to peak amplitude during the recording However since Capmeter uses trigger signal added on top of the command oscillation to synchronize data acquisition the new amplitude must be smaller than the trigger signal By default yo
16. G Podini P Bachi A et al 2002 Regulated exocytosis a novel widely expressed system Nat Cell Biol 4 955 962 Bronk P Deak F Wilson M C Liu X S dhof T C et al 2007 Differential effects of snap 25 deletion on ca2 dependent and ca2 independent neurotransmission J Neurophysiol 98 794 806 Brose N Petrenko A G S dhof T C and Jahn R 1992 Synaptotagmin a calcium 215 sensor on the synaptic vesicle surface Science 256 1021 1025 Brown W J Chambers K and Doody A 2003 Phospholipase a2 pla2 enzymes in membrane trafficking mediators of membrane shape and function Traffic 4 214 221 Burgoyne R D and Morgan A 2003 Secretory granule exocytosis Physiol Rev 83 581 632 Carr C M Grote E Munson M Hughson F M and Novick P J 1999 Seclp binds to snare complexes and concentrates at sites of secretion J Cell Biol 146 333 344 Caumont A S Galas M C Vitale N Aunis D and Bader M F 1998 Regulated exocytosis in chromaffin cells translocation of arf6 stimulates a plasma membrane associated phospholipase d J Biol Chem 273 1373 1379 Caumont A S Vitale N Gensse M Galas M C Casanova J E et al 2000 Identification of a plasma membrane associated guanine nucleotide exchange factor for arf6 in chromaffin cells possible role in the regulated exocytotic pathway J Biol Chem 275 15637 15644 Chakrabarti S Kobayashi K S Flavell R A Marks
17. Graham Carpenter Vanderbilt University Solutions Unless otherwise stated the patches were excised into a solution containing in mM 140 NaCl 1 MgCh 0 3 EGTA 20 HEPES pH 7 3 ATP and GTP were added at final 65 concentration of 2 4 and 0 3 respectively Membrane fusion was triggered by the same solution with 0 5 CaCl i e 0 2 mM free Ca usually without ATP and GTP For whole cell recording with RBL cells both the cytoplasmic and extracellular solutions contained in mM 40 NaCl 90 N methyl D glucamine NMG 1 MgCh 0 01 EGTA 10 HEPES pH 7 3 adjusted with MES Relatively large diameter pipette tips 4 6 um i d were employed in whole cell recording to allow fast exchange of the cytoplasm via pipette perfusion Using this low conductance solution the cell time constants 30 60 us were large enough to use square wave perturbation for capacitance measurements as described subsequently A solution with 0 2 mM free Ca highly buffered with nitrilotriacetic acid was infused into the cell to induce membrane fusion The complete composition was in mM 15 NaCl 90 NMG 3 MgCh 5 CaCh 10 nitrilotriacetic acid 10 HEPES pH7 3 adjusted with MES All free Ca values given in this article were calculated with WEBMAXC http www stanford edu cpatton maxc html Patton et al 2004 Other solutions employed were FVPP solution Huang et al 1998 a phosphatase inhibitor cocktail 110 NaCl 5 NaF 0 1 Na VOu 2 EDTA 10 N
18. IA 4 34 To validate the correction I use the following MATLAB codes MATLAB codes for validating Q correction clear a 10 Speak atb unknown b 5 Sasymptote known tau 100e 6 intervall 10e 6 DAQ speed tl O intervall 3e 3 I1 a exp t1l tau b noise 0 rand length t1 1 0 5 3e 1 Il Il noise Q1 cumsum I1 1l end 1 1 intervall sdirect summation Q1 cat 1 0 Q1 QA a tau l exp tl tau b tl Stheoretical value Qsl Q1 b t1 subtracting DC charges Qs_ corrected Qsl QsA a tau l exp tl tau Stheoretical value after subtracting DC charges Qs corrected Qs corrected tau l exp intervall tau intervall Error Qsl end 1 QsA end 1 QsA end 1 1le2 Error corrected Qs corrected end 1 QsA end 1 QsA end 1 le2 figure plot t1 Qs1 hold on plot t1 QsA Color red hold off figure plot t1 Qs corrected hold on plot t1 QsA Color red 143 hold off The theoretical trace is in red and the charge summation is in blue As shown in Fig 4 8 two traces overlap each other after correction The errors are 5 and 1 5 10 before and after correction respectively I hope I have convinced you that Q SQA has its strong mathematical supports However there is still something in my mind that is if tT is obtained before charge correction and it is then used in charge correction is
19. If the boundary retracts too fast the coating may be too thick and there will be many silicone droplets dispersing along the carbon fiber Similar phenomenon may happen when the sealer is too sticky In this case you may dilute the sealer with some 100 ethanol It usually happens when the sealer is opened and stored for a period of time Cure the sealer at 50 C overnight or at room temperature for at least one day before using The thickness of the insulation is usually 1 um estimated by eyes If it is too thick you may need to improve the coating step move even slower or dilute the sealer a little bit Expose carbon surface and back fill the electrode with 3 M KCl Put the carbon electrode on a clean paper under the stereomicroscope and adjust the magnification as high as feasible Cut the carbon electrode with a scalpel blade under the microscope Be sure not to cut at a silicone droplet if any because you may not be able to move the carbon electrode close enough to the 99 membrane with an enlarged silicone shield around the tip To fill the electrode with KCl you need you make an adaptor first Cut a 10 cm quartz tubing i d 75 um insert it into a 25 G needle and then glue them together using a melted with lighter yellow tip Connect the adaptor to a 0 45 um filter and a syringe filled with 3 M KCl and then insert the adaptor tip into the electrode Push the syringe to fill the electrode with KCl and pull th
20. about 0 5 1 ug ml Fig 2 2 Two other expression constructs were made One is Syt His s and the other one is HBM His s Syt1 The number of histidine was increased in 37 hope that the binding affinity would be higher and more stringent washing conditions could be applied For Syt His s the tag was moved to the C terminus simply because the N terminus tagged construct did not express well For HBM His s Sytl the honeybee secretory signal was added at the very N terminus to ensure that the mammalian type I transmembrane protein e g Sytl can be inserted into the membrane correctly in insect cells and thus increase the final yield As shown in Fig 2 3 the quality and quantity of both expression constructs were significantly increased although still not perfect Using dot blot and serial dilution the concentration of the most concentrated fraction was 300 ug ml C2AB domain for both constructs Fig 2 4 In large scale preparation the protein expression level of HBM His s Sytl was much higher than that of Syt His s 15 mg v s 4 mg C2AB domain however the final yield was similar Fig 2 4 More Ni NTA beads should be added into the crude protein lysate of HBM His s Syt1 in future experiments because about half of the expressed protein was in the flow through Fig 2 4 In addition one more chromatographic purification step may be desired to further purify the product Purification of Synaptobrevin 2 After affini
21. defining patches with capacitance increases smaller than 50 fF as inactive see Fig 3 3A left panel and patches with more robust non SG fusion as active patches see Fig 3 3A right panel Notably the exocytotic response often appeared to be followed by an endocytotic response even in the presence of Ca when ATP and GTP were present on the cytoplasmic side see Fig 3 3A To test whether the non SG fusion is SNARE dependent we treated the patches with tetanus toxin TeTx light chain at a concentration of 200 nM for 2min As shown in Fig 3 3C both the amplitude and the ratio of active RBL patches were decreased significantly 76 by treatment with the wild type toxin compared with the inactive mutant form indicating that the fusion is indeed SNARE dependent Notably however the treatment of patches with 1 mM N ethylmaleimide NEM did not block non SG fusion Table 3 1 implying that SNARE cycling which is blocked by NEM Xu et al 1999 is not required for non SG fusion in excised patches As mentioned in the Introduction it is reported that non SG fusion in bovine chromaffin cells is toxin insensitive Xu et al 1998 A simple explanation for the discrepancy to our data is that RBL and chromaffin cells use different sets of SNAREs for non SG fusion and the SNAREs accounting for non SG fusion in chromaffin cells are toxin resistant Another possibility is that the SNAREs of non SGs in chromaffin cells are complexed su
22. hydroxytryptamine autofluorescence Biophys J 76 1835 1846 Xu J Tang K S Lu V B Weerasinghe C P Tse A et al 2005 Maintenance of quantal size and immediately releasable granules in rat chromaffin cells by glucocorticoid Am J Physiol Cell Physiol 289 C 1122 33 Xu T Ashery U Burgoyne R D and Neher E 1999 Early requirement for alpha snap and nsf in the secretory cascade in chromaffin cells EMBO J 18 3293 3304 Xu T Binz T Niemann H and Neher E 1998 Multiple kinetic components of exocytosis distinguished by neurotoxin sensitivity Nat Neurosci 1 192 200 Yamaguchi T Dulubova I Min S Chen X Rizo J et al 2002 Sly1 binds to golgi and er syntaxins via a conserved n terminal peptide motif Dev Cell 2 295 305 Yaradanakul A Wang T M Lariccia V Lin M J Shen C et al 2008 Massive Ca induced membrane fusion and phospholipid changes triggered by reverse Na Ca exchange in BHK fibroblasts J Gen Physiol In press Yoon T Okumus B Zhang F Shin Y and Ha T 2006 Multiple intermediates in snare induced membrane fusion Proc Natl Acad Sci U S A 103 19731 19736 226
23. including proteins Nevertheless after several years we have not been able to establish conditions that allow routine recordings of neurotransmitter release in excised patches including efforts with several cell types For example bovine chromaffin cells readily allowed seal formation with large diameter pipettes but excised patches are not stable with significant solution flow thereby greatly limiting their use Overall our experience is that the ability to induce neurotransmitter release is very easily lost or destroyed during excision procedures and we did not overcome this limitation for routine work Given the strong inhibition of fusion by cell swelling we strongly suspect that mechanical factors are of most importance but it also remains possible that important soluble factors are lost from the patches 86 Non secretory fusion in excised patches In this article we have described that non SG membrane fusion is both robust and massive in several cell lines when studied by whole cell voltage clamp while responses in excised patches showed a large degree of variability Specifically we found that the ratio of active patches varied substantially from batch to batch of the cells as well as with the length of time after isolation Sometimes no fusion at all was observed in excised patches from an entire batch of cells although whole cell responses were robust and highly reliable in the same cell batch Also individual cell batches w
24. oscillating between p and q volt In this case the command potential can be expressed as Vc 0 Vsi 4 24 where O is the average of p and q and Vsi si is pronounced as letter C is half of the peak to peak amplitude Since O is a constant potential its resulting current is a DC current component After subtracting the DC component from the total current the resulting trace is identical to the one mentioned above Thus all equations derived above are still valid Note that the peak current in Eq 3 8 is a but in Eq 4 21 is a b It is because the definitions for Vc are slightly different In Eq 3 8 Vc is the absolute value of the potential and the baseline current is zero In Eq 4 21 Vc is a relative potential defined as half of difference between two peak potential and in this case the actual baseline current value is b for the raising potential in the fitting routine so the peak current is a b I SQA computer implementation It seems easy to do the curve fitting and extract all cell parameters once we have the equations however it is not true The real world is much more complex and all kinds of unexpected errors and or conditions will occur within the fitting routine if there is a chance for them A tremendous amount of efforts have been made to find test and control these errors conditions in order to make the fitting routine workable and giving reasonable outputs The entire fitting routi
25. 1 Chromatographic purification of untagged Syt1 A Dot blot using anti Syt1 antibody and representative fractions from each column are shown Fraction 12 of HA chromatography was not subjected to Mono S purification simply because I did not know that there were lots of Syt1 in it at that time B Western blot of HA fractions The HA column was able to separate Sytl monomer fractions 11 12 dimer fraction 18 arrow and degraded Syt1 fraction 14 46 Figure 2 2 Purification of His Syt1 Coomassie blue stained gel is shown After affinity purification the quality of Sytl arrow was poor and the concentration was low too In input FT flow through W wash E eluate B beads left BSA bovine serum albumin A Imidazole mM In W 50 100 150 200 250 500 B 66 2 m ab 45 HBM His Syt1 In W B Syt1 His 47 Imidazole mM 50 100 150 200 250 500 B Figure 2 3 Purification of HBM His Sytl and Sytl His Coomassie blue stained gels are shown A HBM His Sytl and B Sytl His The quality and quantity of both expression constructs were significantly increased In input W wash B beads left 48 HBM His Sytl Sytl His Figure 2 4 Estimation of HBM His Syt1 and Sytl His concentration Dot blot with serial diluted samples are shown Sytl C2AB domain with known concentration was used as a standard A HBM His Sytl and B Sytl His
26. 2 Rm Cap tau 1 Ra G 100000 G G 1000 F l exp duration tau2 l exp duration tau2 G2 2 asymp2 asymp2 Ft peak2 V peak2 asymp2 Rm2 1 G2 Ra2 V asymp2 2 Rm2 Cap2 tau2 1 Ra2 G2 100000 G2 G2 1000 if alpha beta 1 1V 10pF if alpha beta 1 1V 1MOhms if alpha beta 1 1V ins errCap Cap StdCap 1 100 errCap2 Cap2 StdCap 1 100 errRm Rm StdRm 1 100 errRm2 Rm2 StdRm 1 100 errRa Ra StdRa 1 100 errRa2 Ra2 StdRa 1 100 Functions SqCF5 and SgQ3 are two separate functions for I SQA and Q SQA respectively They are created for algorithm development and will be implemented into CapEngine4 once the routines are validated In the absence of noise and a filter function the errors of Ra Rm and Cm for both methods in this case are in 10 10 and 10 respectively Square wave perturbation PSD analysis of SQA data As mentioned in the Introduction PSD always gives better signal to noise ratio compared with SQA Since square waves are composed of sine waves with different harmonies and amplitudes in the Fourier space Thompson et al 2001 PSD can also be applied when square pulses are given However since the phase angle might be changing during the experiment it is important to keep in mind that the PSD result might not be valid if the clamping time constant changes a lot To find the optimal phase angle offline
27. 29 GST tag the beads were resuspended in 1 5 ml of the same buffer containing 2 units of thrombin and kept at room temperature for 4 hr with gentle agitation After cleavage the supernatant was collected by centrifugation at 500 g for 5 min and the beads were rinsed again using the same buffer The 0 5 ml supernatant were combined with the previous one 2 ml and then mixed with 20 ml of Mono S loading buffer 50 mM NaCl 25 mM HEPES 0 1 Triton X 100 2 mM B mercaptoethanol pH7 0 For Mono S chromatography the column was equilibrated with the same buffer described above before the sample was loaded After loading the column was washed with 15 ml of buffer containing 100 mM NaCl and Syb2 was eluted by ramping the NaCl concentration gradually from 100 mM to 400 mM in a total volume of 20 ml The elution peak of Syb2 was located at a NaCl concentration of 150 200 mM The concentration of the eluted Syb2 was determined by measuring OD 2so Reconstitution of Syt1 and Syb2 into liposomes The detergent mediated direct incorporation method Rigaud et al 1995 Rigaud and L vy 2003 was used for Syt1 Syb2 reconstitution The protocol for preparing large unilamellar vesicles LUVs is from Avanti polor lipids Alabaster AL To prepare LUVs POPC 1 palmitoyl 2 oleoyl sn glycero 3 phosphocholine and DOPS 1 2 dioleoyl sn glycero 3 phosphoserine were mixed in a glass tube with a molar ratio of 85 15 The solvent chloroform was exp
28. 4 5 P gt microdomains at syntaxin clusters can activate the exocytotic sites Aoyagi et al 2005 PI 4 5 P2 appears to be required for priming of vesicles in pancreatic B cells Olsen et al 2003 and the yeast vacuole fusion process Mayer et al 2000 and it regulates the releasable vesicle pool size in chromaffin cells Milosevic et al 2005 In dense core vesicle fusion PI 4 5 P gt acts via the calcitum dependent activator protein for secretion CAPS to increase the initial rate of fusion Grishanin et al 2004 Loyet et al 1998 Furthermore PI 4 5 P2 is suggested in a liposome liposome fusion system to promote fusion directly via its interaction with the neuronal Ca sensor Synaptotagmin Syt I Bai et al 2004 Finally phospholipase Cs PLCs which cleave PI 4 5 P2 to produce diacylglycerol DAG and inositol triphosphate IP3 are implicated to regulate some 62 vesicle fusion processes Fukami et al 2001 Jun et al 2004 and in general the conversion of large phospholipid head groups to smaller ones is expected to favor fusion of phospholipid vesicles Other phosphoinositides such as phosphatidylinositol 3 4 5 triphosphate PIP3 and phosphatidylinositol 3 phosphate PI 3 P also play important roles in membrane trafficking including events leading up to fusion Lindmo and Stenmark 2006 For example inhibition of a class IA PI 3 kinase PI 3 K which produces PIP3 reduces receptor mediated degranul
29. C B Miyake K et al 2003 Impaired membrane resealing and autoimmune myositis in synaptotagmin vii deficient mice J Cell Biol 162 543 549 Chen X Ara D Wang T M Gilpin C J Zimmerberg J et al 2006 Snare mediated lipid mixing depends on the physical state of the vesicles Biophys J 90 2062 2074 Chen Y A Scales S J and Scheller R H 2001 Sequential snare assembly underlies priming and triggering of exocytosis Neuron 30 161 170 Chernomordik L V Vogel S S Sokoloff A Onaran H O Leikina E A et al 1993 Lysolipids reversibly inhibit ca 2 gtp and ph dependent fusion of biological membranes FEBS Lett 318 71 76 Choi W S Kim Y M Combs C Frohman M A and Beaven M A 2002 Phospholipases dl and d2 regulate different phases of exocytosis in mast cells J Immunol 168 5682 5689 Chow R H and von R den Ludolf Electrochemical detection of secretion from single 216 cells In Single channel recording Sakmann B and Neher E 1995 245 275 Clifford E E Parker K Humphreys B D Kertesy S B and Dubyak G R 1998 The p2x1 receptor an adenosine triphosphate gated cation channel is expressed in human platelets but not in human blood leukocytes Blood 91 3172 3181 Cohen J S and Brown H A 2001 Phospholipases stimulate secretion in rbl mast cells Biochemistry 40 6589 6597 Cool D E and Blum J J 1993 Protein tyrosine phosphatase activity in leishmania
30. Capmeter6v3 can not find the file default values defined in the main program will be used handles aiSamplesPerTrigger it is used to set the AI SamplesPerTrigger property and calculated using code floor 1 handles rSR 0 001 handles aiSR For instence if handles aiSR is 100 kHz and handles rSR is 100 Hz i e 10 ms interval the program will acquire 9 ms of data i e 900 samples and then wait for the next trigger which is 10 ms apart from each other handles SpmCount it is used to set the AI SamplesAcquiredFcnCount property The value 173 is handles aiSamplesPerTrigger round handles rSR 0 5 With the same example shown above the program evokes function process_data every 0 5 s i e 45000 samples to extract and process acquired signals handles filterv2p number of points to be averaged in the background averaging filter The relationships among handles aiSamplesPerTrigger black gray handles SpmCount black gray of the entire time frame and handles filterv2p black are shown below Trigger For1 digitized C G Ra For 1 digitized current Ch4 process_data 0 5 5S handles aidata n by 5 matrix containing all the processed data handles aodata n by 2 matrix containing output signals for AOO and AO1 handles aitime column array containing corresponding time points for handles aidata 4 4 Capmeter 1 What is it Capmeter 1 serves as a plain chart recorder with digital filters and some other
31. Cdiff and Cabs user defined not in standard library and the peak indexes are determined by Cfind using a threshold of 0 15 threshold Variable fc represents number of peaks found and because the last curve is usually incomplete according to my experience it is removed from the data set If no peak is found Sp lt 1 variable quality is set to zero and other fitting procedures will be skipped The next step is to calculate the baseline or middle line more precisely of the current trace This step is important because the DC current is not always or rarely zero Considering that the seal becomes bad during the recording and the liquid junction potential is not compensated perfectly or ion channels open in an experimental condition a DC current will flow into or out of the pipette for sure In these cases the 136 middle line subtraction becomes critical C codes for middle line calculation for 1 0 1 lt fc fmod fc 2 1 zerosum dataA indexpeak i zeroline zerosum double i Variable dataA is an array containing raw current The loops add equal numbers of positive and negative peaks together and the average value is the middle line According to the codes it is important to keep in mind that data clipping shall be avoided because the middle line can not be determined correctly if it happens In Capmeter 6 the PSD I SQA Q SQA pop up menu handles Cm changes its BackgroundColor to red if t
32. Chapter 3 Fusion of endogenous vesicles in excised patches This chapter is basically the paper entitled Ca dependent non secretory vesicle fusion in a secretory cell co authored by only my mentor Dr Hilgemann and I Some more details about the methods are described after the paper and the derivation of the equations is discussed in detail in Chapter 4 57 Ca dependent non secretory vesicle fusion in a secretory cell Tzu Ming Wang and Donald W Hilgemann Department of Physiology University of Texas Southwestern Medical Center at Dallas Dallas Texas 75390 U S A Running Title Characterization of non secretory vesicle fusion Key words mast cell exocytosis wound repair amperometry software lock in amplifier Correspondence Donald Hilgemann Department of Physiology ND13 124 UTSouthwestern Medical Center 6001 Forest Park Dallas TX 75390 9040 email donald hilgemann utsouthwestern edu tel 1 214 645 6031 fax 1 214 645 6049 58 3 1 Abstract We have compared Ca dependent exocytosis in excised giant membrane patches and in whole cell patch clamp with emphasis on the rat secretory cell line RBL Stable patches of 2 4 pF are easily excised from RBL cells after partially disrupting actin cytoskeleton with latrunculin A Membrane fusion is triggered by switching the patch to a cytoplasmic solution containing 100 200 uM free Ca Capacitance and amperometric recording show that large secretory
33. It is critical to select a proper ID especially for the M series boards from NI because they usually have more than one device ID In CapmeterSetting m file you may specify the N device ID you want to use by assigning handles nidagid l or 2 or etc For example if handles nidaqid 1 and the first item in the InstalledBoardlIds property of the board is Dev2 Dev2 is the ID to be used The value of handles nidagid will be pointed to the actual device ID later using code handles nidagid Daginfo InstalledBoardIds 1 handles nidaqid handles dispindex display index used to indicate current channels displayed on the 171 172 panels If handles dispindex 1 5 2 that means the top middle and bottom panels display Chl Ch5 and Ch2 respectively handles sliderIrange slider range in online mode Default value is 120 that means 2 min handles slider2range slider2 range in online mode Default value is 50 that means 50 s handles shiftswitch method for cross correlation used in PSD analysis of SQA data 0 C correlation 1 G correlation 1 both G and C for cross correlation handles aoChI convert command sensitivity for AOO in mV V handles aoCh2convert command sensitivity for AO1 in mV V handles aiSR acquisition speed for AI in Hz handles aoSR acquisition speed for AO in Hz handles rSR frequency for digitizing data in Hz All of the above variables can be adjusted in CapmeterSetting m file If
34. Na Ca exchange current A Effects of cytoplasmic osmolarity on fusion responses The left bar graphs give results for isoosmotic solution the middle bars for cytoplasmic solution with 200 mM sucrose and the right bars for cytoplasmic solution diluted 30 with distilled water B Effects of extracellular osmolarity on fusion responses From left to right the bar graphs are with 1 standard extracellular solution for 2 min after cell opening 2 standard extracellular solution with the NMG aspartate concentration reduced by 80 mM for 2 min after cell opening 3 standard solution reapplied for 2 min after applying hypoosmotic solution for 2 min and 4 as in 3 with 5 uM wortmannin in all cytoplasmic solutions C Capacitance responses of RBL cells for pipette perfusion of cytoplasmic solution with 200 uM free Ca The left bar graph indicates the response magnitude for control cells the middle graph for cells swollen with hyperosmotic cytoplasmic solution 200 mM sucrose for 2 min and the right bar for cells swollen with extracellular solution in which the NMG concentration was reduced by 80 mM 114 115 gt B 140 70 wel I gal wr 4 100 i Pai 50 e 80 g 40 id N Pa a 30L Z wt MEF syt7 MEF f L 1 L f 5 Time 5 5 Time s Cm pF Cm pF Figure 3 9 Synaptotagmin VII is not required for non SG fusion Whole cell recordings from wildtype wt A and syt7 knockout B mouse embryonic
35. Subsequently the membrane was washed again with TBST for 3X10 min with vigorous shaking For chemiluminescent detection the homemade ECL solutions were used The ECL solution A contains 5 mM luminol 200 uM p coumaric acid and 100 mM Tris Cl pH8 5 protected from light The ECL solution B contains 5 4 mM H20 from 30 concentrate and 100 mM Tris Cl pH 8 5 In my experience the quality of H20 is critical for the quality of the prepared ECL solutions and I use H20 from Fluka Equal volume of the two solutions were mixed right before use and dispersed evenly 25 on the membrane for 1 min The excess of ECL mixture was removed by blotting the membrane with task wipers Kimwipes and the exposure and development of the film was performed in a darkroom Dot blot Express For chromatographic experiments with sample of unknown elution volume it might be impractical to run standard western blot for each collected fraction to determine fractions used for the next column To speed up the process a western blot without the gel running and transfer steps was performed Two microliter of 2X sample buffer and 2 ul sample from each fraction were mixed and 3 ul of each boiled sample were dotted on a nitrocellulose membrane To place the dots the nitrocellulose membrane was placed on a paper towel before dotting to avoid excessive spreading of the samples and the plastic insert from a 200 ul EZ Rack tip transfer system Denville S
36. The results define clear differences between non SG and SG fusion processes and provide new insights into the physical basis of non SG fusion 64 3 3 Methods Cell culture Adherent RBL 2H3 cells were cultured in Dulbecco s Modification of Eagle s Medium DMEM Mediatech Herndon VA supplemented with 15 fetal bovine serum FBS Cells were plated on uncoated Petri dishes 1 2 days before experiments and collected by treating them with 0 25 trypsin EDTA solution Serotonin and 5 hydroxytryptophan 0 2 mM each were also added to cells for amperometric recording one day before the experiments to increase the formation of SGs Mahmoud and Fewtrell 2001 Williams et al 1999 After treatment with trypsin the cells were resuspended in the culture medium and left in the CO incubator for 30 min Cells were then treated with Latrunculin A 50 100 ng ml at 37 C for 5 min before experiments as this treatment clearly facilitated the formation of giant excised patches with fusion competent vesicles Mouse embryonic fibroblasts MEFs were cultured in DMEM supplemented with 10 FBS and penicillin streptomycin They were plated on cell culture dish 1 2 days before experiment and collected as stated above The Syt VII deficient MEF cell line was provided by Dr Thomas Siidhof UTSouthwestern the PLC61 and PLC61 54 deficient MEF cell lines were provided by Dr Kiyoko Fukami Tokyo University and the PLCy1 deficient cell line was provided by Dr
37. Y Ch2 X Pulselog structure containing pulse protocol and other information wavept number of AO points for each trigger Length handles aodata 1 data structure array containing information for each pulse train pulse shape of the pulse protocol V NaN is assigned if number of pulses is set to inf Pulseinfo initial step V length AO points interval AO points step increment V number of pulses rounds note reserved for future application 162 trigger trigger time in seconds data array containing processed data Ch1 Ch2 Ch3 Ch4 Ch5 labels cell array containing labels and other information bin at 0 5 s channel applied time s value of each channel Ch1 Ch5 label rawdata generated when ExRaw button is clicked DISABLED by default time s AIO AI1 AI2 rxr if the RXR check box is checked it contains sampled raw data time s AIO AI1 AI2 time array containing time for processed data in s version structure containing version information Shell version of the GUI Engine version of the CapEngine Subtracting the baseline Capmeter6v3 08331 File 080125RBL adenosine 2 mat 0 11 T I T lo x 0 1 1 Left click 7 gt OOuMCa2 3 Left click then right click 200uMCa2 osne i 0 04 a 70 80 90 Y axes 4 479 3071 EAEE A F c e ales s TE f se E suction 2 a pa ce i 100 as
38. a 1 mV trigger signal was added to the sine or square wave usually at 100 Hz For the lock in amplifier function the phase sensitive detector of the program multiplied the current with either an in phase or an orthogonal reference signal The direct current DC component of the product was extracted by averaging to cancel the non DC noise The double of the DC component was assigned to X or Y where the in phase or the orthogonal reference signal was employed respectively The optimal phase angle 0 was determined by small changes of the optimally adjusted capacitance compensation of the patch clamp as follows wX 3 1 0 0 arctan y EO where and Xo Yo and and X Y represent values before and after changing 67 capacitance compensation respectively For square wave perturbation time domain method see Fig 3 1 continuous square pulses were applied and current transients were recorded and analyzed on line using Capmeter 6 The average current i e the DC component was first calculated and subtracted from the total current The resulting trace was then divided into two parts and fitted separately to exponential functions For curve fitting the steady state current I b in Fig 3 1A was determined as the asymptote of current from the averages of three consecutive data sections of equal length A B and C Fig 3 1B dashed sections B AC 2 T An 2 2B C A 2 This equation is the
39. a glutamatergic synapse Pflugers Arch 453 261 268 Olsen H L Hoy M Zhang W Bertorello A M Bokvist K et al 2003 Phosphatidylinositol 4 kinase serves as a metabolic sensor and regulates priming of secretory granules in pancreatic beta cells Proc Natl Acad Sci U S A 100 5187 5192 Osipchuk Y and Cahalan M 1992 Cell to cell spread of calcium signals mediated by atp receptors in mast cells Nature 359 241 244 Ostrowicz C W Meiringer C T A and Ungermann C 2008 Yeast vacuole fusion a model system for eukaryotic endomembrane dynamics Autophagy 4 5 19 Pallen C J and Tong P H 1991 Elevation of membrane tyrosine phosphatase activity in density dependent growth arrested fibroblasts Proc Natl Acad Sci U S A 88 6996 7000 Patton C Thompson S and Epel D 2004 Some precautions in using chelators to buffer metals in biological solutions Cell Calcium 35 427 431 Perin M S Fried V A Mignery G A Jahn R and Stidhof T C 1990 Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein 222 kinase c Nature 345 260 263 Pertile P Liscovitch M Chalifa V and Cantley L C 1995 Phosphatidylinositol 4 5 bisphosphate synthesis is required for activation of phospholipase d in u937 cells J Biol Chem 270 5130 5135 Poole A R Howell J I and Lucy J A 1970 Lysolecithin and cell fusion Nature 227 810 814 Qin W Pappan K and Wa
40. bach erd 175 Running the PRO SAM pas c2 8 ooseh erm dcee dante ssicapare tei he datnisenbulte eomdoseanadats seeded sete Uys deocteon dale 177 Giving pulses and applying NOES c ss5sdarccsscedsaveserciorseeviiiodndtncaa nc uate 178 xii Methods for charge imtesrationys iss sccececsiass ccbacccsvhdvscuaccsstccoveiventtvabs S saebdvscdeancdanstecs coasect 180 Display modes 0nliNe inse OR rn EOP Ee ene eo 181 Displ y MOSS OTE NG aire 8 coreteradth de hncsitsa ast iaine a aaa heel 182 Variables in the WOrkSpaCeinccccczscccuclsesseel iccckucchaninsedsisctea sleeashasstanaedesans Gveardanesevenavess 184 Tg information TOW vista nsey sons costes ate dashnensaney seaveeay ba vaasiovaeds sien ound ea A aea anaa aiiai 186 Main variables in the pro Statics sosstye coxeinuce vet vencnceetansosnee ee caneecustioascines tious eoveyeatatevteuaess 188 4 6 Dynamically linked Subroutines cece ccccccccceeeeeeesteeeeeeeeeeeeseeseneeees 190 PAPE MG Iie WIV ERY FD ace AS Biogas oss co gp E wc oe eg uke EE gs suet ea vo Gaudet AE AE 190 DAlter 0c pe ee eee nee a Oa one Me a 191 D lter2 MEXWI Zonia ie cans un a e serena easy eeu a a twats 192 PIS CHIEN WS Dass ssiases ce eects a a a a a leave eiiaaata a a aa at 193 TOMPCRMCRWSD sso a a co elle anes cael ca Berek al A els a iota ae Goda 194 PhaseMatcher MEXW3 Zared tentei Ee ae aE EE ee a e E 195 SqWaveCale Mex WS ran oea e n aa a A T a a E E Se 196 4 7 Capmodule4 nnen ie A E daa end E eee 197
41. been reported that the wound repair machinery is TeTx sensitive in other model systems Togo et al 1999 and as mentioned earlier non SG fusion is toxin insensitive in bovine chromaffin cells Xu et al 1998 One evidence supporting the notion of wound repair by non SG fusion comes 89 from the low Ca sensitivity of the Ca sensor which is relevant to the ongoing debate about the role of Syt VII in membrane repair McNeil and Kirchhausen 2005 Syt VII is reported to be a high affinity Ca sensor in SGs of PC12 cells Wang et al 2005 The low Ca sensitivity of non SG fusion could in principle ensure that these vesicles are not affected by normal Ca signaling in the cell and that they would fuse with the plasmalemma only when bulk Ca influx comes from the wounded sites In addition to establishing a new approach to study non SG fusion with giant excised patches we have developed computer software that can be useful to other groups to implement both time domain and frequency domain methods Lindau and Neher 1988 In our experience the time domain method is especially useful when clamp time constants are relatively long e g hundreds of microseconds in cardiac myocytes With these approaches we have delineated several new characteristics of non SG fusion First while this type of fusion is SNARE dependent it is not NEM sensitive in excised patches Second while this type of fusion is ATP dependent it is not phosphoi
42. by expressing the open form of Stx Koushika et al 2001 Richmond et al 2001 It is attracting that Stx is blocked by Munc18 and released in the active zone however it cannot explain some controversial experimental observations As mentioned previously neurotransmitter release was abolished rather than increased in Munc18 knockout mice Verhage et al 2000 In addition Secl a Munc18 homologue in yeast binds to Stx in assembled SNARE complex rather than the closed Stx Carr et al 1999 Nevertheless in other vesicular transport systems such as ER and Golgi Stx homologues do not form the closed conformation and the Secl Munc18 homologues bind to the N terminus of Stx Fig 1 4 middle panel Yamaguchi et al 2002 Recently it was reported that Munc18 does bind to the assembled SNARE complex Fig 1 4 lower panel Dulubova et al 2007 and the two different interacting modes open and closed Stx are essential for synaptic vesicle fusion and are coupled by functionally critical binding to Stx N terminus Khvotchev et al 2007 Synaptotagmin 1 Synaptotagmin Syt 1 is a type I transmembrane protein with its N terminus in the lumen of the synaptic vesicle It contains a N terminal transmembrane region and two C2 domains in its C terminus and was proposed to be a potential Ca sensor in regulated exocytosis Perin et al 1990 Further experiments indicated that Sytl binds to negatively charged phospholipids in a Ca
43. cells with 10 uM ATP It is reported that the cytosolic Ca modulates the supply of release competent vesicles in chromaffin cells Smith et al 1998 and low basal Ca concentration may result in the depriming of the vesicles Neher 2006 Smith et al 1998 To ensure that SGs remain primed before the experiment I incubated cells with 10 uM ATP to elevate basal cytoplasmic Ca concentration ATP is the ligand for purinergic P2 type receptors which are also expressed in RBL cells Clifford et al 1998 Osipchuk and Cahalan 1992 An ATP concentration of 30 uM had been used to elevate intracellular Ca concentration of astrocyte to 1 uM Kanemaru et al 2007 I was using 10 uM ATP incubation for 10 min and hoping that the Ca concentration in RBL could fall into a sub micromolar range The outcome was that I still could not preserve SGs in the patches after treating the cells with ATP Pre incubate cells with 100 nM PMA PMA phorbol 12 myristate 13 acetate is a DAG analogue that has been shown to increase the primed SGs in chromaffin cells Gil et al 2001 However the RBL cells became enlarged and transparent after treating them with 100 nM PMA for only a few minutes In addition PMA is a potent carcinogen and will contaminate the recording chamber and might be inhaled by mouth when making the seal Thus I decided to stop this approach 3 Use bovine chromaffin cells 104 I obtained bovine chromaffin cells fr
44. concentration of the mixture should be 5 mM The mixture was kept at room temperature for 30 min for protein incorporation Subsequent dialysis was used to remove OG from proteoliposomes The liposomes were dialyzed against 500 ml of electrophysiological recording buffer at room temperature without stirring for 30 min and then dialyzed against 500 ml and 1 L buffer at room temperature with stirring for 30 min and 1 h respectively The final dialysis step 31 was carried out at 4 C overnight with 1 L buffer and 1 g of Bio Beads Bio Rad added to the buffer to absorb residual OG The detergent free proteoliposomes were stored at 4 C Quality assays of the reconstituted proteoliposomes The size distribution of proteoliposomes was measured by using dynamic light scattering For leakage assay LUVs containing self quenched carboxyfluorescein were made The lipid film was hydrated with buffer containing 100 mM _ 5 6 carboxyfluorescein and proteoliposomes were made using the same procedures as described above The proteoliposomes were diluted 80X in buffer and the leakage of the content i e carboxyfluorescein was measured by means of dequenching of leaked carboxyfluorescein in a fluorescence spectrophotometer At the end of measurement liposomes were lysed by adding 1 Triton X 100 to the cuvette to get maximal fluorescence To test if recombinant proteins were incorporated into liposomes rather than sticking on them urea 4M sto
45. donovani Mol Cell Biochem 127 128 143 149 Coorssen J R Schmitt H and Almers W 1996 Ca2 triggers massive exocytosis in chinese hamster ovary cells EMBO J 15 3787 3791 Cousin M A Malladi C S Tan T C Raymond C R Smillie K J et al 2003 Synapsin i associated phosphatidylinositol 3 kinase mediates synaptic vesicle delivery to the readily releasable pool J Biol Chem 278 29065 29071 De Matteis M A and Godi A 2004 Pi loting membrane traffic Nat Cell Biol 6 487 492 Dennis E A 1994 Diversity of group types regulation and function of phospholipase a2 J Biol Chem 269 13057 13060 Dernick G Alvarez de Toledo G and Lindau M 2003 Exocytosis of single chromaffin granules in cell free inside out membrane patches Nat Cell Biol 5 358 362 Dernick G Gong L Tabares L Alvarez de Toledo G and Lindau M 2005 Patch amperometry high resolution measurements of single vesicle fusion and release Nat Methods 2 699 708 Dougherty P J Davis M J Zawieja D C and Muthuchamy M 2008 Calcium sensitivity and cooperativity of permeabilized rat mesenteric lymphatics Am J Physiol Regul Integr Comp Physiol Dulubova I Khvotchev M Liu S Huryeva I Stidhof T C et al 2007 Munc18 1 binds directly to the neuronal snare complex Proc Natl Acad Sci U S A 104 2697 2702 217 Dulubova I Sugita S Hill S Hosaka M Fernandez I et al 1999 A conformational swit
46. factor to select a region for fitting else the entire data range is used The frequency option is designed to adjust the oscillating frequency and amplitude automatically according to the estimated time constant and peak current so that the cell membrane can be charged properly and the signal is kept in an optimal range However this function in Capmeter function SqAlgo is never perfect under my criteria and I will probably remove it in the future versions It is recommended to keep the Auto check box checked and do not change the default settings 159 Using digital filters There are two digital filters implemented One is the running filter the other is the background averaging filter The running filter does not change the acquired data it simply processes digitized data points that have been shown on the screen Whenever the axes are refreshed all the points are processed again by the filter if the filter mode is not Bypass Two types of running filters are available one is running mean the other is running median Running median filter is good for removing unexpected glitches in the trace You may select one of them from the pop up menu in the Digital filter panel To set a window for calculating mean median you put a number in the edit box next to the pt points button and then press Enter Running filtering can be applied on all channels and it is performed by Dfilter2 mexw32 which will be discussed in l
47. in Capmeter6v3 the program calls a function named PhaseMatcher2 mexw32 which is written in C The PhaseMatcher2 scan and shift the phase angle from n to m and check the cross correlation between phase shifted Y or X or both from PSD and C or G or both from SQA The angle that gives highest cross correlation phase shifted X and Y at the angle and the correlation coefficient are returned from the function The equation for correlation coefficient is YW AYHC C R 1 2 5 1 2 4 40 Er oce 4 which can be found at http local wasp uwa edu au pbourke other correlate index html An example is shown in Fig 4 11 The noise of Q SQA acquired data gray is suppressed after PSD analysis black Part of the MATLAB codes handling offline phase shift are shown below MATLAB codes for offline phase shift Executes on button press in PhaseShift 148 149 function PhaseShift Callback hObject eventdata handles XData get handles plotl XData S size XData if 1 2 lt 2 Sempty plot return end indexl index2 IndexLoc handles aitime XData 1 1 XData l end XData handles aitime indexl index2 1 if handles menuindex 1 1 Y handles PSDofSQA indexl index2 1 sCapacitance at Chl X handles PSDofSQA indexl index2 2 sConductance at Ch2 else Y X s end y Chl Ch2 andles aidata indexl index2 1 SCapacitance a andles aidata indexl index2 2 Conductance a
48. lines are available As shown in Fig 3 9B non SG fusion was still robust in syt7 knockout MEFs 6 observations Unlike results with RBL cells it was notable that non SG fusion in MEFs often involved fusion of large vesicles Fig 3 9 arrowheads Whether such large vesicles can be lysosomes is unclear but it is evident that both types of fusion are still robust when syt7 is ablated We conclude that Syt VII cannot be an important Ca sensor for non SG fusion Relevant to the potential importance of phosphoinosidites phospholipases and alternative possible Ca sensors we tested whether this type of membrane fusion in MEF cells was affected by deletion of three PLCs No evident differences were found for membrane fusion episodes recorded using MEFs and excised patches from MEFs with deletion of PLCy1 PLC81 and both PLC6d1 and PLC64 versus a control MEF cell lines n gt 3 for all observations data not shown Ca dependence of non SG fusion in excised RBL patches To characterize further the Ca sensing machinery of non SG fusion we triggered fusion with different concentrations of free Ca in excised patches from RBL cells The concentration response data for free Ca versus rate of fusion shows a Hill coefficient of 2 an apparent Kp of 71 uM and a maximal rate constant of 2 1 s Fig 3 10 We point out that the two data points at low Ca concentrations were weighted to force the fit through these points Without
49. membrane and red puncta in the cytoplasm which serve as markers for co expression of proteins of interests Theoretically two different colors e g green and red can be targeted to these three cellular compartments i e nucleus plasma membrane and cytoplasm using this approach and cells expressing a maximum of 6 kinds of different proteins can be distinguished from others They have yellow if green and red colors are used nucleus membrane and yellow puncta in the cytoplasm The backbone vector is pIRES2 EGFP Clontech and an Agel site was introduced between the IRES internal ribosomal entry site and the fluorescent protein coding sequence The Agel site was used for later cloning steps To make the backbone vector pIRES2EGFP was digested by BstXI followed by Klenow fill in and NotI digestion The gel extracted vector was then ligated with EGFP sequence which was obtained from 33 pEGFP N1 Clontech by treating them with BamHI Klenow fill in and NotI digestion The resulting vectors are pIRES AgeI EGFP To make pBNG2 bicistronic nucleus is green three tandem repeats of nuclear localization signal NLS from SV40 large T antigen Fanara et al 2000 fused with EGFP coding sequence were generated via PCR and then subcloned into pCR2 1 TOPO vector After sequence confirmation the 3NLS EGFP fragment was excised from the pCR2 1TOPO vector by Agel and NotI digestion followed by gel extraction Vector pIRES Agel EGFP w
50. of the transformed bacteria was inoculated into 100 ml of YTA medium 1 6 tryptone 1 yeast extract 0 5 NaCl and cultured at 37 C overnight with vigorous shaking The overnight culture was added into 2 L of fresh YTA medium and further cultured at 37 C for several hours until the ODs reached 0 8 Induction of Syb2 expression was done by adding IPTG isopropyl B D 1 thiogalactopyranoside to a final concentration of 0 5 mM Cells were then culture at room temperature for additional 6 hr with vigorous shaking before harvest The cells were spun down rinsed with PBS prepared from PBS tablet Sigma and then resuspended in extraction buffer containing PBS 2 mM EDTA 1 mM EGTA 0 05 Tween 20 0 4 Triton X 100 0 5 N lauroylsarcosine 25 ug ml lysozyme 2 mM B mercaptoethanol and protease inhibitors Cells were broken in extraction buffer by sonication and then kept at 4 C for 30 min with gentle agitation to extract membrane proteins Membrane debris were removed by centrifugation at 15 krpm for 10 min with a JA20 rotor followed by additional centrifugation at 35 krpm for 30 min with a Ti70 rotor The supernatant was collected into a 50 ml centrifuge tube and 3 ml of glutathione sepharose 4B beads and additional 1 mM of PMSF were added into the crude protein lysate After overnight binding at 4 C with gentle agitation the beads were washed with 100 ml PBS with added 0 1 Triton X 100 and 2 mM f mercaptoethanol To remove the
51. patches cesceeceeseeteeeeeeeees 101 Chapter 4 Software and algorithm development nA Nah Kolshi a asco aegis oes se soos A owen baie eens 118 4 2 Algorithms for capacitance MeaSureMeNt ccceeeeeessseeeeeeeeeeeeeees 123 PHASES CUS ULV ES detecto onre norarneni e na Guay e A E tetaues 123 Phase sensitive detection online calculation of the phase angle eee 125 Phase sensitive detection offline adjustment of the phase angle cceeee 126 Phase sensitive detection computer implementation c ccccceeseeeeeseeeeeeteeeeeeees 127 Square wave perturbation based on fitting the current transient 00 cece 128 I SQA Computer implementations 1 28 esis cice ates devote lacus nine duel code date vase 134 Square wave perturbation based on fitting the transferred charges 0 c 0e 139 Q SQA computer implementations sscsissccensasecscasstecncdesseadonsiaschessaccdcavsaseceneseacececeses 144 Square wave perturbation validating the algorithms using Simulink 0 0 146 Square wave perturbation PSD analysis of SQA data eececesseeeeesteeeeeeeeees 148 xi 4 3 Capmeter 6 5 sc5 sted oi hee Ia eres ln ec ee 151 AN EDA TSG ainsi sactoa useage tec wusctia ota ame ecm dnc ln vt aaa nasa a ca Na gta a as 151 Systenreg ireMEntS sssrin e aE a E R E A RAE E A 151 Co n ction diagram sonene a cate E A E E E tanta 152 Things you need to know b
52. protein factors DAG itself is enough to promote membrane fusion between synthetic lipid bilayers Go i and Alonso 1999 All of the evidence suggests that the activities of PLC and the production of DAG are deeply involved in regulating and or mediating membrane fusion Among the PLC family PLCS is the one that is most sensitive to Ca activation Rhee 2001 An intracellular Ca concentration of 0 1 10 uM is enough to stimulate the activity of PLC61 Allen et al 1997 and it is also localized to the plasma membrane Lee et al 2004 Thus the hypothesis that PLC61 triggered certain forms of membrane fusion upon Ca dependent DAG production becomes very attractive While the PLC81 knockout mice showed problems of skin stem cell lineage commitment Nakamura et al 2003 knockout of another PLCS isoform PLC464 did show deficient acrosome reaction which is an exocytotic event in sperm required for fertilization Fukami et al 2001 Another PLC that comes into the scope is PLCy PLCy can be activated through several ways It can be activated by receptor protein tyrosine kinases PTKs nonreceptor PTKs and lipid derived messengers like phosphatidic acid PA and AA Rhee 2001 Both PA and AA are implicated in regulating membrane fusion Blackwood et al 1997 Latham et al 2007 Rickman and Davletov 2005 One interesting feature of PLCy is that it hydrolyzes mainly PI but not PI 4 5 P2 Mitchell et al 2001 and the activ
53. second 70 pipette with a sharp unpolished edge Hilgemann and Lu 1998 The patches were positioned in front of a temperature controlled 30 C solution outlet immediately after excision Membrane fusion was triggered by moving the patch to a solution outlet containing 0 2 mM free Ca Capacitance and conductance were measured using the Lindau Neher method Lindau and Neher 1988 Sine waves generated by Capmeter 6 with 20 mV peak to peak amplitude at 2 kHz were applied to the cell The current output from the patch clamp was low pass filtered at 10 kHz When sine wave perturbation was employed the optimal phase angle was determined as described above When patch amperometry was employed a hardware lock in amplifier SR830 Stanford Research Systems Sunnyvale CA was employed as it allowed a higher signal to noise ratio at oscillation frequencies gt 3 kHz Sine waves with Vms of 20 mV at 10 kHz were usually employed The signals were recorded by Capmeter 1 For whole cell recording with 5 um inner diameter pipette tips membrane fusion was initiated via perfusion of Ca containing nitrilotriacetic acid bufferd solution through a quartz capillary with a 40 um outlet manipulated within the patch pipette to a distance of 50 100 um from the cell opening Hilgemann and Lu 1998 Square wave 20 mV peak to peak perturbation at 0 5 kHz was employed in all experiments presented in this article for whole cell capacitance recording wi
54. t and the correction still valid We need some mathematical support to strengthen our belief The summation of the charges can be written as a geometric series j oy a b eP A 4 35 0 The general solution for a geometric series is sole 4 36 l r where r is the factor n is the number of members in the series and A is the first member in the series So Eq 4 35 can be expressed as a b A 1 e Qs 7 nEN 4 37 lao By defining a b A jA 4 38 Eq 4 37 can be re written as Qs A 1 e 4 39 Since nA is exactly an expression of time t Eq 4 39 is still a raising exponential function with the same time constant of T Q SQA computer implementation The Q SQA routine is in a function celled SqQ in CapEngine4 The implementations of Q SQA and I SQA are basically the same except that the integrated charges are used for curve fitting in Q SQA Theoretically the steady state current can also be calculated from the integrated charges that is by estimating the steady state slope of the charges Idt However according to my experience this approach gives noisier outputs compared with values estimated directly from the current Thus the way that the steady state current is estimated is the same in Q SQA and I SQA If the steady state current is valid the fitting routine subtracts it from the total current and then proceeds the integration Eq 4 25 using the following code
55. that biochemical processes are required to restore fusion capability Fig 3 8C presents the equivalent experiments for RBL cells with membrane fusion induced by pipette perfusion of cytoplasmic solution with 200 uM free Ca as in Figs 3 6 and 3 7 Results for hyperosmotic cytoplasmic solution with 200 mM added sucrose and for hypoosmotic extracellular solution with NMG reduced by 80 mM are very similar to results with BHK cells Membrane fusion responses are reduced by 74 and 75 respectively Thus the high sensitivity of the non SG fusion process to inhibition by cell swelling is verified across two cell lines and with different protocols to induce membrane fusion Synaptotagmin VII and PLCs are not required for non SG fusion The non SG pool in RBL cells can almost double the total surface membrane area Figs 3 7A 3 8C and it seems likely that this pool will become involved in the wound repair of the plasma membrane Lysosomes have been suggested to be the major vesicles that undergo Ca dependent exocytosis in nonsecretory cells Jaiswal et al 2002 and in wound repair Chakrabarti et al 2003 Furthermore it is suggested that the Ca sensor in lysosomal fusion is the synaptotagmin VII Syt VII Chakrabarti et al 2003 To test if Syt VII is important for non SG fusion we examined membrane fusion in mouse 83 embryonic fibroblasts MEFs because like RBL cells they also have robust non SG fusion and knockout
56. the Shot button as many times as you want and the stored data will be assigned to the Workspace variable shotdata once Capmodule4 is closed Variable shotdata is a m by 2 matrix and the first and the second columns represent command potential and current respectively If multiple shots are taken NaN is inserted between the shots To store the shotdata you have to use the Save button located at the top of the Workspace Variable shotdata will be cleared if you use the Save button in Capmeter Hot keys Num Lock 10 1 0 1 track 10 1 0 1 198 199 close mute The above diagram shows you the keypad of the keyboard with designated functions You may use the keypad to control the step command potential and other functions of the program Make sure Number lock of the keyboard is on if you want to use the hot keys The information flow start Al never triggered e Al TimerFen 0 05s gt noise _ peekdata Started al stat AO AO SamplesOutputFen 0 05s generate sound sequence Step command panel AO TimerFen 0 25 F scope q peekdata EI store mean current value m iani Saronni Serer 2 ps aaa d i Sto pp ed Wavecaic a frackFeedback get snapshot i i i assign shotdata to the adjustoffsetvalue retrieve mean current i refresh the scope Workspace if available The above diagram shows you the information flow in Capmodule4 Event
57. the membrane targeted EGFP could not been seen clearly under epifluorescence microscope 54 55 oyeUdsOWIOY ueq WI Hg syuouoduoo s214 JJe urezuoo 6 pUe LI OT souoso somgy y ur umoys SY uIoped uorssardxo y Joyo 0 poLojrdw sem gyouNPY pue VSTAYNS VIXIS JSUIeSe sorpoqnue YIM jojq uIasom pue pouojoqns r m sjj 9 JO s ruojoo Burs Jun ooz pue OOF 009 JO 8TYD Jopun PAPS AM S 99 E6TAATH T 8TIUNIN PUE VSTAVNS VIXIS Zurss adx 09 SIUH 29 AQEIS TTT NFH Hg OZ 6L 8L ZL 9LSL VL EL ZL LL HY 0L 68 Z 9S YU L HE lu 6ri ooz 8LyD s w 6r OOF 8LY9 jw 61 009 8LyD 56 A B Syt1 Syb2 Ca blank Ca y a L Syt1 Syb2 Ca Y i E v Syt1 Syb2 Ca _ E E WY ma Syt1 Syb2 2 im i Syt1 Syb2 Ca i blank C 100 Ti 5Y sitt syb2 20 Time s 20 i i Time s Figure 2 12 Perfusion of artificial liposomes to excised patches A After saturating the pipette with blank liposomes Ca it appeared that the speed of capacitance increment became faster after moving the patch to Sytl Syb2 proteoliposomes in the presence of Ca B The pipette was saturated with proteoliposomes without Ca first and then move to proteoliposomes with Ca to see if faster capacitance increment like in A could be observed Unfortunately there was nothing happening when Syt1 Syb2 liposomes were applied with Ca indicating that the result in A could be an artifact Arrowheads starting time points of perfusion
58. the function at all Scaling the data Capmeter6v3 08331 File 080125RBL adenosine 2 mat loj xj 20 T T T T T v i 10 1 Left click 4 2 Left click then right click 0 0415 0 041 1 L L L H 10 E 20 30 179 3071 405 T T 50 T T T T T l 1 EI LptokHz ab 1 YRS aL 4 4 fe re Ta fF wal B Z Bl dz 2 o25 ato 025 2 15 J a 3 A ne tok 1a 1 Set i Ef release s manually puse ao ao EJ po int 3 Ef suction Ej bubble Lock in Show data 0 57 a 1 roo He penri Bypass e P as se a save Sine cece s ja rx Auto lV Ch cha 6 ms geoe a a F231 es OF s 4 0 5 1 1 L 1 1 1 1 fi 0 20 40 60 80 100 120 140 160 O Copyright 2007 Tzu Ming Wang and Donald W Hilgemann 180 PSD only gives relative but not absolute capacitance reading so I usually use the capacitance compensation knob to make a standard peak during the recording and then convert the unit from V to fF afterward using the standard For instance if the standard peak represents 100 fF I put 100 into the edit box next to the Std button in red circle and then click the Std button it might be useful to subtract the baseline using DeDrift first After defining the standard using the mouse cursor 2 points a conversion factor is calculated and stored in the memory Whenever you want to scale the data on the scree
59. the model works let s follow the numbers in the figure Total current flowing through the electrode is driven by voltage difference between the electrode and the membrane Ra is in between In step 1 membrane potential from step 5 is subtracted from command potential and then divided by Ra in step 2 to get the actual current flow Part of transferred charges step 3 are used to charge the membrane step 4 and part of them are leaking out of the cell step 7 Accumulation of the charges on the membrane builds up membrane potential and the value is O Cm step 5 As the membrane potential increases current leaking out of the cell increases and the value is Vm Rm step 6 The leaked charges step 7 is also subtracted step 4 from the total charges step 3 To monitor the net current used to charge the membrane the derivative of total membrane charges is calculated in step 8 213 214 Figure 4 11 Square wave perturbation PSD analysis of SQA data PSD always gives better signal to noise ratio compared with SQA Since square waves are composed of sine waves with different harmonies and amplitudes in the Fourier space PSD can also be applied when square pulses are given An example is shown above The noise of Q SQA acquired data gray is suppressed after PSD analysis black Bibliography Ali K Bilancio A Thomas M Pearce W Gilfillan A M et al 2004 Essential role for the pllOdelta phosphoinositide 3 kinas
60. trigger and arguments PSDref and PSD 90 are in phase and orthogonal reference waves respectively time Ch3 Ch4 Ch1 Y Ch2 X asymptote peak tau CapEngine4 20r3 time AI1 A1I2 fck3 fck4 filterpt aiSamplesPerTrigger PSD ref PSD90 AI10 optional taufactor optional endadj To use SQA the first input argument has to be 2 or 3 and there are three more input arguments Argument AIO is the trigger signal used for locating each individual curve Arguments taufactor and endadj are optional The taufactor is used for determining the region for curve fitting Fig 4 5B solid section Empirically taufactor of 3 is used for I SQA and 1 is used for Q SQA If you do not specify the taufactor the entire curve from 190 the peak to the end will be used for curve fitting which is not recommended The last input argument is endadj It is used for further adjusting the fitted region in earlier versions of CapEngine and the default value in CapEngine4 is zero It was 5 in Capmeter6v3 That means if taufactor determines that the 30 data point is the end of the fitted trace for example endadj tells CapEngine4 to move the boundary by 5 points and the 25 data point is the real end of the fitted trace This argument is no longer critical in the present algorithm and may be removed from the MATLAB code in the future There are three more output arguments for SQA They are steady state current asymptote peak current and time c
61. unchanged by either treatment Non SG fusion in whole cell recording is blocked by cell swelling The lack of a significant effect of these treatments in whole cell recordings suggested that a mechanism becomes important to maintain fusion capability in the excised patch which is not critical under the usual conditions of whole cell recording We reasoned that mechanical forces exerted on the membrane during seal formation and patch excision might disrupt the fusion machinery and ATP hydrolyzing processes would then become essential to restore the fusion capability In other words membrane stretch and or distention i e flattening of invaginations might be an important factor and accordingly we tested whether cell swelling might mimic effects of seal formation and excision Fig 81 3 8 describes two sets of results from BHK cells and one from RBL cells all demonstrating strong inhibition of non SG fusion by cell swelling We note that different BHK batches were employed in the two data sets in A and B and that as often was the case the average capacitance responses were substantially different in the different batches In Fig 3 8A cell swelling was induced in BHK cells by employing a cytoplasmic solution with addition of 200 mM sucrose and shrinkage was induced by employing a cytoplasmic solution diluted by 30 Exchange currents were activated 2 min after opening cells and cell shape changes had clearly occurred As shown in th
62. up when you put the mouse cursor onto the box for few seconds Three examples are shown below y masej f0 fso feo fio fo 1 S Paise f fo fo fio f8 E E Pulse so ao fo fo 1 fe 100 200 300 400 500 600 aoo 0 00 500 600 0 Boo 0 100 40 167 The voltage is set to zero during the interval left panel You may set interval to zero to make the voltage steps continuous middle panel If you want stimulating pulses with the same amplitude you can set step increment to zero and then specify the number of desired pulses right panel In this kind of pulse protocol I recommend you to specify the number of pulses in the rounds of pulses edit box but not in the number of pulses edit box right panel Because calculation of the pulse protocol takes time putting the number in the rounds of pulses edit box avoids redundant calculation for the same pulse and reduces the delay between pushing the Pulse button and the actual pulse generation If you put infin the number of pulses edit box the value in the step increment edit box will be neglected Although you may get the I V curve by using the Pulse panel another program called IQplot might fit your application better IQplot is developed to fulfill my mentor s will and it will be introduced in section 4 5 TTL triggering In case if you need to synchronize the acquisition of Capmeter with other programs e g image acquisition software you may use the T
63. was immersed in staining solution 45 methanol 10 acetic acid 0 05 Coomassie brilliant blue reusable for 2 hr and then destained in buffer composed of 30 methanol and 10 acetic acid for several times After destaining the gel was immersed in solution composed of 35 ethanol and 2 glycerol for 2 3 hr and then dried and framed in cellophane sheets for record 24 Western blot Nitrocellulose membrane was used blotting To make the transfer sandwich the followings were stacked in an order of anode to cathode a sponge pad 2 sheets of Whatman no 1 filter paper nitrocellulose membrane gel 2 sheets of filter paper and a sponge pad The transfer sandwich was immersed in cold transfer buffer for 1 L add 3 g of Tris base 14 4 g glycine and 200 ml methanol to water reusable and the transfer was carried out with a voltage of 60 V for 5 hr at 4 C After transfer the transfer sandwich was disassembled and the membrane was placed in blocking buffer 10 skim milk prepared from powder in TBST pH 8 0 solution which contains 10 mM Tris Cl 150 mM NaCl and 0 05 Tween 20 at room temperature for 0 5 1 hr with shaking The membrane was rinsed with TBST and then incubated with primary antibody which was diluted in 5 skim milk TBST solution for 1 hr at room temperature After washing with TBST for 3X5 min the membrane was incubated with horseradish peroxidase HRP conjugated secondary antibody for another 1 hr at room temperature
64. water saturated isobutyl alcohol was added to the top of the gel The gel was polymerized at room temperature for about 30 min and the isobutyl alcohol was then poured off from the gel The residual alcohol was removed using paper towel For the 3 stacking gel the 23 followings were mixed 2 375 ml water 0 94 ml of 0 5 M Tris Cl buffer pH6 8 37 5 ul of 10 SDS 0 375 ml of 30 acrylamide 0 8 bisacrylamide 55 ul of 10 ammonium persulfate and 5 5 ul of TEMED The mixture was poured onto the separating gel and the comb was then inserted into the stacking gel The stacking gel was polymerized for additional 30 min The electrophoresis module and cell were assembled and filled up with tank buffer for 1 L add 3 g Tris base 14 4 g glycine 1g SDS to water Equal volume of 2X sample buffer for 10 ml mix 4 ml of 10 SDS 3 ml of 0 5 M Tris Cl pH6 8 2 ml of 50 glycerol 1 ml B mercaptoethanol and some bromophenol blue was mixed with desired amount of protein sample and the mixture was heated in boiling water for 3 min The boiled samples were placed on ice for few minutes before loading Electrophoresis was carried out at 90 V before the dye was stacked on top of the separating gel after that the voltage was increased to 120 V After electrophoresis the module was disassembled the stacking gel was discarded and the separating gel was subjected to Coomassie blue staining or western blot For Coomassie blue staining the gel
65. wave the other one is the DC subtracted weighted current The carrier wave is a sine wave and its frequency is determined by the average current The pitch is higher when the current is more positive and lower when the average current is more negative The current frequency relationship is a sigmoidal curve and it is most sensitive when the average current is close to zero i e it is most sensitive during seal formation The amplitude of the carrier wave is enhanced if its frequency is lower than 1 5 kHz The input current wave form is also weighted around its average value using an exponential function The purpose is to magnify little current steps and help the users to hear the seal during seal formation From the quality of the sound users can judge the quality of the seal and from the changing pitch of the sound users will know if the seal is getting better or worse When a seal is perfectly formed you will hear clean and 200 201 peaceful sound else annoying noise If the sound response does not satisfy you you may edit it in function noise Note that the frequency the noise function is evoked also affects the quality of the sound The frequency is determined by handles timerperiod and it also affects the refreshing frequency of function scope 4 8 SlopeScan What is it SlopeScan is used for finding the maximal slope of a trace That s it How to use it SlopeScan z lolx 0 05 T T T
66. weighting these points were not well described by the fit and the Hill coefficient was 3 We stress however that Hill coefficients determined for non SG fusion in BHK cells were also in the range of 2 Yaradanakul et al 2008 It is 84 reported that the Hill coefficient and Ca of Syt VII is 3 6 and 0 9 uM respectively in a liposome binding assay Wang et al 2005 Therefore these results support further a conclusion that Syt VII is not likely to be the sensor for non SG fusion 85 3 5 Discussion In this article we have described some efforts over several years to maintain and to monitor Ca dependent exocytosis in giant excised patches While non secretory vesicle fusion could be routinely monitored in our hands neurotransmitter release was seldom maintained in the excised patches Even in the case of non SG fusion we found it necessary to pretreat cells with agents that disrupt cytoskeleton to maintain fusion That membrane stretch and distention may readily disrupt fusion has been verified in whole cell recording with both BHK and RBL cells We discuss first the methodological and experimental problems encountered and then the data sets on non SG fusion Membrane fusion in excised giant membrane patches In principle the giant patch methods should facilitate multiple types of fusion studies Excised patches allow free access to the cytoplasmic side for a wide range of possible manipulations by exogenous factors
67. you can not give identical pulses in IQplot Since IQplot is used for plotting I V and Q V it doesn t make sense to give pulses with the same amplitude The values in the first two edit boxes in the Pulse protocol panel define the voltage range you are going to scan The sign of the values must be opposite the program will make it different anyway and the program will increase or decrease the voltage from zero toward the direction defined in the first edit box You can define the length of each step the size of the voltage step and how many rounds you want to apply the protocol in the following edit boxes You can always move the mouse cursor onto the edit boxes for few seconds to get the definition of each edit box Clicking the Pulse button triggers the pulse protocol The program acquires 10 ms pre trigger signals to estimate the constant DC current e g caused by junction potential etc and then subtract it from the entire trace before further processing When the protocol is completed raw current with pre trigger signal I V and I Q plots are displayed on axisl axis4 and axis5 respectively axis3 is removed from IQplot2 In addition to giving pulses by clicking the Pulse button you may also ask the program to apply the same protocol automatically in a defined time interval To do so you simply check the check box in the Pulse protocol panel when the program is running and IQplot will trigger the acquisition autom
68. 0 3 1s q 2 0 4 o 40 200 uM Ca 5 eee fe o 5 5 60 Time s F E Figure 3 7 Non SG fusion in whole cell recording is wortmannin adenosine insensitive A Typical whole cell capacitance records and composite statistics for RBL cells during cytoplasmic infusion of 200 uM Ca with control cytoplasmic solution upper and with 5 uM wortmannin and 0 5 mM adenosine lower Differences are not significant B Exchange current densities upper and capacitance responses lower for BHK cells in which membrane fusion was activated by outward Na Ca exchange current with control cytoplasmic solution left bar graph with 0 5 mM adenosine middle bar graph and with 5 uM wortmannin and 0 5 mM adenosine right bar graph are shown 113 A B C BHK Cells BHK Cells RBL Cells a 40 100 E K T 30 Mook l lt 2 amp 20 s 20 80 xxx X 6 10 3 10 gt oa 0 0 S 60 S _ 30 2 T a 100 a Biz xxx xx F kkal f 40 80 4 Q 2 2 a ce roo 5 60 ET z 20 S w 10 40 o i a lt a EK aL Lo o z o 22 EIE we F osc E a s e 3 ou Os 3 ET ew 23 Ge cs ES O65 Ec Es a A o23 3 2 o o oe o0 o v 3 2 a g 2 v vS 2 S zo Z amp g z Figure 3 8 Non SG fusion in whole cell recordings is strongly inhibited by cell swelling A amp B Bar graphs give exchange current densities upper and capacitance responses lower for two batches of BHK cells in which membrane fusion was activated by outward
69. 2 Fig 2 6 column 4 is not suitable for reconstituting Sytl Fig 2 6 column 1 and OG concentration of 0 77 is still the best for Sytl reconstitution Fig 2 6 column 2 This OG concentration was selected for subsequent Syt1 Syb2 reconstitution There was a reason to pick these three OG concentrations I did not choose them randomly The monomeric OG concentration in water is 17 mM Rigaud et al 1995 The OG lipid molar ratio for OG saturated liposomes is 1 3 and when liposomes are totally solubilized the ratio is 3 Rigaud et al 1995 That is when liposomes with 5 mM of total lipids are saturated the OG concentration is 39 17 5X1 3 23 5 mM When liposomes are totally solubilized the concentration is 17 5X2 6 30 mM The molecular weight of OG is 292 4 so 23 5 and 30 mM OG represent 0 69 and 0 88 OG respectively OG concentration of 0 77 is between these two values The size distribution of reconstituted liposome is shown Fig 2 7 Distribution of LUVs made by extrusion method was very homogeneous and the diameter was about 100 nm upper panel When Sytl was reconstituted using 0 67 OG previously established OG concentration for reconstituting Syb2 middle panel the size distribution was very heterogeneous and there seemed to be particles with large diameter which was consistent with previous incorporation assay Fig 2 6 column 5 Using OG concentration of 0 77 the size distribution of Sytl containi
70. 4 5 bisphoshate in carbamylcholine stimulated islets of langerhans J Biol Chem 276 19072 19077 Mousley C J Tyeryar K R Vincent Pope P and Bankaitis V A 2007 The sec14 superfamily and the regulatory interface between phospholipid metabolism and membrane trafficking Biochim Biophys Acta 1771 727 736 Mundroff M L and Wightman R M Amperometry and cyclic voltammetry with carbon fiber microelectrodes at single cells In Current protocols in neuroscience Gerfen 221 C R Holmes A Rogawski M A Sibley D Skolnick P and Wray S 2002 6 14 1 6 14 22 Nagao T Kubo T Fujimoto R Nishio H Takeuchi T et al 1995 Ca 2 independent fusion of secretory granules with phospholipase a2 treated plasma membranes in vitro Biochem J 307 Pt 2 563 569 Nakamura Y Fukami K Yu H Takenaka K Kataoka Y et al 2003 Phospholipase cdeltal is required for skin stem cell lineage commitment EMBO J 22 2981 2991 Nakanishi S Kakita S Takahashi I Kawahara K Tsukuda E et al 1992 Wortmannin a microbial product inhibitor of myosin light chain kinase J Biol Chem 267 2157 2163 Nasuhoglu C Feng S Mao Y Shammat I Yamamato M et al 2002 Modulation of cardiac pip2 by cardioactive hormones and other physiologically relevant interventions Am J Physiol Cell Physiol 283 C223 34 Neher E 2006 A comparison between exocytic control mechanisms in adrenal chromaffin cells and
71. 6 0 6840 17 4 Mg buffer 93 1444 2 13 0 3840 14 13 1 08 0 35 5 neomycin 71422 9 14 0 2940 13 14 0 6240 21 5 Mg buffer 103 1447 3 14 0 4340 14 14 0 92 0 19 6 PIP2Ab 92 6446 8 14 0 4340 14 14 1 1740 13 5 ATP 76 6416 9 14 0 6440 13 14 1 06 0 23 9 staurosporine ATP 50 34 13 8 15 0 4740 13 15 1 25 0 34 7 ATP 62 2413 5 20 0 6 0 11 20 0 8340 17 11 wort adeno ATP 6 943 1 21 0 05 0 05 21 0 88 1 ATP 47 3 26 4 8 0 3840 18 8 1 04 0 32 3 LY294002 ATP 159 1470 9 7 0 5740 2 7 1 49 0 19 4 ATP 104 9475 1 4 0 5 0 29 4 1 8740 51 2 PI TP ATP 202 50 4 4 1 4 1 2840 46 4 TeTx tetanus toxin light chain 200 nM NEM N ethylmaleimide 1mM AMP PNP 2 mM neomycin 500 uM PIP2Ab 1 50 staurosporine 200 nM wort wortmannin 4 uM adeno adenosine 0 5 mM LY294002 100 uM PI TP Pl transfer protein 140 ug ml a counts patches with amplitude gt 50 fF b outliers are removed using Grubbs test P lt 0 05 P lt 0 01 P lt 0 001 Chapter 4 Software and algorithm development 4 1 Introduction There are several ways to measure the membrane capacitance of a cell Depending on the method used to extract the information from the current waveform they generally fall into one of the two categories time domain method frequency domain method Lindau and Neher 1988 The time domain method uses square pulses to stimulate the cell and extract the information by fitting the re
72. AE delta A DNA DOPS DSP EDTA EGTA ER List of Abbreviations and Symbols arachidonic acid analogue input phase shift adenosine 5 B y imido triphosphate analogue output adenosine 5 triphosphate baby hamster kidney bovine serum albumin capacitance coulomb calcium calcium dependent activator protein for secretion channel Chinese hamster ovary membrane capacitance carboxymethyl central processing unit of the computer diacylglycerol data acquisition diethylaminoethyl differences deoxyribonucleic acid 1 2 dioleoyl sn glycero 3 phosphoserine digital signal processing ethylene diamine tetraacetic acid ethylene glycol bis B aminoethyl ether tetraacetic acid endoplasmic reticulum xix FBS FRET GST GTP GUI HA HEPES IP IPTG IRES frequency fetal bovine serum fluorescence resonance energy transfer conductance glutathione S transferase guanosine 5 triphosphate graphic user interface hydroxyapatite 4 2 Hydroxyethyl piperazine 1 ethanesulfonic acid current inositol triphosphate isopropyl B D 1 thiogalactopyranoside internal ribosomal entry site dissociation constant Luria Bertani broth large multilamellar vesicle lysophospholipid large unilamellar vesicle mitogen activated protein kinas mouse embryo fibroblast 2 N morpholino ethanesulfonic acid myosin light chain kinase mammalian homologue of Unc not a number N ethylmaleimide nickle nuclear localizatio
73. Fig 4 6C the time constant is obtained as described previously Variable PeakTau is then adjusted using the time constant and Eq 4 34 As in I SQA all valid data sets a b T are added and averaged to generate one digitized data set in MATLAB using Eq 3 5 and Eqs 3 9 11 Square wave perturbation validating the algorithms using Simulink To validate the algorithms I generate model current first and then check if I SQA and Q SQA can retrieve the cell parameters properly from the current Rather than using an exponential function to generate the signal I use Simulink a MATLAB component to mimic the physical properties of the cell circuits The model diagram is shown in Fig 4 9A Cell parameters can be adjusted in the model Fig 4 9A red circles in an absolute term For example Ra Rm and Cm in Fig 4 9A are 3 MQ 50 MQ and 20 pF respectively The wave protocol square pulses in this case Fig 4 9A a drives the circuit and the total current b in Fig 4 9A black in B current charging the membrane c in Fig 4 9A red in B and current leaking through the membrane d in Fig 4 9A blue in B are displayed The sampled data at 100 kHz are exported e in Fig 4 9A to the Workspace of MATLAB for analysis To explain a little bit more about how the model works let s follow the numbers in Fig 4 10 Total current flowing through the electrode is driven by voltage difference between the electrode and the membrane Ra
74. G concentrations eeeeeseeeeeteeeenees 50 Figure 2 7 Size distribution of reconstituted l1pOSOMES ceeeseeeeeseeeeeeeeeeeeseeeneeaeeeee 51 Figure 2 8 Leakage assay of Sytl containing li1pOSOMES ceeceeseeeeeeseeeeeeeeeeeeaeeeaee 52 Figure 2 9 Orientation tests of Syt1 Syb2 liposomes sseseessesseeseesseseesressesresseessesee 53 XV Figure 2 10 The triple marker transient expression SYStOM cceseeseeeteeteeeteeeeeeeeeeeees 54 Figure 2 11 Stable cell lines co expressing Stx1A SNAP25A and Muncl8 1 55 Figure 2 12 Perfusion of artificial liposomes to excised patches ccccccsceesseeeteetteeees 56 Figure 3 1 Method to determine whole cell capacitance via square wave perturbation wae Abate ol aco inde aaet dda acd eae ts cea E eee ease Aa 107 Figure 3 2 Amperometric and capacitance measurement in RBL cells eee 108 Figure 3 3 Non SG fusion is SNARE dependent ec ceceeceeseeeeeseceeeeeeeeseeeeeeesenneees 109 Figure 3 4 ATP hydrolysis is required for supporting non SG fusiON cesses 110 Figure 3 5 Non SG fusion in excised patches is wortmannin adenosine sensitive 111 Figure 3 6 Non SG fusion is not blocked by antibodies against PI 3 P and PI 4 P 112 Figure 3 7 Non SG fusion in whole cell recording is wortmannin adenosine insensitive was AAEE E E ST ETA coun att eect el Gans TEE EEE a evened a eae il ace vad oD e 114 Figure 3 9
75. In input FT flow through E eluate GST T In FT B1 B2 In FT 7 8 Mono S 49 9 10 14 Pe OMT ete Figure 2 5 Purification of Syb2 Coomassie blue stained gels are shown After affinity and ion exchange chromatographic purification the quality of the purified Syb2 was satisfactory T total protein In input FT flow through B1 beads before thrombin cleavage B2 beads after thrombin cleavage Numbers Mono S fraction number 50 2M urea extraction direct centrifugation la Wye Kreme AM Kesrsin 04 opti omy rlt 00h Cobain tN n h ai i iO p i l no 0 0 ue So 1 2 3 4 5 6 7 Figure 2 6 Reconstitution of Sytl at different OG concentrations OG concentration of 0 67 was used for Syb2 proteoliposome preparation However it is clear that Sytl tends to form aggregation at this concentration column 5 and the best OG concentration for Sytl reconstitution is 0 77 column 6 if total lipid concentration is 5 mM To check if the reconstituted proteins really get integrated into liposomes 2 M urea was used to remove proteins sticking on the liposomes Again reconstitution condition for Syb2 column 4 is not suitable for reconstituting Sytl column 1 and OG concentration of 0 77 is still the best for Sytl reconstitution column 2 This OG concentration was selected for subsequent Syt1 Syb2 reconstitution sup supernatant mem membrane fraction Blank liposome 100 EA z g
76. M Su CY Wang YL Chen S and Tsai HJ 2001 Retina specific cis elements and binding nuclear proteins of carp rhodopsin gene FEBS Lett 508 265 271 xiv List of Figures Figure 1 1 SNAREs and other proteins involved in exocytic and endocytic pathways 11 Figure 1 2 The neuronal SNARE complex s c 3 4ciciccsets i eceeneniieas aeaienivs iene 12 Figure 1 3 The SNARE cycle in synaptic vesicle CXOCYtOSIS ccccceeseeeteeeteeeeeteeeeeeaes 13 Figure 1 4 Different models of SM protein syntaxin interactiOn cccesceeeeeeeeeneeeeees 14 Figure 1 5 PI distribution of endo and exocytic pathwayS ccccccseeeseeeetseeeeetteeeeeees 15 Figure 1 6 Functions of Pl 4 5 Pr cessucesceaasessetes aaa deacncesnteesceaseecmaaadar aan tuanteeasadiiy ate eaattes 16 Figure 1 7 Cleavage sites of four classes of phospholipases c cccsccssecsesceeeeeeteeeeeees 17 Figure 2 1 Chromatographic purification of untagged Sytl oo eeeeeseeeteceeteeeeneeeeaees 45 Figure 2 2 Purification of His o Syt lis cc2egcscowasecaiceiecessccestcoadacsenauoasoenvedaldegesdeveesusncostaaiies 46 Figure 2 3 Purification of HBM His s Syt1 and Syt1 His ceceeeceesceesceeeteeeteeeeseeees 47 Figure 2 4 Estimation of HBM His 3 Syt1 and Syt1 His s concentration 00 48 Figure 2 5 Purification of SY D2 cereus fneta cased cuca scucna oiitng Accntace anes ye caste aucabun stay ueaven cade aiees 49 Figure 2 6 Reconstitution of Syt1 at different O
77. MEASUREMENT AND ANALYSIS OF CALCIUM DEPENDENT EXOCYTOSIS IN GIANT EXCISED MEMBRANE PATCHES APPROVED BY SUPERVISORY COMMITTEE Ege T Kavalali Ph D Committee Chair Donald W Hilgemann Ph D Advisor Thomas C Stidhof M D Josep Rizo Ph D To my parents Hsien Yi and Mei Hsia MEASUREMENT AND ANALYSIS OF CALCIUM DEPENDENT EXOCYTOSIS IN GIANT EXCISED MEMBRANE PATCHES by TZU MING WANG DISSERTATION Presented to the Faculty of the Graduate School of Biomedical Sciences The University of Texas Southwestern Medical Center at Dallas In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY The University of Texas Southwestern Medical Center at Dallas Dallas Texas May 2008 Copyright by Tzu Ming Wang 2008 All Rights Reserved Acknowledgements First I would like to give my thanks to my graduate mentor Dr Donald W Hilgemann for giving me 100 freedom to do what I want to do and insightful suggestions and unlimited support during these years He is more like a good friend of mine rather than a big boss on top of me I would also like to thank Dr Thomas C S dhof for giving me the opportunity to learn with him In his lab I learned not only the techniques but also the cautious attitude a good scientist shall have I must also give my thanks to my thesis committee members Drs Ege T Kavalali and Josep Rizo for their criticism and advice which guided me toward the completion o
78. Qplot2 The AI object is triggered by the first pulse in the protocol Once the desired number of samples calculated and set in Pulse Callback are acquired function process_update is evoked This function calls Olizer to process the data and shows raw data and processed data in the corresponding axes If the Pulse_Callback is evoked by clicking the Set baseline button the process update function also sets the eventcode in handles Pulselog to 1 as a reference Note that although the time constant is not used for any purpose in this program the Qlizer still calculates and returns time constant for each pulse step This feature is reserved for future development of the program 187 generate time array for axis2 Show_update2 update symbol colors on axis2 T li initialize axis2 to Stopped Start_Stop_Caliback e Show_axes2initiai show symbols 4 ie for pulses assign variables to the Workspace ss _ set accessibility for each pulse retrieve raw data for sum up Q for the update axisl axis4 and axis the current pulse train current pulse train There are still many things to deal with after the recording is stopped Besides assigning variables to the Workspace and generating time array for axis2 the Start_Stop Callback calls function Show _axes2Initial to initialize the offline display mode for axis2 The function AccessCtr compares all the pulse protocols in the Pulselog to the current protocol used fo
79. Synaptotagmin VII is not required for non SG fUSION eee eeeeeeeneeeeeeee 115 Figure 3 10 Ca dependence of non SG fusion in excised patches from RBL cells 116 Figure 4 1 Geometrical view of phase sensitive detection ceeeeeeseeeeeeteeenteeeeneees 204 xvi Figure 4 2 Online calculation of the phase angle eecceeceeeseceeeeeeeceteeeeseceteeeeeeeees 205 Figure 4 3 Offline adjustment of the phase angle ec eececsscceteceseeeeeeeesneeeeeeneeeeees 206 Figure 4 4 Demonstration of phase sensitive detection ceeeseeseeeteeseceeeeeeeneeeeneees 207 Figure 4 5 Square wave perturbation based on fitting the current transient 208 Figure 4 6 Square wave perturbation based on fitting the transferred charges 209 Figure 4 7 Correction of the integrated charges cccccccssesseeeseeeeeceeeeeeeeeeseeeseeeneeees 210 Figure 4 8 Demonstration of the charge COrrectiONn ccsccesesseeteceeteeeeneeeeneeeesneeeeaees 211 Figure 4 9 Generation of the model current using Simulink ce eeeeeeteeeeteeeeneeees 212 Figure 4 10 Some more about the diagram in Simulink cee eeeeseeeeeeeeeeeneeeeneeees 213 Figure 4 11 Square wave perturbation PSD analysis of SQA data ee eeeeeeeereees 214 xvii List of Tables Table 3 1 Effects of various reagents on non SG fusion in excised patches xviii AA AI alpha a AMP PNP AO ATP BHK BSA C Ca CAPS Ch CHO Cm CM CPU DAG DAQ DE
80. T T 25 T T T T T Current position Input data ee at 1 9 4 op E ei Slope plot 0 01 L fi 1 i fi 0 1000 2000 3000 4000 5000 6000 B ATE 4 Time m by 1 Data m by 1 Window size pt FigureData 1 FigureData 2 201 1 6 4 cr gt Pick Show Export Developed by Tzu Ming Wang Hilgemann Lab UTSW 2007 15 1 40 L 90 100 110 You put the names of time and data arrays and the window size used for slope calculation in the corresponding edit boxes and then click the Scan button The slope along the input trace is shown on the left panel and the original trace and tangent with maximal slope is shown on the right panel And values of time and slope of the current position are shown in the MATLAB Command Window Note that SlopeScan retrieves input variables from the MATLAB Workspace so both of the variables have to be present in the Workspace and both of them have to be m by 1 column arrays too The averaged time of the window is assigned to the output time array That is if the window size is odd 202 the output time values are identical with the inputs Press lt or gt buttons or left down right up on the keyboard to move along the trace or use the Pick button and then click on either of the panels to select the current position You may generate figures by clicking the Show button and when the Export button is clicked variable SlopeExported is assigned to the Worksc
81. TL triggering function Connect the external TTL source to PFIO on the terminal block and then right click on the Stopped button when Capmeter is stopped Check the TTL option in the context menu and then click the Stopped button to start the program Capmeter is then waiting for the external TTL signal to trigger the acquisition Once the acquisition is started Capmeter does not need any other TTL trigger because the acquisition is continuous Capmeter does not send out TTL signal simply because MATLAB does not support buffered digital output 168 Reader mode You may browse and analyze your data at home without installing a data acquisition board If the program can not detect the board or there is something wrong with the hardware settings it will enter the reader mode If you are trying to modify the program be sure not to use the function isrunning Although the MATLAB recommend the users not to access the running property of AI and AO objects directly Capmeter pretends that there is a variable called running in the reader mode even when AI and AO objects do not exist If function isrunning is used errors may occur in other functions in Capmeter6v3 when reader mode is launched I did not use isrunning at the very beginning of the development simply because there was not such a function at that time The information flow adjustALAO properties Refcaic stated Start wal Caliback Set PSD r
82. What 1S 102 a ocak soa rarei a E a e a a A a a ait 197 Things you need to know before using seessssseessesessressossreserssresessreesssresssrresssre 197 Running the program esesssssesseessessesrossresresressesrrsrtsstesesstsstesseressereesseresssreesssreesse 197 Hot keysni aara arenes A a T E O E T E a 198 Th sinformation NoWe esnea a e R E a E a SESA 199 The sound seguente onea a e e e A E te 200 AD SIGDES CAM r a i ea placa eee oars 202 MYR AS IL Potts cele ce eS SNe stad eke ch Sachi ge ue ai te ide ahaa ee N ade elie oncute E 202 FAO TO USSG tated eet A kN case A ot cinta piace ate Neate SO tet 202 BDH GOT APY cccis esses fans shat vse eocies Seams ces e a Oa E tones yal e ora a a a A EA vel 215 xiii Prior Publications Wang TM and Hilgemann DW 2008 Ca dependent non secretory vesicle fusion in a secretory cell J Gen Physiol In press Yaradanakul A Wang TM Lariccia V Lin MJ Shen C Liu X and Hilgemann DW 2008 Massive Ca induced membrane fusion and phospholipid changes triggered by reverse Na Ca exchange in BHK fibroblasts J Gen Physiol In press Chen X Arac D Wang TM Gilpin CJ Zimmerberg J and Rizo J 2006 SNARE mediated lipid mixing depends on the physical state of the vesicles Biophys J 90 2062 2074 Wang TM Chen YH Liu CF Tsai HJ 2002 Functional analysis of the proximal promoter regions of fish rhodopsin and myf 5 genes using transgenesis Mar Biotechnol NY 4 247 255 Ma GC Wang T
83. a step size of 2Vc let s say from Vc to Vc for the ease of explanation for now the relationship of membrane potential versus time is shown in Fig 4 5C Since the oscillation is too fast the membrane potential can never reach its theoretical steady state value Vss which is i VcRm Rat Rin Pay Recalling that at steady state the circuit is like two resistors in series so part of the Vc is shared by Ra and Vss is a fraction of Vc Eq 3 3 Assuming that the membrane potential is oscillating between Vss and fVss where f is the fraction of Vss across the membrane at the end of the voltage step of duration A the relationship between membrane potential and time can be expressed as Vin SVsst Vss 1 f 1 e 3 4 Solving for f with A 1 p 38 3 5 1 e Ca and membrane voltage at the beginning of the voltage step is _ fVcRm Ra Rm coy From the steady state current 132 Vc 133 eee ee Ra Rm 6 7 and the peak current a Ra 3 8 the solutions for Ra Rm and Cm are Ve 1 f R eS ek oF Eo 3a Vc a b R m b a fb 3 10 1 1 t a bf Cm t 3 11 i ne Rm Vc a b 1 f ee When A is very long f approaches one and Eqs 3 9 3 10 and 3 11 approach exactly Eqs 4 21 4 22 4 23 respectively The derivation of the above equations assumes that the command voltage is oscillating around zero volt What if it is not the case e g
84. a3 YData3 DispCtrl1 15000 XDatal2 YDatal YData2 7500 xXData3 YData3 The above example restricts at most 15000 points to be displayed on top and middle panels and at most 7500 points to be displayed on the bottom panel IQlizer mexw32 Like CapEngine4 for Capmeter6v3 Qlizer is the core processing unit of IQplot2 It is modified from the subfunction SqgQ in CapEngine4 The main difference is that Qlizer does not correct the integrated charges with the time constant as mentioned previously The syntax for OQlizer is asympote Q tau IQlizer AI1 time pulse L pulse NR optional mode optional taufactor optional endadj Argument pulse_L is the number of AI points for each step and pulse_NR is the number of total steps Input argument mode tells Olizer whether to output the direct summation of the charges default mode 1 or to output estimated total transferred charges at steady state mode 0 Arguments taufactor and endadj are used the same way as in CapEngined4 Output arguments include estimated steady state current asmyptote integrated charges Q and estimated time constant tau Note that Q is a column array containing 194 integrated charges for each individual curve asymptote subtracted To plot Q V IQplot2 add them up right before axis5 is updated PhaseMatcher2 mexw32 PhaseMatcher2 is used in Capmeter6v3 when PSD analysis of SQA data is desired Its two functions are 1 lookin
85. according to my experience 1 Keep solution lines as short as possible As mentioned above the solution lines are like an antenna network The longer 93 the lines the stronger the signal Don t get me wrong the signal received by the lines is electrical noise So you have to keep the lines as short as possible To do so you will need to move the solution reservoirs e g syringes close to the recording chamber In addition also keep the volume of solution in the reservoirs as small as possible to further reduce the noise I use lt 0 5 ml for each reservoir 3 reservoirs total Theoretically reducing the chamber size also reduces the noise however it is not practical The solution level in the chamber is increasing during the experiment because of the continuous solution flow If the chamber is too small solution might flow out of the chamber during the experiment and the speed of increasing stray capacitance caused by the increasing solution level will be too fast which might saturate the lock in amplifier during the recording Use negative pressure but not solution switch to control the flow In regular configuration the solution switch is connected between the reservoir and the line to control the solution flow It seems natural and reasonable to connect the system this way however disasters happen 99 of the time if this configuration is used for patch amperometry The inevitable scenario is that when you turn the switch aft
86. ae a i ta dows 37 PULTICATON of Synaptobrevin 2isciasisii ecirar n iieii asi 38 Quality of the liposomes soiin naiaiaee iaa aaa anaE Ea aea aea EE enera Toii 39 Establishment of the triple marker transient expression system seseeeeseeseeseeseeeeee 41 Perfusion of artificial liposomes to excised patches cscceseeseeseeteeeteeeeseeeesneeeeaees 43 Chapter 3 Fusion of endogenous vesicles in excised patches 3 1 ADSIAG ennea eA e tie 59 3 2 troduction e seis ie ane eae ee wes ae ER oe VESNE E A 61 3 3 MethodS aonana en e ude whaitadey entadweestapnieu sss Gelade am N 65 Ce CUES aniar ane ee bch Ae a EERE E EA ems Eaa S EE E ENE eee 65 SOVON Senai a Ta nae tutes E E A E E A E E R E 65 Recording softwareen atirei an i ei i aiai 67 Patch clamp and data ACQuiSitiON isecisserecvescessesstscaensceestuassannneadersenancasecdoedoareusedadevense 70 Pateh ampero meti y eei riirii bons iee er a ee EEEE ne AOE EEA AAF TAERE EOE 71 Recombinant proteins and antibodies ee escesesseeeseeeseeneeeseceeeeeeceaecesaeeeeneeeeeaeeseaees 72 Data analy S E onthe atts E EA a EE 73 34 RESUS aier E E en A E RE E E 75 Distinct vesicle populations in RBL Cell S css eceesencend want vacet lacie ddeeveles tea Uadasy 15 Non SG fusion is SNARE dependent but NEM insensitive in excised patches 76 ATP hydrolyzing processes support non SG fUSION ccceeceesceereceteeeeeeeeteeeneeenaeees 77 ATP dependent generation of PI 4 5 P gt is not critical
87. ape The format is time slope You may show hide the tangent by pressing the h key on the keyboard The scanning process is done by function Rslope mexw32 Rslope uses the specified window size and linear regression to get the slope It moves along the trace and assigns the corresponding slope to the output array Unlike Dfilter2 in Capmeters Rslope does not add extra data points to the original input so the output dimension will become smaller For example If input has 1000 data points and the window size is 11 the number of output points is 1000 11 1 990 The syntax for using Rs ope is newtime slope Rslope time data window The input argument time has to be a m by 1 column array You can only use m by 1 array for input argument data when you call Rslope from the SlopeScan but you may use m by n column array when Rslope is called directly from the Command Window In this case Rslope will calculate running slope for each column and the corresponding slope is assigned to the output column array slope with dimension of p by n p lt m 203 PSD2 Figure 4 1 Geometrical view of phase sensitive detection A If the phase angle is not properly adjusted i e s A 40 according to Eq 4 4 the readout of PSD1 is the projection of G on the x axis X and G also contaminates the readout of PSD2 Y which is 1 2 away from PSD1 Eq 4 5 To adjust the reference phase angle 6r it is just like rotating
88. arge the membrane properly In contrast slower Tt 119 usually gives better curve fitting result using SQA and the noise will be smaller as a consequence PSD using dual frequencies provides a solution for overcoming the drawbacks of single frequency PSD Barnett and Misler 1997 Santos Sacchi 2004 The algorithm is generally based on the fact that capacitance signal is proportional to the oscillation frequency but not the conductance signal There are computer programs available which adopt the dual frequency principle like PULSE www instrutech com and jClamp www scisoftco com etc The single frequency software lock in amplifier can also be found online pClamp www moleculardevices com I don t know if it s using single or dual frequency We don t have pClamp in the lab NI lock in amplifier www ni com and UTiLIA mrflip com papers LIA etc So software packages for measuring capacitance are commercially available and some of them are even free Why did I still develop this program To make a long story short it is because 1 I do not have any commercially available software in the lab 2 my mentor prefers to use MATLAB 3 I love to make my ideas come true and 4 It turns out that the program is more than just a lock in amplifier If you are not interested in this history please skip the next paragraph without feeling guilty Here is the actual story When I jointed the lab we were still using the chart reco
89. arly endosome RE recycling endosome MVB multivesicular body LE late endosome ly lysosome Ph phagosome Ph ly phagolysosome SG secretory granule PGC post Golgi carrier Fig 2 of De Mattis and Godi 2004 Binding site for PH and other domains lon channel activation Production of three second messengers Enzyme activation Cytoskeletal attachment Actin binding proteins Figure 1 6 Functions of P1I 4 5 P2 Phosphatidylinositol 4 5 bisphosphate PI 4 5 P2 is one of the most versatile PIs that has been implicated in a variety of different physiological activities including membrane fusion Fig 1 of McLaughlin and Murray 2005 Figure 1 7 Cleavage sites of four classes of phospholipases Note that the side chains are just for illustration and do not reflect the actual molecular formula Phospholipase B not shown has both PLA and PLA2 activities Fig 1 of Brown et al 2003 Chapter 2 Fusion of artificial liposomes to excised patches 2 1 Introduction For studying a complex biological system or process it is always desired to have a functional in vitro system for experimentation In contrast to the in vivo system which is complicated by the nature of the biological system itself an in vitro reconstituted system is a simplified system with all its components defined and controlled It is complementary to the in vivo system and provides a convenient and powerful platform for testing novel idea
90. as treated the same way and ligated with the 3NLS EGFP fragment NotI is at the 3 end of EGFP For pBMG bicistronic membrane is green the same strategy was employed Fragment containing coding sequence of membrane targeting domain of Src oncoprotein Sigal et al 1994 fused to the 5 end of EGFP coding sequence was generated by PCR and cloned into pCR2 1TOPO vector The fragment was then subcloned into Agel Nofl site of pIRES AgeI EGFP To construct pBPR bicistronic peroxisomes are red coding sequence of peroxisomal targeting sequence 1 PTS1 Clontech was fused to the 3 end of DsRed2 Clontech coding sequence The DsRed2 PTS1 fragment was generated via PCR and cloned into pCR2 1TOPO vector After sequence confirmation the DsRed2 PTS1 fragment was excised from the pCR2 1TOPO vector by EcoRI digestion followed by Klenow fill in and NotI cleavage Vector pIRES AgelI EGFP was treated by BstXI digestion and Klenow fill in followed by NotI cleavage and then ligated with the DsRed2 PTS1 fragment 34 Rat StxlA was cloned into Smal EcoRI site of pBNG2 to generate pBNG2 Stx1A Human SNAP25A was cloned into Smal Pstl site of pBNG2 and Smal PstI site of pBMG to generate pBNG2 SNAP25A and pBMG SNAP25A respectively For rat Munc18 1 it was cloned into Smal EcoRI site of pBPR to generate pBPR Munc18 Liposome perfusion and capacitance measurement in excised patches Since there was only a limited amount of liposomes special per
91. asP20 20 HEPES EDTA buffer solution 140 NaCl 2 EDTA 20 HEPES and protein dialysis solution 140 NaCl 0 3 EGTA 0 3 ZnSO 20 HEPES 2 mercaptoethanol For whole cell experiments presented in Fig 3 7 the standard solutions described previously Yarandanakul et al 2008 were employed For Fig 3 8 results in panel A employed the standard solution with 70 mM NMG substituted for LiOH The cytoplasmic solution was modified by addition of 200 mM sucrose and dilution by 30 to generate the hyper and hypoosmotic solutions respectively In panel B the standard solutions were employed with NMG 66 aspartate reduced by 80 mM to generate the hypoosmotic extracellular solution The control solution was generated by adding 160 mM sucrose to this solution In panel C the solution given above for RBL cells was employed Hyperosmotic cytoplasmic solution was generated by addition of 200 mM sucrose and hypoosmotic extracellular solution was generated by deletion of 80 mM NMG Recording software Capacitance measurement software Capmeter 6 was developed in MATLAB using its data acquisition toolbox R2006b The MathWorks Natick MA Three major on line functions were developed 1 a software lock in amplifier 2 routines for continuous cell parameter determination via square wave voltage perturbation and 3 data smoothening and deglitching routines To synchronize the timing of analog output and input
92. ater section The background averaging filter is designated to process signals received from the data acquisition board i e channels AIl and AI2 After averaging the digitized data from AI1 current are recorded in channel 3 and data from AI2 whatever is connected are recorded in channel 4 channels 1 2 and 5 are not affected by this filter You may set the time window for averaging by putting specified value in ms in the edit box next to the ms button and then press Enter The program will adjust the value automatically so that current resulting from the command oscillation can be cancelled The filter is implemented in CapEngine4 with function name called Dfilter Variables in the Workspace After the recording is stopped Capmeter assigns a number of variables to the MATLAB Workspace They are introduced below 160 161 DAQinfo structure containing hardware settings aiSR analogue input speed in Hz aoSR analogue output speed in Hz aoChI convert command sensitivity for AOO in mV V aoCh2convert command sensitivity for AO1 in mV V starttime initial trigger time of analogue input in year month date hr min s FigureData it contains data in axis and axis2 when Show button is clicked time s data from axis1 data from axis2 PSDiog cell array containing oscillation parameters time frequency kHz amplitude mV phase angle wave form algorithm PSDofSQA it contains PSD analyzed SQA data Ch1
93. atically The pulse protocol is also triggered when you click the Set baseline button in the Pulse protocol panel After the baseline is set you may not change the pulse protocol and the baseline records are subtracted from upcoming records if the Subtract button is clicked You may change the 179 pulse protocol again once the Set baseline button is unclicked To apply notes simply put the note in the first edit box in the Pulse note panel and it will be associated with the next pulse automatically For your convenience there are three more edit boxes in the panel You can put notes that are frequently used in these edit boxes By clicking one of the Apply buttons next to the edit box the note will be placed in the first edit box and associated with the next pulse as mentioned previously Methods for charge integration The current is not used for integration directly For each pulse the steady state current estimated using Eq 3 2 is subtracted from the current and the area shown in Fig 4 6A is the integrated region Since the steady state current is subtracted the integrated charges are independent of the seal condition As shown above the gray areas are added into the transferred charges and the black ones are subtracted The corresponding Q V relationship is displayed on axis5 There are two methods for charge integration in IQplot2 and you may use either one of them in your experiment The first m
94. ation in mast cells Ali et al 2004 PI 3 K C2a which produces mainly PI 3 P is also required for the ATP dependent priming of SGs in neurosecretory cells Meunier et al 2005 From the various methods used to monitor membrane fusion membrane capacitance measurements give the highest signal and temporal resolution and the development of improved excised patch models to analyze and manipulate fusion would have important experimental advantages of high resolution recording with free access to the cytoplasmic membrane side Efforts to date have used chromaffin cells Dernick et al 2003 and the insulin secreting INS 1 cells MacDonald et al 2005 In the present study we describe the use of giant excised membrane patches pipette diameter 10 15 um patch size 2 4 pF to attempt to preserve more fusion capable vesicles in a configuration that allows free access to the cytoplasmic side In short we have found that partial disruption of the actin cytoskeleton facilitates the formation of stable patches from some cell types and preserves more fusion capable vesicles in patches from all cell types tested Nevertheless our experience is that SGs are easily lost from excised patches presumably because they are not docked in a stable fashion at the plasma membrane in RBL cells The non SG 63 fusion is much more robust and we describe here several fundamental characteristics of this type of fusion including its dependence on ATP and Ca
95. atus AO status ie it 0 6 As 0 8 H 2 Aq T ai L 1 J 25 il a 1 1 1 n 1 Fi o 50 100 200 250 300 gq af 40 20 0 20 40 60 Erase Mode Pulse Note 7 Pulse Protec i o S arectintrat Fai ae O ag opogqgmgm 1 Apply z Fiy Erase o R Standby FF Pulse every 3 sec lt gt ud T Gy E 3 Apply Saned _Set basen sma ee sev Developed by Tzu Ming Wang Hilgemann Lab UTSW Dallas 2008 In MATLAB change the Current Directory to the one containing IQplot components and then right click on IQplot2 m file double click on the file will open the M file editor Click Run in the context menu and MATLAB will launch the program Press the Stopped button to start the AI object and the sampled current is shown on axis2 To show the sampled current the function peekdata is evoked every 0 05 s i e 20 Hz The peeked data are displayed on axis2 and the last peeked data is stored in handles datasample Since peekdata function does not extract the data from the MATLAB Engine and the returned data may be missed or repeated please refer to the function references of MATLAB handles datasample does not represent accurate time signal relationship Moreover if errors occur when peekdata is called e g program is 177 busy the program assigns previous value to the current time point In online mode the unit of X coordinate of axis2 is sample and in offline mode the unit is second This c
96. ccccceceeeesessttteeeeeeees 5 PICA SPs and other pias cesceccesucsncavacnsdtaeused a uelacseaueneaietscgsahermcedoislegseeceieda cane O ETAR 5 Phospholipase CS yo cecss annin a Bech AAE AEE EE OE E TAES ones 6 Ph spholipase Dinessrro nii a oasis aa iNet E E E T E EE 8 Phospholipase Aj Tienenie r sii wie a aa aa aae e a E e 9 Chapter 2 Fusion of artificial liposomes to excised patches 2 1 MER OTC COIN oe Aaen ee i aai SEE A EE IREE 18 2 2 Meth d Se ei AEE O E A E R AEEA E EE 22 Small scale plasmid DNA preparation ccccccesccesseeenecesceceseeeseeceseceseeceseceseeseeeeeeees 22 SDS PAGE Analy iSeries 23 Western blot nonnacrir ae E E E E E R E 25 ix Dot bl t Expressa A eae cases eck Cabs EA owe ease pacts cce ahd Sada Sod eet a as 26 Purification of Synaptotagmin 1 from Sf9 Cells eessecceeeceseseccenseesesecceseeeoees 26 Purification of Synaptobrevin 2 from E COLL cceecccecccesecesseeeteeesceeeseeeeeeeseeneneeeeeeaees 29 Reconstitution of Sytl and Syb2 into liposomes cececceeseceeeeeeeeeeteeeeesteeeeeteaeees 30 Quality assays of the reconstituted proteolipOSOMES ccceecceeseesteeeteeeeetteeeeeeeseeeees 32 Triple marker expression system for Stx1A SNAP25A and Muncl 8 1 ee 33 Liposome perfusion and capacitance measurement in excised patches ceeeeeeee 35 2 3 Results and GISCUSSION 4 sssysicicneh see aenecig hed 37 Purification ot Sy aptotagmin 2 82 cinta oiack ou Gets tide iy ee e
97. ch in syntaxin during exocytosis role of muncl8 EMBO J 18 4372 4382 Eberhard D A Cooper C L Low M G and Holz R W 1990 Evidence that the inositol phospholipids are necessary for exocytosis loss of inositol phospholipids and inhibition of secretion in permeabilized cells caused by a bacterial phospholipase c and removal of atp Biochem J 268 15 25 Fan J S and Palade P 1998 Perforated patch recording with beta escin Pflugers Arch 436 1021 1023 Fanara P Hodel M R Corbett A H and Hodel A E 2000 Quantitative analysis of nuclear localization signal nls importin alpha interaction through fluorescence depolarization evidence for auto inhibitory regulation of nls binding J Biol Chem 275 21218 21223 Ferby I M Waga I Hoshino M Kume K and Shimizu T 1996 Wortmannin inhibits mitogen activated protein kinase activation by platelet activating factor through a mechanism independent of p85 p110 type phosphatidylinositol 3 kinase J Biol Chem 271 11684 11688 Fern ndez Chac n R K nigstorfer A Gerber S H Garcia J Matos M F et al 2001 Synaptotagmin i functions as a calcium regulator of release probability Nature 410 41 49 Fern ndez Chac n R Shin O K nigstorfer A Matos M F Meyer A C et al 2002 Structure function analysis of ca2 binding to the c2a domain of synaptotagmin 1 J Neurosci 22 8438 8446 Fix M Melia T J Jaiswal J K Rappoport J Z You D
98. ch that neurotoxins cannot cleave them Chen et al 2001 As described later in whole cell recordings with RBL cells the amplitudes of non SG fusion usually exceed 50 of the basal cell capacitance Figs 3 6 and 3 7 Non SG fusion in chromaffin whole cell recordings is usually substantially smaller in absolute and relative terms Xu et al 1998 indicating that its non SG pool is much smaller and possibly already primed and therefore toxin resistant ATP hydrolyzing processes support non SG fusion It is well documented that ATP is required by the NSF NEM sensitive factor to disassemble the cis SNARE complex in the SNARE cycle Jahn et al 2003 Whiteheart et al 1994 and that the addition of Vam7p a soluble SNARE bypasses the requirement of ATP in the yeast vacuole fusion system Thorngren et al 2004 Since non SG fusion in excised patches is NEM insensitive Table 3 1 one might expect that the non SG fusion would be ATP independent In whole cell recording replacement of ATP with a 77 non hydrolyzable analogue was found to significantly reduce non SG_ fusion Yarandanakul et al 2008 and we describe here that ATP hydrolyzing processes clearly support non SG fusion in excised patches In some cases the absence of ATP caused complete failure of fusion and the ability to fuse was restored when ATP and GTP were added back to the solution see Fig 3 4A That ATP is the critical factor for restoration of fusion was verifi
99. cientific was placed on top of the membrane as a dotting guide Dots were dotted through the holes of the plastic insert Since time but not quality of the blot was the major concern here the lengths of subsequent blocking washing and incubation were all shortened Purification of Synaptotagmin 1 from Sf9 cells The Sytl expression construct contains coding sequences of Syt1 fused with honeybee melittin secretory signal HBM and His tag eight histidine sequences on its N terminus The HBM His s Sytl fragment was generated via PCR and subcloned into pCR2 1 TOPO vector Invitrogen After sequence confirmation the fragment was cut 26 out by Ncol Klenow treatments followed by EcoRI digestion and then cloned into EcoRI Stul site of pFastBac vector Invitrogen The resulting plasmid pFB hhSyt1 was transformed into DH10Bac competent cells Invitrogen which contain parent bacmid and helper plasmid for transposition Gentamicin resistant gene and fusion gene driven by baculovirus polyhedrin promoter on pFB hhSytl were transposed into the parent bacmid and the transposition was verified using PCR To produce recombinant baculovirus Sf9 cells were plated on a 6 well cell culture plate and the recombinant bacmid was transfected into Sf9 cells using Cellfectin Invitrogen according to the manufacture s instruction The P1 recombinant viruses were harvested from the medium for further amplification and the Sf9 cells were scraped from t
100. ck prepared in 40 mM MES pH6 5 was added to proteoliposomes in a final concentration of 2 M to remove proteins that were not incorporated Schwab et al 2000 After 30 min extraction at 4 C the mixture was centrifuged at 65 krmp for 1 hr with a TLA 120 1 rotor Beckman Coulter The supernatant was transferred to a new microtube and the pellet containing incorporated proteins was resuspended in equal volume volume of the supernatant of buffer Dot blot with serial diluted samples was applied to examine the incorporation Orientation test was done by treating proteoliposomes with proteases followed by 32 western blot with designated antibodies To test the orientation of Sytl trypsin and antibodies V216 against cytoplasmic side of Sytl V761 against lumen portion of Syt1 were used For Syb2 chymotrypsin and polyclonal antibody P939 were employed For control experiment proteoliposomes were lysed by adding Triton X 100 to expose all the proteins before enzymatic digestion Western blot was used simply because the protein concentration was not high enough to be seen using Coomassie blue staining Triple marker expression system for Stx1A SNAP25A and Munc18 1 Three bicistronic cloning vectors were engineered They are pBNG2 pBMG and pBPR which express nucleus localized EGFP membrane targeted EGFP and peroxisome localized DsRed respectively When three of them are expressed in the same cell the cell has green nucleus green
101. col 36 2 3 Results and discussion Purification of Synaptotagmin 1 Before the HBM His s Sytl and Sytl His s fusion constructs were made several other expression constructs were used for expressing Sytl One of them co expressed Sytl and Syb2 in Sf9 cells made by Jiong Tang in Dr Siidhof s lab UTSouthwestern and there was no affinity tag fused with Sytl coding sequence To purify the untagged Sytl several chromatographic columns were employed sequentially They were DEAE diethylaminoethyl CM carboxymethyl HA hydroxyapatite and Mono S Amersham columns Pioneer experiments were done to determine the fractions subjected to the next column and the conductance readings of the fractions were used as references Dot blot using anti Sytl antibody and representative fractions from each column are shown in Fig 2 1A The HA column was able to separate Sytl monomer dimer and degraded Syt1 fractions 11 18 14 in Fig 2 1B After further purification using Mono S column the concentration of the most concentrated fraction was only 0 15 ug ml This concentration is 1000 times lower than the practical concentration for reconstitution and the quality is not good either not shown In order to increase the quantity and quality of the purified Sytl a His tag was added to the N terminus of Sytl However Coomassie blue staining of the affinity purified Sytl indicated that the quality was still poor and the concentration was only
102. constituted cell free system the Ca dependent activity of PLA is reported to promote chromaffin SG fusion in a Ca independent manner Karli et al 1990 A similar effect has also been observed by pretreating plasma membrane with PLA and the fusion step was also Ca independent Nagao et al 1995 Furthermore the activities of phospholipases including PLA are reported to stimulate degranulation of permeabilized RBL mast cells Cohen and Brown 2001 If PI 4 5 P gt is the substrate for PLA the resulting free fatty acid is AA which is a precursor of some inflammatory mediators Brown et al 2003 Dennis 1994 As mentioned earlier AA has also been implicated in potentiating membrane fusion Latham et al 2007 and it facilitates SNARE complex formation in the presence of Munc18 Latham et al 2007 Rickman and Davletov 2005 and promotes interaction between SNARE complex and Muncl8 through Stx Latham et al 2007 The other PLA cleavage product is LPL which was thought to be fusogenic Poole et al 1970 and introducing LPL to membrane vesicles prepared from rat parotid acinar cells increased fusion between membrane vesicles and SGs Nagao et al 1995 However direct involvement of LPL in triggering fusion has been ruled out in a hemi reconstituted cell free system Karli et al 1990 and LPL has also been shown to inhibit fusion of biological membranes Chernomordik et al 1993 and synthetic liposomes Che
103. core complex and bring the vesicle and membranes to close proximity Rizo 2003 Rizo and Stidhof 2002 Upon membrane fusion the trans SNARE complex three of its components are in two opposing bilayers becomes cis SNARE complex all three components are in the same bilayer and the complex is then unwound in an ATP dependent process mediated by NSF N ethylmaleimide sensitive factor and SNAPs soluble NSF attachment proteins Rizo and Siidhof 2002 The recycled SNAREs are now ready for the next round of fusion Fig 1 3 Munc18 In addition to SNAREs Munc18 a mammalian homologue of C elegans UNC 18 is also essential for synaptic vesicle fusion In Munc18 knockout mice the Ca triggered release minis and a latrotoxin induced exocytosis were abolished Verhage et al 2000 However it was reported that Munc18 binds to the closed Stx Fig 1 4 upper panel which is incompatible with the core complex and competes with the formation of the complex Dulubova et al 1999 In spite of these controversial results two other active zone proteins Munc 13 mammalian homologue of C elegans UNC 13 and RIM Rab3 interacting molecule were proposed to release Muncl8 from Stx and facilitate the formation of the core complex In UNC 13 mutant neurotransmitter release was completely blocked and RIM mutant in worms showed severe decrease in neurotransmitter release Furthermore the UNC 13 and RIM mutants can be rescued
104. d as an exponential function I b a b e 4 9 where a b and Tt are the peak current steady state current and time constant respectively Fig 4 5A B To estimate b one first takes three equally spaced points A B and C from the decaying curve If the distance between A and B is A then A b a b e 4 10 B b a b e b a bje e 4 11 By defining m a b e n e 4 12 Equations can be re written as A b m 4 13 B b mn 4 14 C b mn 4 15 Solving Eq 4 13 14 15 simultaneously gives Eq 3 2 which is 130 BAC ae ee ce 3 2 2B A C ee However as mentioned in Chapter 3 and as shown in Fig 4 5B we use the averages of section A B and C rather than using three single points By doing this the noise of the estimation is suppressed because of the averaging To proof that Eq 3 2 is still valid when the section averages are used considering that the sum of the sections are i t d i t d A b m 4 16 i t i t i t A d i t d gt B gt b m n 4 17 i t A ist i t 2A d i t d gt C 2 b m n 4 18 i t 2A By defining 1 i t d M m 4 19 i t the section averages A B and C can be re written just like Eqs 4 13 14 and 15 except that the m is replaced by M Accordingly the solution is still Eq 3 2 After subtracting the steady state current b from the half pulse the natural logarithm of the trace is In a b t t 4 20 T
105. data by calling Show_update_Callback calculate wave protocol Pulse fag record AO trigger time ll f Puise_Caliback restat AO protocol completed resume resume AO l set AO StopFcn resume set AO StopFen to NULL set AO TriggerFon Puise Tag set AO TriggerFen to NULL When the Pulse button is pressed the function Pulse Callback calculates the wave protocol and sets AO StopFcn and TriggerFcn Function PulseTag is evoked when AO is triggered and records time point at which the pulse protocol is given When the protocol 170 is completed function resume resets the AO properties and then restart the AO Main variables in the program There are tons of variables in the program and most of them are categorized into groups and have clear descriptions in the codes A few important variables are introduced below handles bufdir tt contains temporary directory used to save CapBuffer daq file If the Raw data check box is checked CapBuffer daq will be renamed to FILENAME daq and moved to the directory indicated by handles current_folder handles current_folder it is the last folder you visited when you use the Save or Load button handles nidaqid device ID for the data acquisition board There might be more than one device ID for the installed board because of different drivers used The device ID can be 1 2 Dev1 or Dev2 etc for boards from National Instruments NI
106. dition B escin has to be prepared freshly and the potency varies among different preparations Thus it is difficult to explain negative results because one can not tell if the cell is not permeabilized enough or the fusion machinery is really jeopardized under testing condition For the same reason it is also difficult to keep the initial Ca concentration in the cell the same in different experiments Because of these reasons I eventually decided not to continue this approach Change the direction of excision As mentioned previously some optical dense materials are detached from the membrane when cells are blew up It is also true when the standard excision procedure is used Since the SGs may be associated with these materials I tried to move excise the patch in different directions in hope that the dense materials might be preserved better It is difficult to describe the actual motion in words and unfortunately I don t have a movie for that either The point is to excise the patch gently and try to keep the dense materials attach to the membrane When RBL cells were used some SGs were indeed preserved in excised patches using this method but the success rate was quite low and there were only few SGs in a patch It is presumably owing to the low abundance of SGs and the heterogeneity of the RBL line will be discussed later To increase the number of secretory granules I have tried the followings 103 l Pre incubate
107. dual lipids by applying specific antibodies in whole cell recording experiments before triggering fusion via Ca infusion Impressively neither anti PI 3 P nor anti PI 4 P antibody was able to block or slow down non SG fusion and a 79 combination of both antibodies was also ineffective Fig 3 6 It seems unlikely that PIP is involved because another PI 3 K inhibitor LY294002 also did not block fusion in excised patches Table 3 1 In fact pretreatment of the cells with a submicromolar concentration of wortmannin which blocks PIP producing PI 3 K and treatment with genistein an inhibitor of tyrosine kinases that are typically activated in this pathway Galetic et al 1999 failed to block non SG fusion in whole cell recording data not shown We also used a PI transfer protein Mousley et al 2007 to remove PI from excised patches to test if there is any role of PIs at all in non SG fusion As shown in Table 3 1 fusion was not inhibited by a concentration that we found to inhibit the ATP dependent stimulation of Na Ca exchange current in excised patches a process determined to reflect phosphorylation of PI Nasuhoglu et al 2002 All of these negative results support the conclusion that non SG fusion is PI independent The actual targets and the specificity of wortmannin and adenosine in this study must therefore be questioned and as described next there is an important difference between excised patches and whole cell respo
108. e adaptor out of the electrode slowly Make sure that the carbon fiber is in contact with KCl solution and also avoid bubbles in the electrode Insert Ag AgCl wire and seal the end Install the carbon electrode to the pipette holder through the infusion line outlet in a direction that is from the holder to the headstage Insert a 76 2 um 0 003 inch Ag AgCl wire into KCI solution in the electrode and then seal it with hot melt glue To seal the junction with hot melt glue push some glue out of the glue gun form a little ball cool the glue a little bit and then immerse the junction into the glue ball Pull the glue gun a little bit away from the electrode in a direction that is perpendicular to the electrode and then further cool down the glue with some airflow Pull away the glue gun quickly and cut the tail with a razor blade If the glue ball is too hot when you immerse the junction into it the seal will be too tight In this case you probably won t be able to reuse the Ag AgCl wire because you may break it when you try to remove it from the electrode However if the glue ball is not hot enough the seal may be too loose and solution will 100 evaporate from the electrode and the electrode will disconnect with the wire in a short period time In addition also note that for convenience the Ag AgCl wire has been soldered to a regular wire that is connected to the headstage Efforts for preserving secretory vesicle
109. e in the allergic response Nature 431 1007 1011 Allen V Swigart P Cheung R Cockcroft S and Katan M 1997 Regulation of inositol lipid specific phospholipase cdelta by changes in ca2 ion concentrations Biochem J 327 Pt 2 545 552 Almers W and Neher E 1987 Gradual and stepwise changes in the membrane capacitance of rat peritoneal mast cells J Physiol 386 205 217 Aoyagi K Sugaya T Umeda M Yamamoto S Terakawa S et al 2005 The activation of exocytotic sites by the formation of phosphatidylinositol 4 5 bisphosphate microdomains at syntaxin clusters J Biol Chem 280 17346 17352 Bai J Tucker W C and Chapman E R 2004 Pip2 increases the speed of response of synaptotagmin and steers its membrane penetration activity toward the plasma membrane Nat Struct Mol Biol 11 36 44 Balla A Kim Y J Varnai P Szentpetery Z Knight Z et al 2008 Maintenance of hormone sensitive phosphoinositide pools in the plasma membrane requires phosphatidylinositol 4 kinase iti alpha Mol Biol Cell 19 711 721 Barnett D W and Misler S 1997 An optimized approach to membrane capacitance estimation using dual frequency excitation Biophys J 72 1641 1658 Blackwood R A Smolen J E Transue A Hessler R J Harsh D M et al 1997 Phospholipase d activity facilitates ca2 induced aggregation and fusion of complex liposomes Am J Physiol 272 C1279 85 Borgonovo B Cocucci E Racchetti
110. e paper try to do it on aluminum foil It sometimes helps Keep moving the quartz tubing to insert the fiber and hold the fiber still with the other hand until the fiber no longer gets inserted or reaches desired length If you can not get carbon fiber inserted long enough into the quartz tubing try to sweep the fiber into the tubing using the forcep made previously Be careful not to break the fiber I usually keep 0 5 cm fiber handing outside of the quartz tubing If the tip portion is too long the recorded amperometric current might be quite noisy If it is too short you may not reuse the electrode since the tip is cut every time before experiment to expose fresh carbon surface step 4 Insulate the protruding carbon fiber Put some flowable silicone sealer on a pipette tip I use yellow tip hold the yellow tip and then adjust the focus of the stereomicroscope so that you can see 98 the ball shaped sealer clearly Under the microscope immerse the protruding carbon fiber into the ball shaped sealer and let it stand for few seconds The sealer goes into the quartz tubing owing to capillary effect Pull the electrode out of the sealer slowly in a direction that is perpendicular to the electrode The carbon fiber is bent in this way but nothing bad is going to happen because it is quite flexible The boundary between the ball shaped sealer and the fiber moves retracts slowly from the quartz tubing to the tip of the carbon fiber
111. e sensitive detection PSD is utilized in both cases If square wave perturbation is selected one can chose to use either integrated charges Q SQA or direct current trace I SQA for calculating the cell parameters Other functions like digital filtering pulse stimulation offline phase angle adjustment baseline subtraction and data normalization are also implemented System requirements Using MATLAB R2006b or R2007a is recommended Versions earlier than R2006b are not supported R2007b is also not recommended because the Foregroundcolor and Backgroundcolor properties of GUI buttons do not work properly However if you need to synchronize the recording with an external TTL signal e g image acquisition software you need to use R2007b because the HwDigital trigger of AO object is not functioning in earlier versions It is reported by my colleague that Capmeter is not compatible with MATLAB R2008a It is always quite painful to deal with this kind of compatibility issues especially when the program is quite complex The even worse thing is that we do not have R2008a license for the lab So you may download R2007 from the Mathworks to run Capmeter if you have the license and there is no solution for the compatibility issues temporarily as of March 2008 151 The program is developed for DAQ boards from National Instruments However you can use other DAQ boards supported by the MATLAB Data Acquisition Toolbox Some ad
112. e surprising therefore that the activities of the kinases as well as other phosphatases also vary among batches of cells and the length of time after isolation Unless AMP PNP is added to the pipette solution the ATP dependency of non SG fusion in whole cell recordings is usually not significant Yaradanakul et al 2008 Thus the excised patch model naturally produces many more sources of variability that do not exist in the intact cells This is at once an advantage for identifying important partial mechanisms of fusion and its regulation and a disadvantage because the reproducibility of experiments is significantly decreased ATP sensitivity of non SG fusion Results from this article further support our conclusion that neither PI 4 5 P gt nor its metabolism modulate significantly non SG fusion when the trigger Ca concentration is high Yaradanakul et al 2008 Depletion of PI 4 5 P2 at the plasmalemma does not block non SG fusion in M1 receptor expressing BHK cells Yaradanakul et al 2008 and we have shown here that multiple PI 4 5 P ligands namely neomycin and PI 4 5 P gt antibodies do not affect Ca induced fusion in the excised patches from RBL cells While the antibodies employed might not have sufficient affinity to block high affinity functions of phosphoinositides in fusion we expect that the high concentration of neomycin employed 500 uM would bind non selectively all phosphoinositides and thereby inhibit thei
113. e upper bar graphs the peak exchange currents were not significantly affected Cell swelling was associated with a 76 decrease of the fusion response p lt 0 01 whereas cell shrinkage with hypoosmotic cytoplasmic solution was without effect Next we tested whether cell swelling by hypoosmotic extracellular solution also reduces membrane fusion As shown in Fig 3 8B placement of cells in extracellular solution with NMG reduced by 80 mM hypoosmotic outside for 2 min prior to activating exchange current caused an 85 decrease of membrane fusion To examine whether the effect of swelling is reversible we placed cells for 2 min in hypoosmotic solution and then moved them back into isoosmotic solution for 2 min prior to activating exchange currents During the protocol swelling and shrinkage of cells was clearly visible As shown for post swelling the fusion response was partially restored In a fourth experimental group we tested whether the restoration of fusion might be blocked by wortmannin Using the same protocol to allow restoration of fusion inclusion of 5 uM 82 wortmannin in the pipette solution significantly decreased the recovery of fusion responses after swelling p lt 0 05 From 3 observations we did not find the adenosine wortmannin combination to be more effective data not shown Overall these whole cell results support the notion that membrane perturbation and or stretch strongly inhibits non SG fusion and
114. ed in several experiments in which ATP was applied without GTP Figs 3 3A 3 4B and data not shown Further experiments supported the notion that ATP hydrolysis is essential to maintain fusion because the non hydrolyzable ATP analogue AMP PNP 2 mM did not substitute for ATP Fig 3 4B Since the decrease of fusion after removal of ATP and with substitution by AMP PNP takes much longer than expected for washout of ATP from the patch ATP hydrolyzing reactions clearly are not part of the final fusion process The ATP mechanism s that support fusion in the longer term could involve the phosphorylation of target proteins lipids and or the use of ATP in other types of energy dependent reactions as in the case of NSF In favor of the former idea we could preserve the fusion capability in the absence of ATP by adding EDTA to the solution see Table 3 1 Since magnesium is the only divalent ion in our recording solution we hypothesized that ATP is used to phosphorylate one or more targets and that activity of the counteracting phosphatases would be magnesium dependent ATP dependent generation of PI 4 5 P2is not critical As mentioned earlier PI 4 5 P2 has been implicated in multiple aspects of fusion processes and we therefore tested if the role of ATP is to maintain PI 4 5 P in the 78 excised patches and as well if the cleavage of PI 4 5 P2 is required for triggering membrane fusion Application of a very high concentration of
115. efore using ssesssssesseeseesressessresressresressrresssrrsssresssre 153 Ru nning THE program eseni a AEE E E a EE eari 154 LOREENA PSD E E T E 157 Usine SOA anan a ee E E N R 159 Using digit l filters aa ak E A E bite ated ik aad 160 Variabl s in the WorkSpace ania sicvsezecanntseasuenshensvosshea tanwsnedtens er e Ee cuenta AE ARNEE 160 Subtracting the DASE Ne x seiceanna a aE E E S 163 Scaling the ata oneens i eal r a a E a 164 SHOW Uo Te ata AAEE E E E S EEEE E EEE S 165 Exporting the datas nie a a E E E a E A A AEA 166 GIving PUSS animeen ia E tages E E A E R at 167 PTE GS CPS E E A E A 168 R ader mode eisian eea SR Ra a E Pn eee 169 Th sinformaton TLOW aasanrssnincwsyaimepsee ss nied anodes a a E o a aR aA 169 Main variables in the program seseesssesersessssessessessressessessressresersrsessseeesssreessressse 171 AA Capmeter Tiniasicaecieuesinnadciemede le ine A a on 174 Wat 18 102 fennen ea sheen s aia tana neretav ona wise E roo a ern Damon 174 Connection AST AE ensendi a a peta Coen TET adalat 174 Things you need to know before USING eeccecceeseeeeeeneeeseeceseeeseeceneeeeeseeecentaaees 174 AS ODIOb E E E ee ee ie 175 WV That isit cas sas soa oleae Gui arte cate oy eb un ies a E AE E ovgunteada tesa E Eat 175 COMME CHOI CIA TAINS ics Faas cea cadiis ea eee sews is sence E E AE aa Os Pee sense ess Etant 175 Things you need to know before usin Giese aieces eteecus oc sogesesvideshediateeae sen iede aaah
116. elationship is shown in B The steady state Qs is also estimated using Eq 3 2 C To get the peak current a and time constant T the trace is inverted first and linear regression of time against the natural logarithm of Y gives t and a b t Since b and Tt are known a can be obtained accordingly 209 a b A Figure 4 7 Correction of the integrated charges Direct summation of the product of the current and time interval will over estimate the transferred charges because of the gray areas as shown in A The phenomenon is simulated in MATLAB and shown in B The back trace shows the summation and the gray trace represents the actual transferred charges C Current acquired in an acquisition interval of A is shown The ratio of the square and the white area is used to correct the transferred charges Eq 4 34 210 211 AJOA ds WONIIIIOD JOY PUL BOJ9Q i NTxS PUB G IL SIONO IL Joued IU oy UI UMOYS SE 19040 Youd deyAdAo soven OM pep by SUISN UOTD9IIOS 19y ONG Ul SI uonewwns SB1eYO JOOIIP IY pue Por UT ST 9921 BOT OIONY OY oued Yo oy UI GV TLV OU JO JOYS UAIS UOYIIIIOI IS AvYI IY JO UOV AYSUOWIG S p IMZA s eatdure 4 1 Ula T sP2B ed OelBol s SWvowv 1 SeRea daH mopu doyseq soo y sur Mal WPI d oH ET oal anl l leav l eangu deH mopu dopseq sool mesur Mem IPI Ad KE ot
117. ell simulations using the MATLAB component Simulink as well as our own routines In the absence of noise and a filter function the algorithm retrieved simulated cell parameters with errors of 1 ppm With cell parameters that would be considered experimentally unacceptable e g 200 pF a Ra of 20 MQ a Rm of 50 MQ and voltage oscillation at 200 Hz the algorithm still retrieved the parameters with an accuracy of 99 9 Signals were usually acquired at 100 kHz and digital filtering was performed by averaging signals in an adjustable time window Data were usually digitized at 100 Hz and a running mean median filter was applied to the digitized data when data smoothening deglitching was desired Program Capmeter 1 was used with the hardware lock in amplifier serving as a plain data recorder with digital filtering and data smoothening deglitching functions The programs are available for download at http capmeter googlepages com Patch clamp and data acquisition We used National Instruments Austin TX board PCI 6052E to generate the command potential and collect signals and we used an Axopatch 1D Molecular Devices Sunnyvale CA for patch clamp Electrode tips were dipped in molten hard dental wax Kerr Corporation Romulus MI before cutting and polishing to reduce stray capacitance For excised patches electrodes with 15 um inner diameters were employed The giant patch was excised by essentially aspirating the cell into a
118. elled from the lipids under N gt flow and the lipid film was further dried under vacuum for an additional hour An appropriate amount of buffer buffer for electrophysiological recording was added into the glass tube to make 30 the final lipid concentration 15 mM and the tube was agitated strongly using Vortex for at least 5 min to hydrate the lipid film The hydrated lipids were moved to a 15 ml centrifuge tube and subjected to 5 freeze thaw cycles using liquid nitrogen and warm water to increase entrapment of water soluble compounds The lipids formed large multilamellar vesicles LMVs after these steps The extrusion method was used to make LUVs from LMVs The mini extruder was assembled according to the manual Avanti polar lipids and a polycarbonate membrane with 80 nm pore size was employed After several times of extrusion the LUVs were collected in a 1 5 ml microtube and stored at 4 C The diameter of LUVs made by this method is about 100 nm To make Syt1 Syb2 proteoliposomes 1 volume of liposomes total lipid concentration 15 mM was mixed with desired amount of Sytl and Syb2 recombinant proteins contains 1 OG The total volume was adjusted to 3 volumes and the OG concentration was adjusted to 0 77 Note that if buffer needs to be added into the mixture add buffer prior to adding proteins because LUVs may be solubilize if the OG concentration which is present with proteins is too high After mixing the total lipid
119. embrane binding through electrostatic interaction with acidic phospholipids Proc Natl Acad Sci U S A 91 12253 12257 Smith A J Pfeiffer J R Zhang J Martinez A M Griffiths G M et al 2003 Microtubule dependent transport of secretory vesicles in rbl 2h3 cells Traffic 4 302 312 Smith C Moser T Xu T and Neher E 1998 Cytosolic ca2 acts by two separate pathways to modulate the supply of release competent vesicles in chromaffin cells Neuron 20 1243 1253 Spudich A and Braunstein D 1995 Large secretory structures at the cell surface 224 imaged with scanning force microscopy Proc Natl Acad Sci U S A 92 6976 6980 Siidhof T C and Rizo J 1996 Synaptotagmins c2 domain proteins that regulate membrane traffic Neuron 17 379 388 Tang J Maximov A Shin O Dai H Rizo J et al 2006 A complexin synaptotagmin 1 switch controls fast synaptic vesicle exocytosis Cell 126 1175 1187 Thompson R E Lindau M and Webb W W 2001 Robust high resolution whole cell patch clamp capacitance measurements using square wave stimulation Biophys J 81 937 948 Thorngren N Collins K M Fratti R A Wickner W and Merz A J 2004 A soluble snare drives rapid docking bypassing atp and sec17 18p for vacuole fusion EMBO J 23 2765 2776 Togo T Alderton J M Bi G Q and Steinhardt R A 1999 The mechanism of facilitated cell membrane resealing J Cell Sci 112 Pt 5 719 731 T
120. eoretically two different colors e g green and red can be targeted to these three cellular compartments and cells expressing a maximum of 6 different proteins can be distinguished from others using the combination of color localization information I further targeted cytoplasmic fluorescent proteins to peroxisome to ensure that membrane targeted fluorescent proteins can be distinguished from them Ideally cells co expressing Stx1A SNAP25A and Muncl18 1 look like Fig 2 10A The idea was great but the outcome was sad The major problem was that the membrane targeted EGFP could not been distinguished clearly from nucleus targeted EGFP under epifluorescence microscope Fig 2 10 B C I am sure that they can be distinguished under confocal microscope however I do not have a confocal with my patch clamp setup and it is also impractical to scan every cell to locate the right one Another problem was the transfection efficiency Even when the transfection efficiency looked great on the culture dish it was still not easy to find a cell that expresses two different colors after trypsinization Presumably because the transfection efficiency was over estimated when the cells were still attached to the culture dish As a last resort I started to make cell lines co expressing all three components 42 Expression constructs pBNG2 Stx1A pBPR Munc18 and pBNG2 SNAP25A were used I did not use pPBMG SNAP25A simply because at the time of transfect
121. er estimated Variable astmax represents the last maximal point of the curve I use the last maximal point because if data clipping happens or when the seal is gone many points around the real peak will be with the same value which is 10 volts Variable SPC stands for samples per curve If the range for asymptote estimation astmax to SPC 1 is less than 12 points determined empirically a point at SPC 12 is assigned to astmax The size of each section is then adjusted so that no memory leakage will occur The asymptote is calculated using Eq 3 2 and if the estimation cannot be done variable quality is set to zero to skip the coming fitting procedures To calculate the peak current and the time constant the routine uses Eq 4 20 and linear regression C codes for peak and tau estimation fladdl firstmin lastmax tl1 for i 0 i lt SPC it dataB i asymp It is 1n a b temp double mxRealloc temp fladdl sizeof double for i 0 1 lt fladdl it temp i dataB lastmax ti Cfind temp 10 0 fladdl amp ftemp amp fc if fc 0 firstmin lastmax ftemp 0 so that there won t be negative in log linear regression fladdl firstmin lastmax 1 for i 0 i lt fladd1l i lny log dataB lastmax i sx dataBtime lastmaxti 138 sx2 dataBtime lastmax i dataBtime lastmaxti sy lny sxy
122. er everything is ready the potential difference presumably between the reservoir and the chamber blasts your precious seal It does not help to use other types of solution stopper e g metal clamp as long as they are placed between the reservoir and the chamber The only way I found useful is to apply negative pressure to hold the solution and release it to start the solution flow This special reservoir is made by two 3 ml syringes One of the syringes is cut at 0 5 94 ml scale mark and the other one is cut very close to the tip The tip portions of the syringes are glued together using a hot melt glue gun One end of the reservoir is connected to the solution line and the other end is connected to a solution switch This switch is used to hold the air pressure and when it s turned on the pressure is released Shield everything Since the solution lines behave like an antenna network it is import to shield the exposed lines as much as possible You may make a Faraday cage around the perfusion system using aluminum foil or plate and make sure that the Faraday cage is grounded If the cage is not grounded it might introduce even more noise into the system In addition to shielding the solution lines you may want to shield the temperature control system as well Although the circulating water in the temperature control system is not electrically connected to the recording chamber it may still carry detectable noise into the reco
123. ere encountered in which fusion did not run down in the absence of ATP and EDTA data not shown while the loss of fusion over time in ATP free solution was highly reliable in other batches For these reasons all experiments were done in a one control vs one test result pattern as pointed out in Methods and results can only be compared with data collected at the same time using the same batch of cells Our overall interpretation is that in our routine whole cell configuration all available vesicles eventually fuse to the plasma membrane as there are no inactive cells and the percentage increase of cell area so impressively large in relation to the number of docked vesicles observed Yaradanakul et al 2008 Since the fusion process is strongly inactivated by cell swelling it seems likely that membrane stretch or flattening of invaginations disrupts the fusion machinery with restoration requiring ATP dependent processes of an unknown nature Remodeling of cytoskeleton is an attractive but unproved possibility Our results on run down in patches Fig 3 4 and Table 3 1 are consistent with ATP being used to phosphorylate one or more targets that maintain the 87 fusion capability whereby dephosphorylation evidently occurs by a magnesium dependent phosphatase In this regard it is known that the enzymatic activities of tyrosine phosphatases vary with culture conditions and ages Cool and Blum 1993 Pallen and Tong 1991 It would not b
124. es from tens to hundreds of microseconds For a data acquisition board acquiring data at 100 kHz the time interval between two sample points is 10 us which is in the same time scale of the clamping time constant Direct summation of the product of the current and time interval will over estimate the transferred charges in this condition because of the gray areas shown in Fig 4 7A To show it mathematically it is gt 1 Azf Idt 4 31 According to simulation unless the A is 40 times faster than the clamping time constant the summation of the product can not approach the actual transferred charges properly not shown That is if t is 40 us the acquisition speed for a single channel has to be at least 1 MHz and for 3 channels the speed has to be at least 3 MHz This kind of board is not available as of March 2008 The phenomenon is simulated in MATLAB and shown in Fig 4 7B The black trace shows the summation and the gray trace represents the actual transferred charges To solve this problem without waiting for new technologies I derived an equation to correct the estimated Qs Current acquired in an acquisition interval of A is shown in Fig 4 7C White area represents real transferred charges which is 141 142 m t 1 e 4 32 The rectangle area used for charge summation is m A 4 33 1 Thus the transferred charges can be corrected by multiplying with the ratio of Eq 4 32 and Eq 4 33 Qs Qs t 1 e
125. et al 2004 Imaging single membrane fusion events mediated by snare proteins Proc Natl Acad Sci U S A 101 7311 7316 Fukami K Nakao K Inoue T Kataoka Y Kurokawa M et al 2001 Requirement of phospholipase cdelta4 for the zona pellucida induced acrosome reaction Science 292 920 923 218 Galetic I Andjelkovic M Meier R Brodbeck D Park J et al 1999 Mechanism of protein kinase b activation by insulin insulin like growth factor 1 revealed by specific inhibitors of phosphoinositide 3 kinase significance for diabetes and cancer Pharmacol Ther 82 409 425 Geppert M Goda Y Hammer R E Li C Rosahl T W et al 1994 Synaptotagmin 1 a major ca2 sensor for transmitter release at a central synapse Cell 79 717 727 Gil A Viniegra S and Guti rrez L M 2001 Temperature and pma affect different phases of exocytosis in bovine chromaffin cells Eur J Neurosci 13 1380 1386 Go i F M and Alonso A 1999 Structure and functional properties of diacylglycerols in membranes Prog Lipid Res 38 1 48 Grishanin R N Kowalchyk J A Klenchin V A Ann K Earles C A et al 2004 Caps acts at a prefusion step in dense core vesicle exocytosis as a pip2 binding protein Neuron 43 551 562 Hilgemann D W and Lu C C 1998 Giant membrane patches improvements and applications Methods Enzymol 293 267 280 Hu C Ahmed M Melia T J S llner T H Mayer T et al 2003 Fusio
126. ethod summates all At fragments and outputs the 180 final value The second method further estimates the asymptote of the integrated charges Fig 4 6B using Eq 3 2 which is also used in Q SQA However note that in Q SQA the amount of transferred charges is corrected according to the time constant using Eq 4 34 In IQplot the value is not corrected because time constant from a single curve every curve is considered different from each other is not quite accurate and will introduce excessive noise to Q if Eq 4 34 is used Also these two methods are interchangeable during the experiment so you might want to make a note to specify the method you used The program does not keep track of it Display modes online When the recording is started axis2 shows you the sampled current After the first pulse protocol is given axis shows you the corresponding current the number of points displayed is reduced 10X to relieve CPU load in online mode and axis4 and axis5 show I V and Q V plots respectively If the pulse protocol s is given more than once the old traces will be shown in gray and the new one is placed on top of them unless the Erase mode is selected To erase the old traces you may either click the corresponding Erase button in the Erase mode panel every time when needed or you may check the corresponding check boxes in the panel and the old traces will be erased every time when the new one is plotted You may adjus
127. etically but as you may know all kinds of unexpected conditions and or errors may occur in MATLAB Anyway axis2 and the corresponding variable datasample in the Workspace should not be considered as a real experimental data It is just a reference for you to monitor the seal condition etc as 182 mentioned at the very beginning If you have clicked the Set baseline button in the Pulse protocol panel during the experiment the given pulse train will be represented by a star shaped mark in the offline mode You may also set the baseline offline simply by clicking the Set baseline button Data for current pulse train will be used for subsequent subtraction and the color of the mark becomes yellow Note that you may set the baseline to data from any pulse train in the record not just to one that has been used during the experiment When the baseline is set offline you can only access pulse trains which used the same pulse protocol as the baseline used The color of symbols for pulses using different protocol s becomes gray in this case You may navigate the pulse trains by using the Navigate panel Click the gt button to see the next record and click the lt button to check the previous one You can use the Pick button to jump to the desired record directly too You may also specify the time range in the edit boxes and then press Enter or use the to button to select a region to be shown on axis2 To show all
128. f my dissertation I would like to thank my friends in Don and Tom s labs especially Chengcheng Shen Marc Llaguno Alp Yaradanakul and Vincenzo Lariccia Chengcheng helped me a lot in computer programming Marc gave me many critical suggestions for making carbon electrode and Alp and Vincenzo are the persons who make the lab like a family I would also like to thank my sister and her husband for taking care of my parents over these years Most of all I would like to give my thanks to my parents and my wife Without their love and encouragement they have given to me it would be impossible for me to complete my dissertation They give me the courage and strength to face any coming challenges in my life MEASUREMENT AND ANALYSIS OF CALCIUM DEPENDENT EXOCYTOSIS IN GIANT EXCISED MEMBRANE PATCHES Publication No Tzu Ming Wang Ph D The University of Texas Southwestern Medical Center at Dallas 2008 Advisor Donald W Hilgemann Ph D Ca dependent exocytosis was studied in both excised and whole cell patch clamp with emphasis on the rat secretory cell line RBL Capacitance and amperometric recordings show that secretory granules SGs containing serotonin are mostly lost from excised patches Small vesicles that are retained non SGs do not contain substances detected by amperometry Non SG fusion is reduced by tetanus toxin light chain treatment however it is unaffected by N ethylmaleimide implying that SNARE cycling
129. fibroblasts MEFs Fusion remains robust in syt7 knockout MEFs Unlike results with RBL cells non SG fusion in MEFs often involves fusion of large vesicles arrowheads Rate constant 1 s a 0 200 400 600 800 1000 Ca uM Figure 3 10 Ca dependence of non SG fusion in excised patches from RBL cells The concentration response data are best described by a Hill equation with a slope coefficient of 2 0 a Km of 71 UM and a maximal rate constant of 2 1 s 116 Table 3 1 Effects of various reagents on Paired control Testing condition Amplitude fF mean SEM n Ratio of active patches mean SEM n non SG fusion in excised patches Rate constant 1 s mean SEM n TeTx E234Q 209 2460 9 0 8940 11 9 0 3 0 04 7 TeTx WT 10 9 8 4 10 0 1 0 1 10 0 36 1 ATP GTP 160 8442 2 18 0 67 0 11 18 1 52 0 27 12 NEM ATP GTP 138 5443 7 18 0 5 0 12 18 1 0140 21 9 ATP 119 8431 8 12 0 58 0 15 12 0 8 0 11 7 AMP PNP 35 349 4 13 0 15 0 1 13 1 04 0 27 3 Mg buffer 53 3 19 9 10 0 3 0 15 10 1 1242e 3 2 EDTA 168 6470 7 10 0 8 0 13 10 1 50 34 8 EDTA 1min 221 34106 2 5 0 8 0 2 5 0 72 0 11 4 neomycin EDTA 102 4438 5 5 0 8 0 2 5 0 4740 1 3 FVPP 1min 267 3461 8 1 8 0 75 0 29 8 PIP2Ab FVPP 118 3 25 6 7 0 8640 14 7 1 12 0 3 6 FVPP 2min 78 2439 6 0 5 0 22 6 0 32 0 09 2 PIP2Ab FVPP 128 9440 4 6 0 67 0 21
130. functions It is modified from Capmeter 6 and inherits most of the functions from Capmeter 6 except the ability to extract cell parameters You may record at most 4 channels at the same time using Capmeter 1 Connection diagram Capmeter1 ALO Al1 Inputs a trigger ANS AO peat Chit f TTL in Al4 AlS optional PFLO CTRO AO 1 AQ 0 Y EXT Command front switched EXT Command rear switched Although Capmeter 1 does not send out sine or square waves it still uses trigger signals from AOO to trigger acquisition so that signals can be extracted and processed during recording using MATLAB function ge data please refer to Introduction for detail Things you need to know before using 1 Please cite the paper Wang and Hilgemann 2008 2 Please remember to adjust the command sensitivities 3 Please refer to section 4 3 Capmeter 6 for detailed description and discussion 174 4 5 IQplot What is it IQplot is a program to probe current voltage I V and transferred charge voltage Q V relationships in voltage clamp configuration It is developed to fulfill my mentor s will I don t have any idea about the differences between IQplot and other commercially available software because I have never used any of them please refer to the Introduction if you are interested in the history Connection diagram IQplot2 ALO Al1 a b Inputs a trigger Al2 e b current Al4 AL5
131. fusion system were made Two quartz tubings with 40 um inner diameter were stuck together to make a mini O tube The mini 6 tube was fixed into a glass pipette and the orientation of the mini tube was marked on the glass pipette under stereomicroscope The perfusion system was then mounted to the microscope and positioned according to the marker on the glass pipette If liposomes were not loaded with carboxyfluorescein a tiny amount of carboxyfluorescein was mixed with liposomes prior to experiments so that solution flow could be monitored under fluorescence microscope An amount of 30 ul liposomes was enough to use for an entire day and air pressure from a syringe was used to control the solution flow as used in the intra pipette infusion system Temperature was set to 37 C by a heating fan with its sensor stuck on the stage of the microscope Patches were excised from INS 1 cells or cells co expressing Stxl1A SNAP25A and Muncl18 1 Two different protocols for triggering liposome fusion were tested The first one mixed ImM Ca with liposomes first and then moved the patch from blank liposomes Ca to proteoliposomes Ca In the second protocol patches were placed under blank liposome Ca to trigger endogenous vesicles fusion and then incubated with 35 Syt1l Syb2 proteoliposomes for 1 min The patches were then moved back to blank liposomes Ca again to trigger fusion This protocol was referred to as the docking priming proto
132. g for the optimal phase angle using cross correlation and 2 calculating correlation coefficient between two input traces For detail please refer to section 4 2 Square wave perturbation PSD analysis of SQA data To scan the phase spectrum the syntax is P C sft G sft R c R g PhaseMatcher2 switch Csqa Gsqa Cpsd Gpsd step degree Input argument switch determines the channel s used for cross correlation If switch 1 PhaseMatcher 2 returns an angle so that PSD and SQA from Ch2 conductance have the highest correlation If switch 0 PSD and SQA from Ch1 capacitance are used For switch lt 0 both Chl and Ch2 are used and the sum of cross coefficients between PSD and SQA from Chl and Ch2 is the highest at the given angle Input arguments Csqa Cpsd Gsqa Gpsd denote capacitance from SQA from PSD conductance from SQA and from PSD respectively The last argument step defines the scanning step size in degree in PhaseMatcher2 Scanning step size of 0 1 degree is used in Capmeter6v3 The output argument P is the obtained phase angle and C_sft and G_sft are phase shifted Chl and Ch2 data at the given angle P respectively Output arguments R_c and R_g are the corresponding correlation coefficients at angle P To get the correlation coefficient between two traces simply use the following syntax 195 R PhaseMatcher2 tracel trace2 The correlation coefficient R is returned SqWaveCalc mexw32 The on
133. ghtman 2002 and the one on the Protocol Online website http www protocol online org The mast cell buffer used in the protocol contains in mM 140 NaCl 5 KCl 10 glucose 2 CaCl and 1 MgCl pH7 4 One mouse is used for each preparation and the mouse is anesthetized by inhalation of a mixture of CO and O2 Clean the abdomen of the anesthetized mouse with 70 ethanol and then inject about 3 ml of the mast cell buffer and 2 ml of air into the peritoneal cavity using a syringe with 25 G needle It is important to inject some air into the peritoneal cavity as it increases the yield in the following shaking step After injection retract the needle from the mouse and the injected buffer and air is retained inside the cavity Shake the mouse 10 15 times and also massage its abdomen for about 2 min Shaking the mouse harshly or for too many times may result in contaminating mast cells with excessive amount of red blood cells Use a forcep to hold the skin and cut a little hole with sharp scissors Do not make the incision too large because the buffer containing mast cells may flow out Collect buffer in the peritoneal cavity with a plastic or glass pipette and pour the cell into a 15 ml centrifuge tube Wash the cavity several times with a total amount of about 15 ml buffer and collect the buffer in the same tube Centrifuge the tube at 200 g for 5 min discard the supernatant and then resuspend the cell pellet in 8 ml RBL culture medium
134. granules SGs containing serotonin are mostly lost from patches Small vesicles that are retained non SGs do not release serotonin or other substances detected by amperometry although their fusion is reduced by tetanus toxin TeTx light chain Non SG_ fusion is unaffected by N ethylmaleimide phosphatidylinositol 4 5 bis phosphate PI 4 5 P2 ligands such as neomycin a PI transfer protein that can remove PI from membranes the PI 3 kinase inhibitor LY294002 and PI 4 5 P gt PI 3 P and PI 4 P antibodies In patch recordings but not whole cell recordings fusion can be strongly reduced by ATP removal and by the nonspecific PI kinase inhibitors wortmannin and adenosine In whole cell recording non SG fusion is strongly reduced by osmotically induced cell swelling and subsequent recovery after shrinkage is then inhibited by wortmannin Thus membrane stretch that occurs during patch formation may be a major cause of differences between excised patch and whole cell fusion responses Regarding Ca sensors for non SG fusion fusion remains robust in synaptotagmin Syt VII mouse embryonic fibroblasts MEFs as well as in PLC51 PLC 51 64 and PLCy1 MEFs Thus Syt VII and several PLCs are not required Furthermore the Ca dependence of non SG fusion reflects a lower Ca 59 60 affinity Kp 71 uM than expected for these C2 domain containing proteins In summary we find that non SG membrane fusion behaves and is regulated subs
135. h the command voltage at the same frequency Eq 4 3 becomes a mixture of DC and AC components After removing the AC component using low pass filtering the double of the DC component of Eq 4 3 is X V cos 0 0 4 4 123 After proper adjustment of 9 so that it equals to i e the conductance component oscillates in phase with the command voltage X in Eq 4 4 represents the actual magnitude of the signal the conductance component By adding 1 2 to 9 which is the oscillating phase angle of the capacitive component the double of the DC component of Eq 4 3 is Y V sin 0 0 4 5 Again if 0 is adjusted properly Y in Eq 4 5 represents the actual magnitude of the capacitive component For signals oscillating at angular speed other than i e background noise the DC component of Eq 4 3 is basically zero beacuse 40 thus they are sequestered after low pass filtering It is also very helpful to view how PSDs work geometrically First let s consider only the conductance component G for now In Fig 4 1A assuming that the phase angle is not properly adjusted i e 6 040 according to Eq 4 4 the readout of PSD1 is the projection of G on the x axis X and G also contaminates the readout of PSD2 Y which is 7 2 away from PSD1 Eq 4 5 To adjust the reference phase angle it is just like rotating the coordinates by an angle of 6 0 as shown in Fig 4 1B After the adjustment X reflects t
136. hat sliderl also retrieves original data for displaying To 165 show the entire trace again you need to put 0 zero into the edit box on the right of the to button and then press Enter By clicking the Show button in the Show data panel a MATLAB figure is generated The figure contains only traces displayed in axis and axis2 and the axes settings and color settings are the same with axes 1 and 2 If you would like to show only 1 channel in the figure the fastest way is to select the same displayed channel for both axes and then click Show Variable FigureData is assigned to the Workspace and the format is time s data from axisl data from axis2 Note that values in FigureData are exactly the displayed data If you use DeDrift and or running filter baseline subtracted and or filtered data are exported Exporting the data Using the Show button is the only way to export processed data to the MATLAB Workspace as introduced in the previous paragraph To export raw data within the specified time frame you may use the ExRaw button This function is disabled by default because the MATLAB function daqread is EXTREMELY slow when there are lots of NaN in the file You may enable it by editing the ExRaw_Callback in Capmeter6v3 m file and you will see tons of warnings about NaN when you click it The exported raw data is in variable rawdata Be sure to check the Raw data check box under the Save button before sav
137. he calculated peak is larger than 10 volts using the following MATLAB codes MATLAB codes in function SqAlgo try if isequal get handles Cm ForegroundColor 0 0 0 amp amp isnan tau 1 1 set handles Cm ForegroundColor black elseif isequal get handles Cm ForegroundColor 0 0 0 amp amp isnan tau 1 1 set handles Cm ForegroundColor red end if isequal get handles Cm BackgroundColor 1 0 O amp amp max peak isnan peak lt 10 set handles Cm BackgroundColor get handles figurel Color elseif isequal get handles Cm BackgroundColor 1 0 O amp amp max peak isnan peak gt 10 set handles Cm BackgroundColor red end end The function SqAlgo is evoked periodically to calculate cell parameters using peak current steady state current and time constant estimated by CapEngine4 In SqAlgo if data clipping happens the background color of the pop up menu becomes red In addition if CapEngine4 can not get the time constant NaN not a number is assigned 137 the ForegroundColor property of the list menu is set to red After subtracting the middle line value from the raw current the fitting routine processes each curve individually Starting from the steady state current asymptote estimation the codes are listed below C codes for asymptote estimation signB Csign Cmean amp dataA inde
138. he actual amplitude of G and nothing contaminates PSD2 When 6 is properly adjusted PSD1 and PSD2 give the actual G and C respectively The mixture of the two components is a new vector with an amplitude R of C and a phase angle 0 of tan C G Fig 4 1C It is important to note however by knowing R and does not mean that you can extract the actual G and C There are 124 infinite ways to get a vector with R and 0 Fig 4 1D three of them are shown and the combination of G and C is only one of them The hardware SR830 DSP dual phase lock in amplifier Stanford Research Systems Sunnyvale CA calculates and outputs the values of R and too The user s manual of SR830 is also a very good introduction of PSD which is at http www thinksrs com downloads PDFs Manuals SR830m pdf Phase sensitive detection online calculation of the phase angle To test whether the current 0 is valid or not one can change the amount of compensated capacitance from the patch clamp and see if the change AC is solely reflected on the readout of PSD2 If 9 is not correct changes of capacitance compensation result in signal changes both on PSD1 X X and PSD2 Y as shown in Fig 4 2 The correct phase angle 0 can be calculated by rotating the PSD1 PSD2 coordinate by an angle of a given that is tan X X0 Y Yo and Xp Yo and X Y represent values before and after changing capacitance compensation respectively The
139. he plate for western blot to confirm the production of recombinant protein For short term storage 2 FBS was added into medium containing recombinant viruses kept at 4 C and protected from light For long term storage the viral stock was stored at 80 C For construct containing Sytl and His tag fused to its C terminus Sytl His s fragment was also generated by PCR cloned into pCR2 1 TOPO vector and then subcloned into pFastBac vector for producing recombinant bacmid The fragment was excised from pCR2 1 TOPO by NcoI digestioin Klenow treatments and then by HindIII digestion It is cloned into HindIII Stul site of pFastBac vector The procedures for producing recombinant baculovirus were the same as described above For large scale expression of Syt1 in Sf9 cells 40 ml recombinant viruses were added into 1 6 L of Sf9 cells at a density of 2X10 cells ml Cells were cultured at 27 C with vigorous shaking and harvested after 2 days The harvested cells were broken using one 27 freeze thaw cycle followed by several times of sonication The lysate was subjected to centrifugation using a JA20 rotor Beckman Coulter at 15 krpm for 15 min and the membrane pellet was resuspended in extraction buffer 50 NaH2PO 300 NaCl 2 B mercaptoethanol 1 PMSF 2 imidazole in mM and supplied with 2 Triton X 100 Extraction was carried out at 4 C for l hr with gentle agitation After extraction membrane debris were removed by centrifuga
140. he recording configuration either of the methods might be better than the other in that specific case The phase angle of the PSD depends on the clamping time constant T and the oscillating frequency of the wave Santos Sacchi 2004 The t equals to RC where R is RaRm Ra Rm That is if Ra and or Rm change the phase angle will change For experiments using excised patches since Ra is almost non existing charging of the membrane is superfast i e time constant is very small and PSD apparently is the best method to use Actually PSD is the only way to measure Cm in excised patches because the current transient is too fast to be fitted by SQA Potentially the triangular wave could be used to calculate absolute Cm in excised patches however since the actual Cm is always contaminated by the stray capacitance from the electrode knowing the absolute C is actually not quite meaningful If PSD is to be used for whole cell recording it is important to keep in mind that the phase angle might be changing during the recording It might be due to the reseal of the opening changing of the seal resistance or opening of the ion channels on the membrane e g Ca activated chloride channels etc SQA might be a good choice if the tT is not too fast For cells like cardiomyocyte the clamping Tt can be as long as several hundreds of us In this case high resolution PSD could not be achieved because slow oscillating frequency is used in order to ch
141. he slope and the intercept at time zero of Eq 4 20 represent 1 t and n a b 131 respectively Since b is known a is then obtained from the intercept To estimate all three cell parameters namely Ra Rm and Cm from a b and q let s consider two situations 1 when the membrane is not charged at all and 2 when the membrane is fully charged In the first condition since the membrane is not charged current flowing through Ra is driven by a full voltage step of 2Vc where Vc is half of the command voltage The relationship is written as _2Ve R a b 4 21 When the membrane is fully charged the current flow through Ra first and then flow across Rm directly without charging the membrane Current flowing through two resistors in series is described as Vc b Ra Rm Cy The solution for Rm according to Eqs 4 21 3 7 is _ Vce a b aT 4 22 and membrane capacitance is always presented by Eq 3 11 which is eet oa t a b oe soe Rm 2Vc a b 4 23 The above solutions for Ra and Rm are valid only when the membrane is properly charged If the oscillation is too fast that is the step voltage changes its direction before the membrane is fully charged which is one of the assumptions the situation becomes much more complex We approach this issue by formulating the membrane potential during the fast oscillation Considering that the command square pulse oscillates with
142. hol 25 24 1 was added and mixed thoroughly with the crude plasmid DNA and then centrifuged at 12000 g for 2 minutes The aqueous layer upper portion of the mixture was transferred to a new tube and the plasmid was precipitated by adding 1 10 volume of 3M sodium acetate pH 5 2 optional and 2 volumes of anhydrous ethanol If higher yield was needed the mixture was kept at 20 C for 20 minutes else it was subjected to centrifugation directly Centrifugation was performed at 12000 g for 20 min at 4 C and the supernatant was discarded The plasmid pellet was washed in 1 ml of 70 ethanol optional and centrifuged at 12000 g again for 5 minutes The supernatant was discarded and the pellet was dried either by air or vacuum The plasmid was dissolved in desired volume of either double distilled water or TE buffer 10 mM Tris Cl 1 mM EDTA pH 8 0 RNA was removed by RNaseA digestion before DNA sequencing no further purification was performed SDS PAGE analysis The mini gel casting stand and frame Bio Rad were assembled and all glass plates were cleaned and dried with ethanol before use For a single 10 separating gel the following ingredients were mixed 1 5 ml water 1 5 ml of 50 glycerol 1 85 ml of 1 5 M Tris Cl buffer pH8 8 75 ul of 10 SDS 2 5 ml of 30 acrylamide 0 8 bisacrylamide 55 ul of 10 ammonium persulfate and 5 5 ul of TEMED N N N N tetramethyl ethylenediamine The mixture was poured into the casting module and
143. i jes x ae o Sate 490 Paai Brew Foret aie CEC ea 0 5 L L 4 80 ulin l 0 20 40 60 100 120 140 160 af ff Copyright 2007 Tzu Ming Wang and Donald W Hilgemann lV chae cha 6 ms Module gt masse 180 When the solution flow is turned on solution level in the recording chamber increases and the increased stray capacitance will be detected if higher oscillation frequency is used e g for excised patch In this case subtraction of the increasing baseline capacitance is desired To do so you can use the DeDrift button in red circle After clicking the button you use the mouse cursor to select multiple points from the axis to define sections for linear regression and then right click the mouse to subtract the baseline using section specific slopes For example baseline between points and 2 and baseline on the left of point 1 are subtracted using slope of points 1 to 2 baseline between points 2 and 3 and baseline on the right of point 3 are subtracted using slope of points 2 to 3 You may select as many points as you want but make sure that you use this function only when 163 164 you are certain about the origin of the drift If the first click is not on axis2 data in axis will be processed Also you don t have to point to a real data point on the trace because the function only gets the X coordinates from your clicks Y coordinates are not used in
144. inal X and Y on the PSD1 axis which is Xcos and Ysin q respectively Again the new PSD2 readout Ya is the sum of the projections of the original X and Y on the PSD2 axis which is Xsin and Ycos Q respectively The equations are summarized below X X COS a Ysin a 4 7 Y a X sin a cos a 4 8 Offline adjustment of phase angle is particularly useful for whole cell records As mentioned previously phase angle might shift if the clamping time constant shifts It is usually caused by changing of Ra Rm and or Cm which happen all the time during whole cell recordings 126 127 Phase sensitive detection computer implementation The implementation of PSD is relatively simple as mentioned in the Introduction In Capmeter the program times the acquired current in a given time interval with either an in phase or an orthogonal reference wave The product is then averaged to get the DC signal and the 2DC value is assigned to PSD1 or PSD2 An example is shown in Fig 4 4 and the actual codes are shown below MATLAB codes for setting references Reference wave calculation function Refcalc FH handles guidata FH PPS handles aiSR handles PSDfreq 1000 Spoints per sine wave L floor handles aiSamplesPerTrigger PPS PPS Sto make sure that the DC noise can be cancled T L 1 handles aiSR P handles PSDphase pi 180 F handles PSDfreq 1000 handles PSDref sin linspace P pi 2
145. ing Set is not necessary MATLAB sets the limits automatically if the Auto button is pressed By pressing the Lock button the scale of axis2 is locked to axis It is useful to use Lock when PSD is selected because one can check the phase angle by adjusting capacitance compensation and then compare the relative changes in channel 1 Y and channel 2 X Also notice that if all of the data are out of the range of axis settings the X axes of axis and axis2 become 0 to 1 without updating To correct the situation you can either adjust the Y axes settings for axisl or simply press the Auto button to reset axis1 Similar effect also happens to axis3 and the solution for it is the same The X axes are controlled differently online and offline by two sliders Sliderl controls axis and axis2 and slider2 controls only axis3 In the online mode the values let s say n for example of the sliders indicate that the last n seconds of data are displayed If the value is zero all of the data are shown The default maximal values for slider and slider2 are 120 and 50 seconds respectively You may change it from CapmeterSetting m In the offline mode the sliders represent the entire data set You may navigate through the entire data set by sliding the slider and the length of the data shown on the screen is defined in Show data panel which will be introduced in later section To make notes you can use the Label panel Button
146. ing the file if you want to retrieve raw data later The file daq will be very large and probably useless I recommend you not to use it unless necessary 166 An alternative way to save and export raw data is to check the RXR run time exporting raw data check box during the experiment By checking the check box the program saves raw data for only two consecutive square pulses or 2 sine waves for PSD in every processing cycle 0 5 s and the sampled raw data will be assigned to the Workspace in variable rxr after recording You can check and uncheck the RXR check box during the recording and only the desired sections of raw data are stored The drawback is that you need to locate the section of interests by yourself after the recording It is usually not too difficult but you can always automate the processes by making a M file for these routine works You know my major is neuroscience not computer science and my goal should be advancing the science but not developing a program So please understand that I only develop functions that will speed up my research and data analysis Giving pulses You may need to trigger membrane fusion using voltage pulses for certain cell types or you may want to probe the current voltage relationship using increasing voltage steps In these cases you can use the Pulse panel which is above the Lock in control panel The definition for each edit box within the panel will show
147. ion I did not have kit purified pBMG SNAP25A by my hands After transfection HEK293 cells were diluted and plated onto cell culture dishes Mediums with 600 400 and 200 ug ml G418 were applied to select cells containing expression vectors Single colonies of cells were subcloned and western blot with antibodies against Stx1A SNAP25A and Munc18 1 was employed to check expression pattern As shown in Fig 2 11 clones 16 17 and 19 contain all three components Perfusion of artificial liposomes to excised patches To make a long story short I did not observe any convincing liposome fusion and this project was suspended However there were still something to be reported or discussed It is critical to saturate the pipette with liposomes before perfusing proteoliposomes because capacitance increases and gets saturated even when blank liposomes without Ca are perfused not shown There are two explanations one is that the increase of capacitance is caused by fusion of blank liposomes to the patch the other one is that lipids stick onto the glass pipette and then diffuse into the patch membrane The answer is still unknown and amperometry is probably required to answer this question Anyway after saturating the pipette with blank liposomes Ca it appeared that the speed of capacitance increment became faster after moving the patch to Sytl Syb2 proteoliposomes in the presence of Ca Fig 2 12 A It seemed that I have got
148. ion Nat Rev Neurosci 3 641 653 223 Rizo J Chen X and Ara D 2006 Unraveling the mechanisms of synaptotagmin and snare function in neurotransmitter release Trends Cell Biol 16 339 350 Sagi Eisenberg R 2007 The mast cell where endocytosis and regulated exocytosis meet Immunol Rev 217 292 303 Sakaba T Stein A Jahn R and Neher E 2005 Distinct kinetic changes in neurotransmitter release after snare protein cleavage Science 309 491 494 Santos Sacchi J 2004 Determination of cell capacitance using the exact empirical solution of partial differential y partial differential cm and its phase angle Biophys J 87 714 727 Sarantopoulos C McCallum J B Kwok W and Hogan Q 2004 Beta escin diminishes voltage gated calcium current rundown in perforated patch clamp recordings from rat primary afferent neurons J Neurosci Methods 139 61 68 Schuette C G Hatsuzawa K Margittai M Stein A Riedel D et al 2004 Determinants of liposome fusion mediated by synaptic snare proteins Proc Natl Acad Sci U S A 101 2858 2863 Schwab R B Okamoto T Scherer P E and Lisanti M P Analysis of the association of proteins with membranes In Current protocols in cell biology Bonifacino J S Dasso M Harford J B Lippincott Schwartz J and Yamada K M 2000 5 4 1 5 4 17 Sigal C T Zhou W Buser C A McLaughlin S and Resh M D 1994 Amino terminal basic residues of sre mediate m
149. is in between In step 1 membrane potential from step 5 is subtracted from command potential and then divided by Ra in step 2 to get the actual current flow Part of transferred charges step 3 are used to charge the membrane step 4 and part of them are leaking out of the cell step 7 Accumulation of the charges on the membrane builds up membrane potential and the value is O Cm step 5 As the membrane potential increases current leaking out of the cell increases and the value is Vm Rm step 6 The leaked charges step 7 is also subtracted step 4 from the total charges step 3 To monitor the net current used to charge the membrane 146 147 the derivative of total membrane charges is calculated in step 8 The sampled model current is analyzed using the following MATLAB codes MATLAB codes for algorithm validation cle V 10 Sin mV duration 0 0025 thalf pulse duration in sec StdCap 2 10pF 1 StdRa 3 1MOhms 1 StdRm 50 1MOhms 1 time simout time 1000 end trigger simout signals values 1000 end 1 current sSimout signals values 1000 end 2 current2 current 0 03 rand length current 1 0 5 asymp peak tau SqCF5 trigger current time 3001 3 5 asymp2 peak2 tau2 SqQ3 trigger current time 3001 1 5 F l exp duration tau 1t exp duration tau G 2 asymp asymp F peak V peak asymp Rm 1 G Ra V asymp
150. itter release in neurons Tang et al 2006 1 2 Lipids and lipases implicated in fusion P1 4 5 P2 and other lipids Phosphatidylinositides PIs and their derivatives are presently thought to be critical regulators of membrane trafficking in the cells Fig 1 5 De Matteis and Godi 2004 Phosphatidylinositol 4 5 bisphosphate PI 4 5 P2 is one of the most versatile PIs that has been implicated in a variety of different physiological activities including membrane fusion Fig 1 6 McLaughlin and Murray 2005 Evidence from PC12 cells suggested that formation of PI 4 5 P microdomains at syntaxin clusters can activate the exocytotic sites Aoyagi et al 2005 In addition PI 4 5 P2 appears to be required for priming of vesicles in pancreatic B cells Olsen et al 2003 and the yeast homotypic vacuole fusion system Mayer et al 2000 In dense core vesicle fusion PI 4 5 P2 promotes the binding of CAPS calcitum dependent activator protein for secretion to the plasma membrane and increases the initial fusion rate Grishanin et al 2004 Loyet et al 1998 and it is also reported that PI 4 5 P regulates the releasable vesicle pool size in chromaffin cells Milosevic et al 2005 Moreover PI 4 5 P2 is suggested to promote fusion directly via its interaction with Sytl in a liposome liposome fusion system Bai et al 2004 and it also modulates many enzymes that are implicated in regulating fusion see next section including phos
151. ity is blocked by the PI 3 K inhibitors wortmannin and LY294002 Mitchell et al 2001 Phospholipase D PLD is a class of phospholipases that cleaves phosphatidylcholine PC and produces PA and choline Fig 1 7 PA itself might be fusogenic because it is cone shaped Dephosphorylation of PA by PA phosphohydrolase produces DAG side chains are saturated mono unsaturated which is fusogenic too PLD is regulated by PI 4 5 P gt Pertile et al 1995 Ca Qin et al 1997 and small G proteins Caumont et al 1998 and its activity is also implicated in regulating exocytosis Choi et al 2002 In chromaffin cells activation of PLD near the exocytotic sites is found to be important for catecholamine release Caumont et al 2000 and a similar finding has also been reported in PC12 cells Vitale et al 2005 Moreover blocking PLD activities by 1 butanol or by inactive mutant PLD suppressed secretion in mast cells Choi et al 2002 and the PLD isoforms PLD1 and PLD2 were found to regulate different phases of exocytosis Choi et al 2002 Phospholipase A2 PLA is a phospholipase superfamily that cleaves phospholipid and produces lysophospholipid LPL and free fatty acid Fig 1 7 It can be found in both secretory or cytoplasmic forms Brown et al 2003 and their activities may also require Ca depending on the subgroups Dennis 1994 The activity of PLA has been reported to facilitate membrane fusion In a hemi re
152. justments of the codes may be required The minimal AI and AO speed for the default setting is 300 kHz and 200 kHz respectively I use PCI 6052E from NI AI 333 kHz AO 333 kHz but I recommend using the M series board like PCI 6251 AI 1 25 MHz AO 2 8 MHz since it is cheaper and faster The program works fine with Pentium4 2 53 GHz 1GB RAM 25 GB hard drive free space and Window XP SP2 Connection diagram Capmeter6 Inputs a trigger b current c Ch4 d TTL in optional PFLO CTRO O AO 1AD0 v EXT Command front switched EXT Command rear switched Analog output AO0 is used to send out either sine or square pulses It is also connected to AIO to trigger the acquisition Output channel AO1 is used to generate stimulating pulses when the Pulse button is clicked After connecting AO channels to external command inputs of the patch clamp be sure to check the command sensitivities in the M file The default values for AOO and AO1 are 20 mV V and 100 mV V respectively You may edit the values of handles aoChI convert for AOO and handles aoCh2convert for 152 AO1 in the CapmeterSetting m file or change the default values directly in line 155 of Capmeter6v3 m Input AIl receives current from the patch clamp The acquired data are used for estimating cell parameters I recommend you to low pass filter the input at 10 20 kHz Although all the algorithms works fine even when the hardware fil
153. l D Holroyd P et al 2001 Snares are concentrated in cholesterol dependent clusters that define docking and fusion sites for exocytosis EMBO J 20 2202 2213 Latham C F Osborne S L Cryle M J and Meunier F A 2007 Arachidonic acid potentiates exocytosis and allows neuronal snare complex to interact with munc18a J Neurochem 100 1543 1554 Lee S B Varnai P Balla A Jalink K Rhee S et al 2004 The pleckstrin homology domain of phosphoinositide specific phospholipase cdelta4 is not a critical determinant of the membrane localization of the enzyme J Biol Chem 279 24362 24371 Lindau M and Neher E 1988 Patch clamp techniques for time resolved capacitance measurements in single cells Pflugers Arch 411 137 146 Lindmo K and Stenmark H 2006 Regulation of membrane traffic by phosphoinositide 3 kinases J Cell Sci 119 605 614 Loyet K M Kowalchyk J A Chaudhary A Chen J Prestwich G D et al 1998 Specific binding of phosphatidylinositol 4 5 bisphosphate to calcitum dependent activator protein for secretion caps a potential phosphoinositide effector protein for regulated exocytosis J Biol Chem 273 8337 8343 MacDonald P E Obermiiller S Vikman J Galvanovskis J Rorsman P et al 2005 Regulated exocytosis and kiss and run of synaptic like microvesicles in ins 1 and primary rat beta cells Diabetes 54 736 743 220 Mahal L K Sequeira S M Gureasko J M and S l
154. l be described in later sections This program is never being popular in the lab probably because people tend to fix on what they used to use As the time passed by two out of three lock in amplifiers in the lab showed signs of malfunctioning To save money for my mentor I decided to add PSD to my program which is quite easy as mentioned earlier The resulting Capmeter versions started to be accepted in the lab and the current version is Capmeter6v3 as of Feb 2008 In addition to PSD Capmeter6v3 uses two SQA methods to extract the information The I SQA fits the current transient directly while the Q SQA integrates the current first and then fits the resulting time charges trace 121 To synchronize analog input and output in the MATLAB one could either do it at the very beginning of the acquisition or do it repetitively If the first method is to be used the MATLAB function peekdata rather than getdata has to be used because the Logging property of the analog input remains ON during the acquisition and data can not be extracted As of its name the peekdata function does not extract the data from the MATLAB Engine and the returned data may be missed or repeated please refer to the function references of MATLAB The raw data have to be retrieved and processed again after the acquisition and there is always a risk to overload the RAM during recording too Capmeter uses the second approach The timing of analog output and input is synchro
155. labeled with two different fluorophores i e green and red membrane fusion and its intermediates were resolved both by monitoring the change of fluorescence intensity and the change of FRET efficiency during membrane fusion Yoon et al 2006 Although the TIR FM approaches are very informative and the speed of image 20 acquisition could be very fast too inevitably the temporal resolution of these systems are limited by lipid mixing process which happens after membrane fusion hemi or complete and is restricted by the speed of lipid diffusion Furthermore the dilation of the fusion pore could not be monitored either From the various methods used to monitor membrane fusion membrane capacitance measurement gives the highest signal and temporal resolution and the fusion intermediates can also be resolved In this project I was trying to reconstitute the fusion system in giant excised membrane patches and monitor fusion using capacitance measurement when liposomes containing full length Syt1 purified from insect cells and Syb2 were applied Unfortunately after about 1 year of protein purification and half a year of protein reconstitution and capacitance measurements no convincing liposome fusion was observed This project was suspended at the end of 2004 The following sections describe the methods and discuss the results in some more detail 21 2 2 Methods Small scale plasmid DNA preparation Everyone likes to
156. lf of the smaller one into the large one and then glue them together using a hot melt glue gun Do not use too much glue You may disperse just a tiny amount of glue evenly by spinning the tubing at the hot metal opening of the glue gun for a while and then pull the tubing away quickly from it Insert carbon fiber into the quartz tubing This is the most difficult part and you will need to make some tools for manipulating the fibers first The first tool is used to separate one single carbon fiber from a bundle of fibers I found the best tool is the quartz tubing You can use two old carbon electrodes made by quartz tubing too for this purpose The 97 second tool is for inserting the carbon fiber Cut a 0 5 cm piece of PE tubing and then stick one of the sharp ends of a forcep into it The protruding PE tubing is used for inserting the fiber To isolate a single carbon fiber put a small bundle of fibers 4 cm long on a white paper and then separate a single fiber using two quartz tubings under a stereomicroscope After a single fiber is isolated hold the quartz electrode prepared in step 1 in one of your hands and hold the forcep in the other hand Press the protruding PE tubing of the forcep on the carbon fiber to hold it and then move the quartz tubing toward the tip of the fiber with an angle of 30 degree You may find it difficult insert the fiber because of some electrostatic force If it s difficult to do so on th
157. light chain TeTx for 2 min blocks fusion while the mutant toxin E234Q has no effect indicating that non SG fusion is SNARE dependent 109 gt w B 200 12 25 7 ATPIGTP lo 1 0 c a 200 uM free Ca 9 150 p20 p buffer pe a T E v g g 1 5 S S100 506 3 rs oO oO e g a 8 1 0 a Ej E 2904 o S 2 5 g 5 4 g X 0 5 00 E a 0 2 L E ATPIGTP i oo ll2 MBE L L L J 30 1 1 f f D Uz Dy WU WD WU 10 Time s 10 Y Y y AN w Za Figure 3 4 ATP hydrolysis is required for supporting non SG fusion A Without ATP Ca application fails to trigger exocytosis in this patch but exocytosis was restored by placing the patch in ATP GTP containing solution for an additional minute B AMP PNP can not preserve fusion indicating that the hydrolysis of ATP is required to support non SG fusion 110 200 1 2 2 5 21 0 2 0 _ 150 5 2 80 8 5100 50 6 2 2 5 9 1 0 a 90 4 g 50 5 Bs 0 2 0 oo H20 rat oo MLN Hy PELS Dy Pals Me Gp K Op 7 Og X Og Be De Figure 3 5 Non SG fusion in excised patches is wortmannin adenosine sensitive Incubation of patches with the PI kinase inhibitors wortmannin wort 4 uM and adenosine 0 5 mM significantly decreases the average fusion magnitudes and the ratio of active patches 111 gt Y a b0 e j d e ytet a a fitted curve e original data w Capacitance pF w O 2 NODA OAON Su 2uejonpuog
158. lner T H 2002 Calcium independent stimulation of membrane fusion and snarepin formation by synaptotagmin i J Cell Biol 158 273 282 Mahmoud S F and Fewtrell C 2001 Microdomains of high calcium are not required for exocytosis in rbl 2h3 mucosal mast cells J Cell Biol 153 339 349 Mayer A Scheglmann D Dove S Glatz A Wickner W et al 2000 Phosphatidylinositol 4 5 bisphosphate regulates two steps of homotypic vacuole fusion Mol Biol Cell 11 807 817 McLaughlin S and Murray D 2005 Plasma membrane phosphoinositide organization by protein electrostatics Nature 438 605 611 McNeil P L and Kirchhausen T 2005 An emergency response team for membrane repair Nat Rev Mol Cell Biol 6 499 505 Metcalfe D D Baram D and Mekori Y A 1997 Mast cells Physiol Rev 77 1033 1079 Meunier F A Osborne S L Hammond G R V Cooke F T Parker P J et al 2005 Phosphatidylinositol 3 kinase c2alpha is essential for atp dependent priming of neurosecretory granule exocytosis Mol Biol Cell 16 4841 4851 Milosevic I S rensen J B Lang T Krauss M Nagy G et al 2005 Plasmalemmal phosphatidylinositol 4 5 bisphosphate level regulates the releasable vesicle pool size in chromaffin cells J Neurosci 25 2557 2565 Mitchell C J Kelly M M Blewitt M Wilson J R and Biden T J 2001 Phospholipase c gamma mediates the hydrolysis of phosphatidylinositol but not of phosphatidylinositol
159. lso be massive in CHO and 3T3 cells Coorssen et al 1996 as well as BHK and HEK293 cells Yaradanakul et al 2008 and as described in some detail in this article in RBL MEF and INS 1 cells The prevalence of large scale Ca activated non SG fusion processes in both secretory and non secretory cell lines suggests that the non SG fusion may be important for cell survival Since the non SG pool can exceed 50 of the total surface membrane area and the requirements for cytoplasmic Ca are rather high Yaradanakul et 61 al 2008 it seems reasonable that this membrane pool is involved in wound repair of the plasma membrane Present understanding of membrane fusion relies strongly on studies of transmitter hormone release from neurons Bronk et al 2007 Sakaba et al 2005 and endocrine cells Burgoyne and Morgan 2003 and the homotypic fusion of yeast vacuoles Ostrowicz et al 2008 In these cases the SNARE soluble N ethylmaleimide sensitive factor attachment protein receptor proteins are clearly implicated to initiate fusion and in general are thought to do so by associating and perturbing the two membranes involved As introduced in an accompanying article Yaradanakul et al 2008 phosphatidylinositides and their derivatives are presently thought to importantly modify SNARE dependent fusion processes De Matteis and Godi 2004 Evidence from PC12 cells suggested that formation of phosphatidylinositol 4 5 bisphosphate PI
160. ly function of SqWaveCalc is to generate AO data for square wave perturbation in Capmeter6v3 The reason to make a C rather than a MATLAB function is because SqWaveCalc might be evoked frequently when the SQA auto frequency option is selected Generating a wave form takes a lot of loops in the program and MATLAB does not handle loops efficiently If the wave form is not generated in time the acquisition is jammed That is why the function is in C output SqWaveCalc totalAOpt SamplesPerWave amplitude Input argument totalAOpt is the total number of AO points per trigger It is determined by AO speed handles aoSR and the recording frequency handles rSR Argument SamplesPerWave tells the function the number of AO points for a single wave It is determined by AO speed and oscillation frequency Note that totalAOpt has to be the multiple of SamplesPerWave and the trigger signal is added in MATLAB function Wavecalc but not in C function SgWaveCalc 196 4 7 Capmodule4 What is it Capmodule4 is a tool for you to make the seal patch clamping It generates step command tracks the current displays the current and makes sound noise according to the current Axopatch 200 Molecular Devices Capmodule4 Axopatch 1D Things you need to know before using 1 Please cite the paper Wang and Hilgemann 2008 2 Please adjust the net gain a from the GUI 3 To save the snapshot data please use the Save button in
161. n you just click Scale next to the Std button and the previously calculated factor will be used to scale the data Note that the Scale function only works for Chl now and the first point is defined as zero Also if you click the Scale button twice the data will be scaled twice too and of course it is wrong When defining the standard the function only gets the Y coordinates from your clicks so the X coordinates are not important at all It is particularly useful to do so when the trace is noisy because you may define precise Y coordinates by putting the mouse in the middle of the noisy trace like doing low pass filtering by eye Showing the data You can use the to button in the Show data panel or the edit boxes next to it to specify a region to be displayed These two methods are slightly different When the to button is used it only crops the data which have been displayed on the screen when you put values into the edit boxes and press Enter the program retrieves original data within the specified time frame and then update the displayed data It will make a difference after you use the DeDrift function For example you may want to subtract the baseline first and then enlarge a portion of the baseline subtracted data In this case you need to use the to button but not the edit boxes After selecting the desired region you may drag slider to move along the trace as introduced previously Notice t
162. n are shown Red synaptobrevin yellow syntaxin 1 blue SNAP25 N terminus green SNAP25 C terminus orange Habc domain of syntaxin 1 Fig 2 of Rizo and S dhof 2002 Figure 1 3 The SNARE cycle in synaptic vesicle exocytosis Upon membrane fusion the trans SNARE complex three of its components are in two opposing bilayers becomes cis SNARE complex all three components are in the same bilayer and the complex is then unwound in an ATP dependent process mediated by NSF N ethylmaleimide sensitive factor and SNAPs soluble NSF attachment proteins Fig 1 of Rizo and Siidhof 2002 a Neuronal exocytosis Syntaxin 1 z Vesicle Munc18 1 SM protein Priming _ Synaptic ee Endocytosis Recycling Plasma Membrane Figure 1 4 Different models of SM protein syntaxin interaction Unlike neuronal exocytosis upper syntaxin homologues in other vesicular transport systems such as ER and Golgi do not form the closed conformation middle and the SM proteins bind to the N terminus of syntaxin Fig 5 of Rizo and Stidhof 2002 Recently it was reported that Munc18 does bind to the assembled SNARE complex lower and the two different interacting modes open and closed syntaxin are essential for synaptic vesicle fusion and are coupled by functionally critical binding to Stx N terminus Fig 4 of Dulubova et al 2007 Figure 1 5 PI distribution of endo and exocytic pathways CP coated pit EE e
163. n et al 2006 In general it is now believed that LPL inhibits membrane fusion by preventing the formation of fusion intermediates such as stalks Chernomordik et al 1993 If LPL plays any regulatory role in membrane fusion in vivo is still not clear SV PM regulated CV PM i Qa syntaxin 1 LPM a aot cen j Qb SNAP 25 N t rm a ee Qc SNAP 23 C term ET SES 9g Qb Sec9p Spo20p N term R cellubrevin synaptobrevin 4 Qc Sec9p Spo20p C term Munc18b c R Snct 2p rab Secip Gece S Qa syntaxin13 16 TGN PV O Qb vtita Qa Pept2p 95 Qc syntaxin6 m GONE ea Qb membrin LE LE ORENK Qa syntaxin7 ie hers Qb vtitb slyt Qc syntaxing R VAMP8 renee vps33a b rab7 ER Zi Mammalian Cell Yeast Figure 1 1 SNAREs and other proteins involved in exocytic and endocytic pathways SNAREs forming the four helix bundle Qa Qb Qc R SM Secl Munc18 like proteins and rab proteins are shown EE early endosome CV constitutive vesicle SV secretory vesicle LE late endosome Lys lysosome TGN trans Golgi network CGN cis Golgi network ER endoplasmic reticulum PV prevacuolar compartment corresponding to late endosome Vac vacuole corresponding to lysosome Fig 6 of Jahn et al 2003 Core complex Figure 1 2 The neuronal SNARE complex The crystal structure of the SNARE complex and the NMR structure of syntaxin 1 Habc domai
164. n et al 2006 there are more than 80 90 of Syb2 incorporated correctly in Syb2 only liposomes when 0 67 OG were used In conclusion to reconstitute Sytl and Syb2 into liposomes at the same time the detergent mediated direct incorporation method with 5 mM total lipids and 0 77 OG gives proteoliposomes with satisfactory quality in the aspects of size distribution leakage of content protein incorporation and orientation Establishment of the triple marker transient expression system The original plan was to excise giant patches from cells transiently expressing Stx1A SNAP25A and Muncl8 1 and then perfuse Sytl Syb2 proteoliposomes to the cytoplasmic side of the membrane One immediate technical challenge was to identify cells co expressing all three proteins under regular epifluorescence microscope It is common to locate cells co expressing 2 different proteins using green and red fluorescence as markers and it is also possible to use a 3 color as a marker for the 3 41 protein of interests However identifying cells with correct color mixture among other fluorescent cells is also quite challenging and the microscope also needs to be equipped with different excitation light sources and filter sets which is very unusual for a microscope used for patch clamping To overcome this limitation I designed a marker system that also uses the localizations i e nucleus membrane and cytoplasm of fluorescent proteins as markers Th
165. n of cells by flipped snares Science 300 1745 1749 Huang C L Feng S and Hilgemann D W 1998 Direct activation of inward rectifier potassium channels by pip2 and its stabilization by gbetagamma Nature 391 803 806 Jahn R Lang T and Siidhof T C 2003 Membrane fusion Cell 112 519 533 Jaiswal J K Andrews N W and Simon S M 2002 Membrane proximal lysosomes are the major vesicles responsible for calcium dependent exocytosis in nonsecretory cells J Cell Biol 159 625 635 Jun Y Fratti R A and Wickner W 2004 Diacylglycerol and its formation by phospholipase c regulate rab and snare dependent yeast vacuole fusion J Biol Chem 279 53186 53195 Kanemaru K Okubo Y Hirose K and Iino M 2007 Regulation of neurite growth by spontaneous ca2 oscillations in astrocytes J Neurosci 27 8957 8966 219 Karli U O Schafer T and Burger M M 1990 Fusion of neurotransmitter vesicles with target membrane is calcium independent in a cell free system Proc Natl Acad Sci U S A 87 5912 5915 Khvotchev M Dulubova I Sun J Dai H Rizo J et al 2007 Dual modes of munc18 1 snare interactions are coupled by functionally critical binding to syntaxin 1 n terminus J Neurosci 27 12147 12155 Koushika S P Richmond J E Hadwiger G Weimer R M Jorgensen E M et al 2001 A post docking role for active zone protein rim Nat Neurosci 4 997 1005 Lang T Bruns D Wenzel D Riede
166. n signal N methyl D glucamine N ethylmaleimide sensitive factor nitrilotriacetic acid XX OD OG PA PAGE PBS PC PCR PH PI PI 3 K PI 3 P PI 4 5 P gt PIP PKC PLA PLC PLD PMA PMSF POPC PSD PTK PTS1 Q Qs Ra optical density octyl B D glucopyranoside phosphatidic acid polyacrylamide gel electrophoresis phosphate buffered saline phosphatidylcholine polymerase chain reaction pleckstrin homology phosphatidylinositol PI 3 kinase phosphatidylinositol 3 phosphate phosphatidylinositol 4 5 bisphosphate phosphatidylinositol 3 4 5 triphosphate protein kinase C phospholipase A phospholipase C phospholipase D phorbol 12 myristate 13 acetate phenylmethylsulphonyl fluoride 1 palmitoyl 2 oleoyl sn glycero 3 phosphocholine phase sensitive detection or detector protein tyrosine kinase peroxisomal targeting sequence 1 charges charges steady state current subtracted access resistance random access memory rat basophil leukemia reference Rab3 interacting molecule xxi Rm SDS SG SQA SM SNAPs SNAP25 SNARE Stx Syb Syt omega t tau T TBST TEMED TeTx theta O TIR FM Tris t SNARE membrane resistance sodium dodecylsulfate secretory granule square wave algorithm Sec1 Munc18 like soluble NSF attachment proteins Synaptosome associated protein of 25 kDa SNAP receptor Syntaxin Synaptobrevin Synaptotagmin angular speed time time constant Tris buffered saline with T
167. ne is in a function called SgCF in CapEngine4 mexw32 The first step for the fitting routine is to find the positions of all the peaks Since both the data acquisition and the command oscillation is at a fixed speed it seems that the difference among peak indexes is a fixed constant e g acquire at 100kHz and oscillate at 1kHz one half pulse is represented by 50 points theoretically however it is not true Even when the acquisition speed is the multiple of the oscillating frequency the distance between two half pulses is always one or two points away from each other e g represented by 49 points or 51 points So the fitting routine has to locate the peak indexes from the trigger signals that are acquired from AIO 134 C codes for peak detection for i 0 1i lt L it t dataA i data n L 1 i dataTrig i trigger n L 1 i dataTime i time n L 1 i Cdiff dataTrig L amp diffabs For i O i lt L 1 it diffabs i Cabs diffabs i Cfind diffabs 1 thresholdl L 1 amp indexpeak amp fc if fc 0 for i O i lt fc it indexpeak i indexpeak i 1 adjust the index Sp fc 1 remove the last curve bcz it s usually incomplete if Sp lt 1 quality 0 poor quality if quality other fitting procedures Variable dataTrig is an array containing the command output recorded by AIO The absolute difference between each points is calculated by
168. ne4 mexw32 which is written in C C codes for PSD multiplication and averaging void PSD double data double ref int Mref int L int ppch double output ine izj double A L 1 1 is the NaN A double mxMalloc Mref sizeof double for i 0 i lt ppch it data point index for j 0 j3 lt Mref j data ref D _ u ll data i L j ref j output i 2 Cmean A Mref It is clear from the codes that the function allocate the memory for array A first and then assign A the product of the current data and reference wave ref in a element by element manner The DC component is calculated by averaging the entire array A and the 2DC value is the output of the function Square wave perturbation based on fitting the current transient Equations in this section are derived by my mentor Dr Hilgemann and I so I use we rather than I in this section The whole cell patch clamp can be considered as an 128 129 R C circuit Current flowing through the electrode is determined by access resistance Ra membrane capacitance Cm and membrane resistance Rm Equations for extracting cell parameters from peak current steady state current and time constant have been introduced in Chapter 3 In this section I will discuss and derive the equations again in more detail starting from the estimation of the steady state current Current flowing through an R C circuit can be describe
169. neomycin 500 uM to bind PI 4 5 P2 Eberhard et al 1990 and probably other anionic phospholipids in a magnesium free solution did not block non SG fusion Furthermore application of anti PI 4 5 P2 antibodies in FVPP solution did not block fusion Table 3 1 although the concentrations of antibody employed potently blocked PI 4 5 P senstive currents in excised patches e g outward Na Ca exchange current not shown These results suggest that neither the synthesis nor the hydrolysis of PI 4 5 P2 can be a requirement for non SG fusion As shown in Table 3 1 no protecting effect of these agents was observed when they were applied in a magnesium containing solution Clearly magnesium dependent hydrolysis of PI 4 5 P2 cannot be the mechanism of run down of the fusion process Non SG fusion is probably phosphatydialinositide independent Since the ATP mechanism does not appear to involve PI 4 5 P2 we used other inhibitors to probe potential phosphorylation targets As shown in Table 3 1 treating the patches with staurosporine a broad spectrum protein kinase inhibitor was not able to block fusion Interestingly treating the patches with high concentrations of wortmannin 4 uM and adenosine 0 5 mM which inhibit multiple classes of PI 3 Ks and PI 4 Ks Balla et al 2008 significantly decreased non SG fusion Fig 3 5 implying that PI 3 P and or PI 4 P might be responsible for the ATP dependency Therefore we tested for roles of indivi
170. ng X 1997 Molecular heterogeneity of phospholipase d pld cloning of pldgamma and regulation of plant pldgamma beta and alpha by polyphosphoinositides and calcium J Biol Chem 272 28267 28273 Rhee J Li L Y Shin O Rah J Rizo J et al 2005 Augmenting neurotransmitter release by enhancing the apparent ca2 affinity of synaptotagmin 1 Proc Natl Acad Sci U S A 102 18664 18669 Rhee J S Betz A Pyott S Reim K Varoqueaux F et al 2002 Beta phorbol ester and diacylglycerol induced augmentation of transmitter release is mediated by munc13s and not by pkcs Cell 108 121 133 Rhee S G 2001 Regulation of phosphoinositide specific phospholipase c Annu Rev Biochem 70 281 312 Richmond J E Weimer R M and Jorgensen E M 2001 An open form of syntaxin bypasses the requirement for unc 13 in vesicle priming Nature 412 338 341 Rickman C and Davletov B 2005 Arachidonic acid allows snare complex formation in the presence of munc18 Chem Biol 12 545 553 Rigaud J and L vy D 2003 Reconstitution of membrane proteins into liposomes Methods Enzymol 372 65 86 Rigaud J L Pitard B and Levy D 1995 Reconstitution of membrane proteins into liposomes application to energy transducing membrane proteins Biochim Biophys Acta 1231 223 246 Rizo J 2003 Snare function revisited Nat Struct Biol 10 417 419 Rizo J and Siidhof T C 2002 Snares and munc18 in synaptic vesicle fus
171. ng proteoliposomes was improved tremendously Fig 2 7 lower panel although it was still not as homogeneous as Syb2 only proteoliposomes Chen et al 2006 Sytl containing proteoliposomes reconstituted using 0 77 OG did not show significant sign of leakage within a two hour period Fig 2 8 The orientations of incorporated proteins were tested by enzymatic digestion followed by western blot with antibodies against cytoplasmic or lumen portion of the proteins With antibody against Sytl lumen portion V761 the smear on western film indicated correct orientated Sytl Fig 2 9 A As shown in the boxed region there were probably more than 70 of Sytl were inserted in the correct orientation To further confirm the 40 result antibody against Sytl cytoplasmic portion V216 was employed Signals remained on the film indicated inversely incorporated Syt1 Fig 2 9 B As indicated in the boxed region there were probably less than 30 of Sytl compared with total input that were in inverted orientation when reconstituted in this fashion The orientations of Syb2 in Sytl Syb2 prepared in 0 77 OG and Syb2 only prepared in 0 67 OG proteoliposomes were also tested Fig 2 9 C The ratios of correctly incorporated Syb2 were similar in both cases According to this blot Fig 2 9C probably 60 of Syb2 were in correct orientation Note that the bands in this blot were saturated so the estimation might not be accurate According to Che
172. nized by embedding a mV trigger signal if the command sensitivity is 20 mV V on top of the generated sine or square wave every 10 ms if digitized at 100 Hz The data are extracted from the MATLAB Engine between triggers and then processed digitized by Capmeter Since data are extracted piece by piece problems mentioned above are avoided In the following sections I will show you how to use the programs and explain how the programs work 122 4 2 Algorithms for capacitance measurement Phase sensitive detection The lock in amplifier is invented by physicist Robert H Dicke at Princeton University source wikipedia org PSD gives superior signal to noise performance because it only detects signals oscillating at a specific frequency Since from the Fourier theorem s point of view the background noise can be considered as a combination of periodic waves oscillating at the entire frequency spectrum noise oscillating at frequencies other than the designated one is eliminated and thus superior noise performance is achieved Mathematically considering a signal is oscillating at an angular speed of a phase angle of 0 and with amplitude of V ic the signal can be expressed as V gsin w t 0 4 1 The product of the signal and a reference wave at an angular speed of a phase angle of O sin w t 0 4 2 is 5 V al C05 w w t 0 0 cos w w t 0 6 4 3 If equals to i e signal oscillates wit
173. nositide sensitive Thus the ATP sensitivity comes about by a novel mechanism that might prove to be relevant to other types of membrane fusion Third the non SG vesicles are presumably pre docked at the plasmalemma to remain attached to excised patches and in this regard more detailed ultrastructural analysis will be of paramount importance for further progress Fourth the Ca dependence of this mechanism is unlikely to represent the function of the putative Ca sensor Syt VII And finally the non SG fusion mechanism strongly inhibited by cell swelling and presumably membrane stretch Together these results have established multiple potentially novel directions for future 90 studies of non SG fusion which is likely to be an important partial reaction of the ubiquitous membrane wound repair response Acknowledgements We thank Vincenzo Lariccia for assistance and discussions Marc Llaguno and Alp Yarandanakul for advice and criticism We thank Dr Thomas Sidhof UTSouthwestern Dr Kiyoko Fukami Tokyo University and Dr Graham Carpenter Vanderbilt University for generously providing MEF cell lines Chengchen Shen Mei Jung Lin for assistance and Dr Vladislav Markin UTSouthwestern for discussions of mathematical methods 91 3 6 Some more methodological details Isolation of rodent peritoneal mast cells The protocol for isolating mouse peritoneal mast cells is modified from the standard protocol Mundroff and Wi
174. nput argument fwindow is the window size used for filtering and wswitch defines the way Dfilter2 handles the data time relationship wswitch 1 F wswitch 0 p WSWIICR 1 a m a Je As shown above in order to keep the dimension of the output data the same with the input gray Dfilter2 adds fwindow 1 points white to the original data set If wswitch is 1 Dfilter2 repeats of the last data point fwindow 1 times at end of the input data and to is on the left hand side of the filter window solid section For wswitch of 1 the first data points are repeated fwindow 1 times at the beginning of the data set and the to point is on the right hand side of the window The default value for wswitch is 0 that means the first and the last data points are repeated fwindow 1 2 times at the beginning and the end of the data respectively and the to point is in the middle of the window If fwindow is even let s say 8 for example the first point is repeated 4 times and the last data point is repeated 3 times To get the median Dfi ter2 uses build in C function qsort to sort the data and then get the median If the window size is even e g 20 Dfilter2 takes the average of the middle 2 sorted points i e 10 and 11 as the median The gsort function uses Quicksort sorting algorithm and you may find the introduction about it at http en wikipedia org wiki Qsort Dfilter2 does not do summation sorting for every loop instead i
175. nses in this regard Wortmannin adenosine insensitivity of non SG fusion in whole cell recording The pronounced inhibition of non SG fusion in excised patches by the wortmannin adenosine combination was unexpected as we had tested these agents in whole cell recording of Ca induced fusion in BHK fibroblasts and found no effect Therefore we reexamined this issue in whole cell recordings from both RBL cells and 80 from BHK cells as shown in Fig 3 7 Individual records from RBL cells are shown in Fig 3 7A with the composite statistics for the data set Pipette perfusion of solution containing 200 uM free Ca as in Fig 3 6 was initiated 2 min after opening cells In control cells the average increase of cell capacitance was 119 12 and it occurred with a time constant of 2643 1 s n 5 In cells that were perfused with 5 uM wortmannin and 0 5 mM adenosine for 2 min the average increase was 114 5 2 and the time constant was 303 1 s Fig 3 7B shows the equivalent results for whole cell BHK recording using NCX1 to initiate membrane fusion In these experiments we tested adenosine 0 5 mM alone as well as the same adenosine wortmannin combination used in RBL cells After 2 min cytoplasmic infusion of the respective solutions the peak exchange currents were modestly reduced 20 at just the level of significance but the percent increment of cell capacitance upon activating exchange currents 65 in these experiments was
176. ntegrate the gray area as shown in Fig 4 6A The integrated charge value Qs is expressed as 139 140 t Qs f I b dt a b t 1 e 4 25 0 Without subtracting b from the current before integration the trace Fig 4 6B will keep going up without reaching a steady state To get the time constant using Qs I use Eq 3 2 to estimate the asymptote a b t for Qs first and then invert the trace by subtracting Qs from the estimated asymptote Fig 4 6C black trace The resulting trace is Y a b re 4 26 Again linear regression of against the natural logarithm of Y gives the time constant and a b t Since b and Tt are known a can be obtained accordingly To show you that Eq 3 2 is still valid for a function like Eq 4 25 consider that three equally spaced points A B and C can be expressed as A a b t m 4 27 B a b t mn 4 28 C a b t mn 4 29 where n e and the solution for a b t is still Eq 3 2 Also note that Qs is only part of the charges that are used to charge the membrane the total membrane charges are Q Qs 2be dt a b t 1 e 4 30 I understand that O V is Cm however in order to use the same MATLAB codes for Q SQA and I SQA I decided to output the peak current a rather than the charges Q in Q SQA It seems that we have got every equation for Q SQA however it is not true The clamping time constant in whole cell recording rang
177. of pulses rounds note affiliated note 185 trigger AO trigger time in year month date hr min s dataindex the last voltage step in the protocol is the dataindex step in variable data axis2index pulse is given when there are axis2index data points in variable datasample rawfileinfo indexes for locating pulse records in raw files in file number trigger number data array containing processed data in J O no summation yet T datasample sampled current It does not represent accurate time signal relationship version structure containing version information Shell version of the GUI Engine version of the Qlizer The information flow log start time reset variables process_update Ojlizer Started Start_Stop_Caiiback gt start Al waiting for AO Al SamplesAcquiredFen NCI Ones adjustAl properties if ii the protocol is changed start AO P Pulse Tag update_axes2 end wm i Puise_Caliback write pulse log getAo trigger time Set baseline Set_base_Caliback calculate wave protocol Auto pulse start timer object 1 t Autotrigger This section and the following one introduce how IQplot2 works The above diagram shows you the information flow when the Stopped button is clicked Function names are in italic and names of buttons and check box are in black square text boxes When you start the recording function Start_Stop Callback is evoked It resets some variables and adjusts pro
178. ol panel _ A A il 0 8 0 8 0 4 0 2 B a a ATT O LP10kHz ab 1 025 fo z 025 2 release EJ manually I y Lock in control panel oof er slider2 7 y 4 L 1 L 1 1 1 1 1 1 us S S SS E R Module Fi ua sat 0 Copyright 2007 Tzu Ming Wang and Donald W Hilgemann Before starting the acquisition you might want to adjust the sampling frequency points per second to be digitized from the raw data The default setting is 100 Hz you can modify the setting in CapmeterSetting m to change the start up value After selecting the desired algorithms PSD I SQA Q SQA from the pop up menu you may adjust parameters frequency amplitude for command potential generation in the Lock in control panel and then start the recording by clicking the red Stopped button In case if you forget the definitions for the edit boxes you can always move the mouse cursor onto the box for few seconds and the description will pop out The scale of the Y axes is controlled by Y axes control panel To change the setting 155 for axis you first select Top Middle for axis2 Bottom for axis3 from the pop up menu and then adjust the values The first row is for the upper limit and second row is for the lower limit You may also specify the settings by putting values into the edit boxes and then press Enter press
179. om Dr Joseph P Albanesi s lab in UTSouthwestern As mentioned in the Discussion previously it is easy to get giant excised patches from bovine chromaffin cells However the patches were not stable under significant solution flow making it impractical to use them for other excised patch experiments Use rat chromaffin cells Rat chromaffin cells were isolated according to paper published by Dr Frederick W Tse in University of Alberta Canada Xu et al 2005 and his supervision Unlike bovine chromaffin cells chromaffin cells from rat are quite small I did not have any success with them Use mouse peritoneal mast cells Mouse peritoneal mast cells can be prepared easily and they have tons of SGs However they are quite small and difficult to patch The success rate for me to excise a giant patch from them is zero So I stopped this approach Use rat peritoneal mast cells The rat peritoneal mast cells are substantially bigger than the mouse ones But unlike mast cells from mouse their shape is flat It is possible to make small excised patches from them but for giant patches it is not quite easy I did not continue this approach both because of the low success rate and because knockout rat does not exist 7 Subclone RBL cells 105 After extensive amperometric recordings in cell attached configuration I found out that the RBL cell line I was using is quite heterogeneous and does not contain many SGs To overc
180. ome this problem I subcloned several RBL lines and tested their abundance of SGs There was not much luck either I could not get a RBL line that has lots of SGs and the abundance of SGs varies from batch to batch too After all I still could not establish the method to study SG fusion in giant excised patches If someone is going to try this difficult project again although not recommended I will suggest him to semi reconstitute the system using patch from RBL cells and SGs from rat peritoneal mast cells If it works then try SGs from mouse peritoneal mast cells and then liposomes filled with dopamine None of the above is easy What I can say is good luck 106 a O 200 400 600 800 1000 time us Figure 3 1 Method to determine whole cell capacitance via square wave perturbation Model current for whole cell recording is shown in A Peak current a and the projected steady state current b were determined as described in the text B Half of the current from a real recording dots with the fitted exponential function used to determine cell parameters is shown The asymptote of current was determined using the averages of three equally spaced data sections dashed sections according to Eq 3 2 given in Chapter 3 and 4 The asymptote was subtracted and the data range from the peak to a point located at 3t estimated as peak current times e was used to determine the exponential constants via linear regression of
181. on changes are in the range of a few tens of femtofarad see Fig 3 3A Thus we typically calculated a ratio of active patches for a group of experiments as well as the average capacitance changes and rate constants were collected and compared from the patches that met the active criterion For whole cell experiments phase sensitive detection was also used off line to improve the signal to noise performance of the exponential fitting routine with square wave perturbation as follow The phase angle was determined at which C the absolute capacitance determined by the exponential analysis and Y had the highest cross 73 correlation Offline phase angle adjustment was done using equations X a X cos 0 Y sin 0 3 13 Y X sin 0 Y cos 0 3 14 a The whole cell capacitance traces were fitted with a delayed mono exponential function C a b 1 e 1 e ct 3 15 where a and b represent initial cell capacitance and vesicle pool size respectively The constants k and n reproduce reasonably the observed delays and the constant kz is the rate constant used for statistical comparison The Ca activated conductance increase was used as a reference for determining the t point For all bar graphs of amplitude and ratio of active patches panels numbers in the bars give the total number of patches for other panels numbers represent the numbers of valid data after removing outliers with Grubbs test Statis
182. onstant Note that even when SQA is selected CapEngine 4 still runs PSD for the input signals The returned PSD results are assigned to variable handles PSDofSQA in Capmeter6v3 Dfilter mexw32 Dfilter is the background averaging filter used in Capmeterlv3 and its function has been integrated into CapEngine4 which is used for Capmeter6v3 It can process multiple channels and the syntax is output Dfilter fcks matrix aiSamplesPerTrigger filterpt Argument fcks is a 1 by n array that tells Dfilter if the channels are going to be filtered or not Argument matrix is a m by n matrix where m is the total number of points for each channel and n is the number of channels For example if fcks is 1 0 0 1 and the matrix is Ch1 Ch2 Ch3 Ch4 all the channels are digitized according to aiSamplesPerTrigger but only Chl and Ch4 are filtered using fi terpt points The output 191 is a p by n matrix where p is equal to m 1 aiSamplesPerTrigger 1 1 is the NaN Dfilter2 mexw32 Dfilter2 is a running mean median filter used in Capmeters The syntax is output Dfilter2 fswitch data fwindow optional wswitch Input argument fswitch is a filter switch that tells the function what to do Values of 0 1 and 2 indicate bypassing the filter running average filter and running median filter respectively Argument data must be a m by 1 array processes 1 channel at a time and the output argument is also a m by 1 array I
183. onversion is done by using MATLAB code _handles datasample 1 etime clock handles starttime handles datasample end 1 after you stopped the acquisition As you may have seen it just scales the array using the elapsed time time between clicking the Start Stop button and total number of samples and it is not accurate either Axis2 is designed for monitoring the patch condition but not for providing accurate time current relationship If accurate time current relationship is desired please use Capmeterlv3 or Capmeter6v3 Giving pulses and applying notes mle ts ot 40 20 40 Op on nnn nnn nnn nnn nb nn nnn nnn nnn nnn E 20 20 ooo 40 20 604 4 40 Mira fo s of 80F 60 o 10 20 aD ao 60 70 Go 0 100 110 TAD 190 40 O 10 29 G0 40 80 60 70 80 GO 100 110 120 190 140 The pulse protocol used in IQplot is different from that used in Capmeter in several ways First pulse protocol in Capmeter is a one way scanning protocol but in IQplot it is a loop like protocol as shown above i e the same voltage is applied twice in the entire protocol The advantage of this kind of protocol is that one can examine if hysteresis exists which usually happens when the charging time is not long enough according to my mentor Second the pulses in IQplot are continuous The voltage will not go back to 178 zero between pulses and there is no interval between pulses either Third unlike Capmeter
184. orrelation coefficient at the angle are displayed in the MATLAB Workspace else sdisp entered handles shiftvalue PS PS PS pi 180 Cap X sin PS Y cos PS Cond X cos PS Y sin PS if handles menuindex 1 1 R_c PhaseMatcher2 handles aidata indexl index2 1 Cap R_g PhaseMatcher2 handles aidata indexl index2 2 Cond disp CorrMethod N A P num2str handles shiftvalue R_c num2str R_c R_g num2str R_g end r r end guidata hObject handles If the user modified the value in the Shift edit box the function adjust the angle as specified Function PhaseMatcher2 is called to calculate the correlation coefficients for C and G at the specified angle if square pulses were used Notice that for cross correlation between X and conductance I did not take Ra into account I understand that the overall conductance is determined by both Ra and Rm however if Ra fluctuates a lot during the experiment the phase angle must also varies a lot In this case it does not make sense to find a fixed phase angle to fit the overall conductance trace so I use only Rm for correlation Again please keep in mind that the PSD analysis may not be valid if the clamping time constant changes a lot during the experiment 4 3 Capmeter 6 What is it Capmeter 6 is a tool for measuring membrane capacitance It can use either sine waves or square waves to estimate cell parameters Phas
185. ot predocked at the membrane in RBL cells Smith et al 2003 in a stable fashion and or because the docking is disrupted by membrane suction We also attempted to develop the INS 1 cell line and bovine chromaffin cells for excised giant patch studies Our experiences with the INS 1 cells were similar to those reported for RBL cells Batch to batch variability was even greater 15 and we were not able to identify a reliable line Bovine chromaffin cells readily allowed seal formation with large diameter pipettes but excised giant patches were not stable with significant solution flow thereby greatly limiting their use In short the giant patch approaches did not facilitate in our hands excised patch analysis of SG fusion processes Occasionally high resolution recordings were indeed obtained with clear capacitance steps and amperometric spikes as shown in Figs 3 2C D for RBL patches The fact that only non SGs were preserved on the great majority of excised patches implies that the non SGs are in close vicinity to the plasma membrane and might be associated with the membrane physically Non SG fusion is SNARE dependent but NEM insensitive in excised patches The physical characteristics of non SGs are not well established and we therefore used the giant patch approach to analyze this process in some detail in particular to manipulate the cytoplasmic membrane side Capacitance traces were fitted as described in Methods Y in Fig 3 3B
186. p the cell with a second pipette theoretically can avoid mechanical force applied on the cell during the excision procedure and the cells were indeed opened in this way and exposed the cytoplasmic side to the solution flow Surprisingly SGs were still not preserved in this way Under the microscope detachment of optical dense materials from the plasma membrane were observed clearly when the solution was injected into the cell Presumably the cytoskeleton is included in the optical dense materials and the detachment of it may result in pulling off the SGs which are likely to be associated with it Permeabilize the cell using 40 uM B escin The B escin is a detergent that makes holes on the membrane and allows molecules with a molecular weight of up to 10 kDa to pass through the pores Fan and Palade 1998 It has been used to make perforated patches in different cell types like neuron Sarantopoulos et al 2004 and myocyte Dougherty et al 2008 As a continuous effort to reduce the mechanical force applied on the cell during the excision procedure I applied 40 uM B escin from the extracellular side for 30 s to create pores for Ca entry Using mouse peritoneal mast cells Ca did enter the B escin treated mast cells and triggered massive SG fusion 102 However it is often observed that SGs started to fuse before Ca was applied indicating that the ER and or other internal Ca stores were also permeabilized In ad
187. perties of GUI components Variable handles starttime is logged here and AI and timer object 2 are then started Timer object 2 evokes its TimerFcn every 50 ms to refresh axis2 Once the AI object is started it is waiting for the trigger signal i e the first pulse from AO There are three ways to trigger the AO object when the program is running The first one is to press the Pulse button the second one is to click the Set baseline button and the last one is to check the Auto pulse check box When the Auto pulse check box is checked timer object 1 is started and its ZimerFcn calls function Pulse_Callback periodically to trigger the AO object The Pulse Callback does several things When it s evoked it first compares the current pulse protocol with the previous 186 one If the two protocols are different it generates new AO data for the current pulse protocol and adjusts AI properties SamplesAcquiredFcnCount SamplesPerTrigger LogFileName TriggerCondition and TriggerConditionValue If the protocols are the same the original AO data and AI settings are used Besides calculating the pulse protocol and adjusting AI properties the function also prepares information for handles Pulselog Once everything is ready the function queues AO data into memory and then trigger the AO object The TriggerFcn of the AO object PulseTag records the initial trigger time of each pulse although this information is not used anywhere else in I
188. pholipase D PLD phospholipase A PLA2 etc Rhee 2001 Finally metabolites of phospholipase Cs PLCs cleavage of PI 4 5 P2 i e diacylglycerol DAG and inositol triphosphate IP3 are also implicated in regulation of some vesicle fusion processes Jun et al 2004 Other PIs such as phosphatidylinositol 3 4 5 triphosphate PIP3 and 5 phosphatidylinositol 3 phosphate PI 3 P are also implicated in regulating membrane trafficking including events leading up to fusion Lindmo and Stenmark 2006 For example inhibition of a class IA PI 3 kinase PI 3 K which mainly produces PIP3 reduces receptor mediated degranulation in mast cells Ali et al 2004 In addition a synapsin I associated PI 3 K is implicated in mediating delivery of synaptic vesicles to the readily releasable pool in neurons Cousin et al 2003 Furthermore PI 3 K C2a which produces mainly PI 3 P is also required for the ATP dependent priming of secretory granules SGs in neurosecretory cells Meunier et al 2005 and when PI 3 P was sequestered by PI 3 P binding motifs tandem repeats of FY VE motif secretion was also abolished Meunier et al 2005 Some other lipids and lipid derivatives are also implicated in regulating the fusion process Cholesterol is reported to be critical for the clustering of SNAREs in the fusion hot spots Lang et al 2001 DAG a metabolite of PI 4 5 P2 can activate Munc13 mammalian homologue of UNC 13 and potentia
189. plemented In summary using the software I developed non SG fusion were characterized and found to be regulated substantially differently from SG fusion An ATP dependent vii process is probably required for restoring non SG fusion capability after it is perturbed by membrane stretch and dilation viii Table of Contents Committee Signatures sh soso ees usados aT cada uhd xa ets Caan Niece Gata cada A cutee aaapeans Ooi tee A i Dedi canon as ot brn ad bia ane aa oly nek anes atin A a laccadh akan ieee he at ctan a eeate tes ii MUAY ge ionu i E R E E E E dd E a E dich hal dee diols ill COPYT Sitera a E E woot gi duelahcs probe oie eastencase E eso ts iv Acknowledgements 5s cavccaivatigate acneeacassiveeeacticass E EE E EATE OE AE v Abstracta nen R a a E a E A oe e T vi Table OF COntEntS issin aaaeeeaa aaa a EE E REAA ARA T ix Prior PUA CANIS nsss nea E A dentues eau pier EEE a Ea EEOAE EENG ATEA xiv List Of gures m n tr his E E TE AA A E RA E E ER xV IES OT A ASS A E E E A A T N ETS xviii List of abbreviations and symbols sssssesesseessseseessessseserssessresrtssessressrsseesssreesserrssseeesse X1X Chapter 1 General introduction 1 1 Proteins involved in membrane fusion 0 0 eee eects eeeeeeees 1 SNARES and SNARE cyclen ens a ET Ea TTE Ea eS 1 Alera ted Ce EE E ee PRY ty Tey pana E E EE 2 Synaptotacmin lissen e RET a a a a e e CREE eR OEP 3 1 2 Lipids and lipases implicated in FUSION 0 cece ccc
190. purified from bacteria BL21 according to the manufacture s protocol GE Healthcare Piscataway NJ Proteins were eluted from the beads using reduced glutathione and then dialyzed against buffer containing ZnSO final concentration 200 nM Anti PI 4 5 P antibody 1 50 was kindly provided by Dr Kiyoko Fukami The University of Tokyo Tokyo Anti PI 3 P 1 100 and anti PI 4 P 1 100 antibodies were purchased from Echelon Biosciences Salt Lake City UT The PlI transfer protein 140 72 ug ml was generously provided by Dr Vytas A Bankaitis The Univeristy of North Carolina Chapel Hill Data analysis Except for experiments done with MEFs all experiments were performed in a one control vs one test result pattern For excised patch records the capacitance traces were well described by a mono exponential function with a small linear drift component Y a b 1 e ct 3 12 where b represents the theoretical maximal amplitude k is the rate constant used in statistical analysis and c represents the slow component For patches with b lt 0 the amplitude of zero was assigned To calculate the ratio of active patches we used a threshold of 50 fF to define active and inactive b lt 50 fF patches We mention that we were not able to determine the capacitance of excised patches routinely so that normalization of results to patch size is impossible Also we mention that methodologically induced capacitance changes during soluti
191. q 2 amp 0 001 100 10000 10EM4 10E 6 Rem Pa Sytl reconstitution with 0 67 OG EA i 20 S 10 amp 0 001 010 1 00 10 00 100 00 1 0E 3 1 0E 4 Rn Sytl Syb2 proteoliposome ie 30 20 10 amp 0 0 01 1 00 100 00 10EH4 10E 46 R nm Figure 2 7 Size distribution of reconstituted liposomes Dynamic light scattering was used for estimating the size distribution of liposomes Distribution of LUVs made by extrusion method was very homogeneous and the diameter was about 100 nm upper panel When Sytl was reconstituted using 0 67 OG previously established OG concentration for reconstituting Syb2 middle panel the size distribution was very heterogeneous and there seemed to be particles with large diameter Using OG concentration of 0 77 the size distribution of Sytl containing proteoliposomes was improved tremendously lower panel 51 52 Irtersity o8 8888 8s888 Add TritonX 100 Scan every 20 min for 2 hr 500 510 520 530 540 550 Emission wavdengh Figure 2 8 Leakage assay of Sytl containing liposomes Proteoliposomes loaded with 100 mM carboxyfluorescein were diluted 80X in buffer and the leakage of the content i e carboxyfluorescein was measured by means of dequenching of leaked carboxyfluorescein in a fluorescence spectrophotometer At the end of measurement liposomes were lysed by adding 1 Triton X 100 to the cuvette to get maximal fluorescence No significant sign of leakage was detected
192. qQSq Mou oy QLUIPIOOO ZASd 1ASd oy ZUNLIJOI AG Q pUe Y YM 107994 amp sodwo Wy X pue Y SIOJOIA eUOSOYLO OM OI SIS JOIpITdure ur y90 9Y yM yey SULIOPIsUOD YW pue y JO JOS MOU L PNYSUODI 0 0 YIYS seyd porlsop e YM IPUIPIOOID ZUSd IdSd OU 23101 ULed uo suryo BUL seyd y Isnfpe op Sue seyd y Jo yvwysnfpe IMO Ep 3M LdSd lt 207 0 0 002 0 004 0 006 0 008 0 01 I 2sin 2Mft ssin 2mft 7 2 1 N 1 i j S f 05 L 4 0 5 Hy BE S E JE E ff 4 j i j hb 4 a o A j f L IJ 4 fo of fy Poy 05 oH 05 Log fl hy J Vi W Y U a A Vi ty 0 002 0004 0 006 0 008 001 K 0 002 0 004 0 006 0 008 0 01 Refl sin 27ft Ref2 sin 2mft 7 2 BAADALA AAMAAANUAI iii K 0 002 0 004 0 006 0 008 0 01 4 0 002 0 004 0 006 0 008 IxRefl IXRef2 Figure 4 4 Demonstration of phase sensitive detection Current Z black composed 0 01 of conductive blue and capacitive red components are shown on the top In phase Refl and orthogonal Ref2 reference waves are shown in the middle The products are shown on the bottom The value of the DC component black line is exactly half
193. r baseline subtraction If the protocol is different from the baseline protocol the symbol for the pulse train is set to gray and you will not be able to access it unless the Set baseline button is unclicked Once the initialization of axis2 is completed function Show_update2 is called This function simply sets the symbol color for current pulse train to red and sets symbol color for current baseline to yellow if the Set baseline button is clicked Function Show_update_ex2 updates all the axes except axis2 It retrieves raw data using MATLAB function daqread subtracts the data if desired sum up charges from each individual pulse response and then update the plots Main variables in the program As usual there are tons of variables in the program and most of them are categorized into groups and have clear descriptions in the codes A few important variables are introduced below and many of them are also used in Capmeters 188 189 handles bufdir it contains temporary directory used to save raw files The default names for the raw files are IQraw numbers daq The raw files will be renamed to FILENAME numbers daq and moved to the directory indicated by handles current_folder handles current_folder it is the last folder you visited when you use the Save or Load button handles nidaqid device ID for the data acquisition board Please refer to section 4 3 for more details handles aoChI convert command sensi
194. r functional role Only one positive result concerning 88 phosphoinositides was obtained namely that non SG fusion was blocked by the combined application of wortmannin and adenosine However both reagents may have non specific effects At high concentrations wortmannin inhibits mitogen activated protein kinase MAPK Ferby et al 1996 and myosin light chain kinases MLCK Nakanishi et al 1992 Since non SG fusion is not blocked by staurosporine Table 3 1 although staurosporine blocks the activities of MLCK MAPK might be an interesting target to be tested in future work In conclusion it does not seem surprising that non SG membrane fusion which is probably used for cell wound repair is regulated in substantially different fashion from the release of neurotransmitters and the ATP dependence of non SG fusion likely represents a unique regulatory mechanism that comes into play after mechanical perturbation of the fusion system Wound repair and non SG fusion While we infer that the membrane fusion process examined in this study is related to the membrane wound response the membrane compartment s involved in non SG fusion are still enigmatic In addition to lysosomes a novel organelle named the enlargosome is proposed to mediate membrane repair Borgonovo et al 2002 Fusion of the enlargosome in rat PC12 cells is TeTx insensitive which is different from non SG fusion in RBL cells as described here However it has also
195. rders Although LabView from National Instrument was already a data acquisition and analysis platform my mentor was never happy about the graphic functions of it Why didn t we try pClamp from Molecular Devices or PULSE from HEKA It s clear that to use PULSE the entire lab had to switch from Axopatch to HEKA amplifiers for pClamp 120 I don t know the actual reasons but I guess my mentor wanted to have more control over the functions of the software so he decided to develop a program based on the MATLAB platform The first acquisition software in the lab is called program2 developed by Chengcheng Shen who became a good friend of mine after he jointed the lab The program2 served as a plain data recording software and everyone was happy about it However human beings always want more At that time I was trying to do carbon fiber amperometry and one of the desired function is the digital filtering To make real time digital filtering the core of program2 had to be redesigned In addition I also wanted more functions like pulse generation and some changes of the graphic user interface GUI of program2 Everyone has his own business I could not always ask Chengcheng to do something for me As a fearless biologist I decided to learn MATLAB and design new program which satisfies my needs The first developed program is called Capmeter 1 which still serves as a plain data recorder but has the ability to do real time digital filtering wil
196. rding system Use aluminum foil to wrap all the tubings and again make sure that the foil is properly grounded There is still something more to be shielded Tubings for hydraulic micromanipulator if used the headstage for recording amperometric current and the carbon electrode except the portion that is inserted into the pipette holder also need to be shielded It is to reduce the noise in amperometric current 4 Ground yourself 95 Human body is conductive and receives lots of electrical noise from the environment too If you put your hands into the Faraday cage e g turning on the solution flow or moving the patch without grounding yourself huge noise signals are introduced into the system and sometimes may even bast the seal The best way to solve this problem is to ground yourself electrically by keeping one of your hands on the Faraday cage during the experiment If you ground yourself to the cage right before moving your hands into it charges discharged from your body may still result in spike like artifacts in the record 5 Do not let anything shaking in the Faraday cage Anything shaking in the cage may also give spike like or low frequency noise in the record Possible objects include tubings of hydraulic micromanipulator suction line carbon electrode and aluminum foil etc Also avoid strong air flow around the cage to minimize potential source of vibration 6 Do not move your chair during recordings I kno
197. s C codes for charge integration dataB 0 0 for i 1 i lt SPC i t notice that i starts at 1 dataB i dataB 1i 1 dataA indexpeak np i 1 signB asymp interval Qs int I asymp dt estimate peak tau from Q subtracted dataB s1 0 s2 0 s3 0 for i 0 i lt w i sl dataB sp1 i s2 dataB sp2 i s3 dataB sp3 i 144 145 sl w s2 w s3 w if 2 s2 s3 s1 0 PeakTau s2 s2 s1 s3 2 s2 s3 s1 else quality 0 Variable interval is calculated at the very beginning using the following code when CapEngine4 is called interval time int aiSamplesPerTrigger 1 time 0 aiSamplesPerTrigger 1 The value is then passed to SgQ with other data and parameters After summation the value of steady state charges a b t is estimated using Eq 3 2 and then assigned to variable PeakTau C codes for peak and tau estimation Reverse the Q subtracted curve for fitting for i 0 1 lt SPC itt dataB i PeakTau dataB i adjust lastmax and firstmin etc linear regression of lny v s x fenmu double fladdl sx2 sxxsx if fenmu 0 amp amp fladdl sxy sx sy 0 get peak and tau tau 1 fladd1l sxy sx sy fenmu PeakTau tau 1l exp interval tau interval correct int Q peak PeakTau tautasymp else quality 0 After inverting Qs Eq 4 26
198. s 1 5 put notes on axisl and 156 buttons 6 0 put notes on axis2 You may also put notes by pressing the numbers on the keyboard during the recording Other Keypress functions are calls the Module if the program is stopped and t puts time constant on axis when SQA is selected The Module button by default calls Capmodule4 m which is a tool for making seals If the Auto start check box is checked Capmeter starts automatically when the module is closed Using PSD Capmeter6v3 08326 peal 3 1 Press PAdj 2 Change Cm 3 Press 4 PAdj 4 Change Cm compensation to check phase angle j2 i compensation i 1 1 1 i c 2 4 4 o 00115 1 1 6 LP1OkHz ab 1 0 012 0 25 Top 0 25 gt ajaa E eal oo paei ee 8 ite iess oars EE eaS 4 release a manual Jock the scale y ey 7 lt 0 CACHEN c sf suction uani 70 014 le pect Press E lee sald od eee Sine feia fs Feet ale 2 fv 28 44 Set fies 23 i 0 015 gt 4 6 8 10 12 14 16 18 Vashon 6 F Rawdaia Started Neste SS nan 8 o Copyrigt 2007 Tzu Ming Wang and Donald W Higemann If PSD is selected the first thing you need to do after starting the recording is to adjust the phase angle Capmeter can calculate the phase angle for you when you use the PAdj phase adjustment in red circle button in the Lock in control panel as shown 157 above You first click the button PAdj becomes
199. s Wavecaic l Stopped start Al M for AO start AO p TTL trigger Al pi cn AO started Al started ote i O Al SamplesAcquiredFen Al TimerFen assign variables Pas J J ey to the Workspace i process_data update_piot Show_update_Caliback i CapEnginet Disp Ctr Ofiter 2 This section and the following one introduce how Capmeter6v3 works The above 169 diagram shows you the information flow in the program Function names are in italic and functions that are evoked periodically during the recording are in the dashed box When you start the recording function Start_Stop Callback is evoked It resets some variables and adjusts properties of GUI components AI and AO objects etc Then function Set PSD is called This function further calls Wavecalc to calculate the oscillation wave form and calls Refcalc to generate reference waves for PSD AO is started after the output waves are queued into the memory Note that AO is started in function Set PSD but not in function Start _Stop_Callback If TTL trigger is selected AO will not start sending out signals unless a TTL trigger is detected AI is started in Start Stop Callback however the acquisition will not start until it receives trigger signals from AO Once the AI is triggered its SamplesAcquiredFcn and TimerFcn will be evoked periodically to process the data and update the plots When the recording is stopped the AI StopFcn will assign the data to MATLAB Workspace and shows all the
200. s and hypotheses Membrane fusion seems to be a simple biophysical phenomenon however it is critical in almost every aspect for an organism to survive and its regulation is also behind many fundamental biological processes with huge scientific and clinical interests e g neurotransmitter release and information processing insulin secretion and diabetes release of cytokines and modulation of the immune system viral fusion and pathogenesis etc Reconstitution of the fusion machinery in vitro presumably would allow researchers to manipulate each component in the system and define the roles of them in an unambiguous way Early attempt to reconstitute neurotransmitter release had been done in the early 90 s Karli et al 1990 Secretory granules SGs were purified from bovine chromaffin cells and labelled with fluorescent dye and membrane vesicles were prepared from bovine adrenal medulla homogenate In this semi reconstituted system fusion of SGs to membrane vesicles was monitored by fluorescent lipid dequenching and the role of phospholipase A PLA was explored Karli et al 1990 A totally and artificially reconstituted fusion system was reported in the late 90 s Weber et al 1998 Artificial proteoliposomes with defined lipid compositions and v SNARE Syb2 t SNAREs SNAP25 Stx1A were made and dequenching of fluorescent lipids was also utilized to monitor membrane fusion Weber et al 1998 In this reconstituted
201. s happening periodically are in dashed boxes and function names are in italic Once the program is launched AI and AO objects start automatically Actually AI is never triggered however its ZimerFcn is still evoked periodically to generate the noise Function scope locates the positions of the pulses refreshes the oscilloscope and gets the snapshot when the Shot button is clicked When the Track button is clicked the TrackFeedback function adjusts the holding potential to make average current close to zero TrackFeedback adjusts the holding potential in a stepwise manner every 0 2 s and the step size is determined by the average current The smaller the absolute value of the average current the smaller the voltage step If you feel the tracking is too slow i e taking a long time or too fast i e jumping around you may adjust the step size in TrackFeedback Whenever the absolute value of the average current is smaller than a certain threshold defined in the function a negative feedback is applied to reduce the holding potential by 0 005 mV in every cycle The reason to implement a negative feedback here is that when seal is formed suddenly the average current is dropping close to zero and in this circumstance it does not make sense to keep the same magnitude of the holding potential This design is more reasonable rather than critical The sound sequence The sound sequence is composed of two components One is the carrier
202. s in excised patches The original plan for my thesis was to study regulated exocytosis in giant excised membrane patches Sadly after doing amperometric recording it turned out that the membrane fusion I was studying for these days are not fusion of large SGs To accomplish the original goal without much luck though I addressed this issue from two directions One is to change the way the patch is excised and the other direction is to use cell lines with more SGs They are summarized below 1 Use air bubbles to excise the patch The idea came from the literature that people lift the cell in the air for 1 s to make sure that the patch is in inside out but not in vesicle like configuration Dernick et al 2003 Special apparatus was made to control the attacking strength of bubbles However technically it was difficult to excise the patch this way and the method itself did not help to preserve SGs in the patches either 2 Use electrical pulses to excise the patch To avoid mechanical force that might disrupt the fusion machinery I also tried to open a hole on the cell using electroporation with a platinum wire It did not help either 3 Inject solution to blow up the cell 101 In my standard a good scientist should be able to solve the problem creatively As a good scientist to be I decided to use a creative way to excise the patch and entertain myself at the same time to relieve my stress in these days Blowing u
203. solution for the asymptote b of the three simultaneous functions A b Y e B b Y e and C b Y e The steady state current was then subtracted from the trace and the data range from the peak current to a point located at 3t estimated as peak current times e Fig 3 1B solid section was used for fitting via linear regression to determine the slope and intercept at zero time namely 1 t and n a b respectively Our routine to calculate cell capacitance Cm membrane resistance Rm and access resistance Ra can be derived as follows Membrane voltage V approaches a steady state Vss Vex VcRm Ra Rm 3 3 as a function of the command voltage Vc peak to peak step 2Vc with a time 68 69 constant t of Cm 1 Ra 1I Rm For square wave perturbation membrane voltage during the pulse is given by the exponential function Vy fVss Vss 1 f 1 e 3 4 where f is the fraction of Vss across the membrane at the end of the voltage step of duration 4 Solving for f with t 4 e 3 5 1 e oe and membrane voltage at the beginning of the voltage step is _ fVcRm Ra Rm 2 6 From the steady state current Ve pS Ra Rm Ca and the peak current Ve V 0 3 8 ae ys 3 8 the solutions for Ra Rm and Cm are Ve 1 f Ra _ Oat bf CO Vc a b b a fb Cm 1 2 3 11 Our algorithm was verified by using it to retrieve cell parameters from model c
204. sulting current transient with a decaying exponential function Thompson et al 2001 The obtained parameters are then used to calculate membrane capacitance Cm membrane resistance Rm and access resistance Ra In the frequency domain method a technique called phase sensitive detection PSD is used By using PSD superior signal to noise ratio is achieved Two different approaches have their own advantages and drawbacks over the other The time domain method I used to call it square wave algorithm SQA is much noisier than the PSD especially when the time constant Tt is fast Nevertheless the curve fitting procedure is not always working and takes a lot of CPU power A beautiful exponential decay is usually required for the calculation However unlike PSD SQA gives you the absolute values of Cm Rm and Ra and most importantly the calculation is independent of the phase angle which might be changing during the experiment will be discussed later The PSD uses a reference wave with a shift of phase angle to extract the information Thus setting the angle at different values result in different readouts for the same input In addition by using two references signals proportional to C and G are 118 exported Since no information about Ra is obtained the absolute value of C and G cannot be calculated The advantages of PSD are its superior noise performance and the ease of computer implementation Depending on t
205. system however the kinetics of membrane fusion was extremely slow which took hours to reach the plateau and Ca was not required to trigger fusion In hope to recapitulate the fast Ca dependent membrane fusion Mahal et al purified full length Sytl from bacteria and reconstituted the machinery using the same method Mahal et al 2002 Although membrane fusion was stimulated in the presence of Sytl surprisingly it was not Ca dependent and the kinetics was still very slow Mahal et al 2002 Later another group reconstituted Ca dependent membrane fusion in the test tube with similar slow kinetics Tucker et al 2004 Rather than using full length Sytl they used only C2A C2B domains from Syt1 Tucker et al 2004 They claimed that the Ca independent manner of fusion reported previously Mahal et al 2002 was because the bacterial expressed full length Sytl was not functional Ironically this group also used C2A C2B domains purified from bacteria Tucker et al 2004 The diffusion time required for liposome docking was always blamed to be the cause of slow kinetics in this system However when liposomes were clustered using streptavidin biotin mediated docking system Schuette et al 2004 it clearly indicated that the slow kinetics of liposome liposome fusion was not due to the preceding docking step Schuette et al 2004 In spite of these controversial results and the debates about the validity of these
206. t only does it once in the first loop For subsequent loops Dfilter2 removes one old data point from the sum series and adds the new one to the sum for running mean or to the appropriate position of the series for running median In this way the processing speed of Dfilter2 is greatly enhanced The calculation of running mean becomes superfast and the running median filter becomes at least 75 times faster in my computer For data containing NaN Dfilter2 removes NaN in the window and then calculate the mean median If there is no real number in the filter window NaN is assigned to that time point Since Dfilter2 removes NaN before calculation the total number of NaN will be reduced after filtering For instance if the longest consecutive NaN in the input data set is less than the filter window there will be no NaN in the output array The original data gaps composed of NaN are filled by data average median surround the gaps DispCtrl mexw32 Because showing data takes a lot of CPU power Capmeters use function DispCtr to reduce the displayed data in the online only called in AI ZimerFcn update_plot but not 193 offline mode It simply samples equally spaced points from the input array and the space is determined by the total length of the data and the number of points to be displayed If the number of total points is less than the setting all points are displayed An example is shown below XDatal2 YDatal YData2 XDat
207. t the scale of axis2 by dragging the slider bar next to it 181 Display modes offline 0 25 lo xi 0 2 0 15 0 1 0 05 0 0 05 0 1 0 15 L a p k 5 L 1 5000 10000 mes 10 par was the baseline 15 oet 4 eer 0 5 o2 o 7 O AD E 0 5 ool Yy 4 0 4 E 1 5 current position different pulse protocol current baseline 06 0 8 l m 1 L L fi Jes n i j i o oe is 10 15 20 25 30 35 g0 60 40 20 o 20 40 60 Erase Mode or Pulse Protocol _________________ Nevigete rere x toad direct intg Aene Lg Avot an p E L 4 Module Civ Erase o2 A ely d Pulse every 3 sec we E 3 Awn Stoned 2 ey 2n J hex ESI Developed by Tzu Ming Wang Higemann Lab UTSW Dallas 2008 The online and offline display modes are very similar except that you may adjust the scale of X axis of axis2 offline and there are many symbols plotted on axis2 in offline mode Each symbol represents a pulse train and the current pulse train displayed is in red color If there is a note associated with the current pulse the note will also be displayed in the first edit box in the Pulse note panel Symbol for pulses with notes associated with them are in square shape I would like to emphasize that the recording of variable handles starttime may be delayed occasionally and makes the estimated time for axis2 very inaccurate It should not happen theor
208. tantially differently from SG fusion and we have identified an ATP dependent process that restores non SG fusion capability after it is perturbed by membrane stretch and dilation 3 2 Introduction Mast cells of hematopoietic origin play a central role in inflammatory responses by releasing numerous substances that modulate immune responses Metcalfe et al 1997 Fusion of large secretory granules SGs to the plasma membrane underlies degranulation of mast cells and much experimental effort has focused on the regulation of this exocytotic process Burgoyne and Morgan 2003 Sagi Eisenberg 2007 In addition to exocytosis of SGs another type of fusion that does not result in clear step wise changes of membrane capacitance non SG was reported in rat peritoneal mast cells twenty years ago Almers and Neher 1987 While the step size of non SG events is not readily resolved by capacitance recording the increase of cell capacitance mediated by such fusion events can be of very large magnitude Almers and Neher 1987 The sources of membrane involved in non SG fusion as well as the underlying fusion mechanisms remain rather enigmatic Data from chromaffin cells indicate that non SG fusion requires relatively high Ca 100 uM and is ATP dependent but neurotoxin insensitive Non SGs in chromaffin cells are not likely to represent acetylcholine containing synaptic like microvesicles Xu et al 1998 and it is striking that non SG fusion can a
209. te membrane fusion Rhee et al 2002 Finally arachidonic acid AA which is also a PI 4 5 P metabolite is reported to facilitate SNARE complex formation in the presence of Munc18 Rickman and Davletov 2005 and potentiate exocytosis in chromaffin cells Latham et al 2007 Phospholipase Cs Phospholipase C is an enzyme family that contains X and Y catalytic domains and depending on the subtypes it also contains other domains like C2 EF hand SH and PH domains etc Rhee 2001 The classical substrate of PLCs is PI 4 5 P2 and the resulting products DAG and IP Fig 1 7 are utilized to regulate a variety of different cellular activities While IP increases intracellular Ca concentration via Ca release from internal stores DAG also plays several critical roles in regulating membrane fusion In yeast homotypic vacuole fusion system sequestering DAG by applying DAG binding CIB domain blocked vacuole fusion Jun et al 2004 Thorngren et al 2004 and similar effects were observed when the PLC inhibitors 3 nitrocoumarin and U73122 were added into the reaction mixture Jun et al 2004 In addition to the well known DAG target protein kinase C PKC DAG also interacts with Muncl3 and augments neurotransmitter release Rhee et al 2002 and its analogue PMA phorbol 12 myristate 13 acetate has also been shown to increase the primed SGs in chromaffin cells Gil et al 2001 Furthermore without interacting with other
210. tens of fF indicating that the diameter of the granules is close to micrometer range A typical fusion event with fusion pore dilation is marked between two broken lines A gradual increase of capacitance transient increase of conductance and the amperometric foot signal is observed Here and in all subsequent figures numbers given between axis ticks indicate the tick interval 108 A hi O ee 4 be 200M free Ca 9 9 o Re lz Q a Q ATP o 1s i amp Q G ar 10 T 15 8 3 8 3 g jo JE A 3 Of es 2 2 J 50 Ca 4 50 Ca 4 2 Time s 2 Time s i oem 400 1 2 1 0 sei a an iiias a y iiis 1 0 SF 300 5 a f k B08 Y at b l e tet o a 50 6 xe o si 200 06 Z O a 00 4 20 4 j lt ao j 100 oc 50 Dja 0 2 fitted curve original data Ai 3 a 7 l4 2 Time s E234Q TeTx E234Q TeTx E234Q TeTx Figure 3 3 Non SG fusion is SNARE dependent A Two typical recordings from excised patches are shown In the left panel the amplitude is smaller than 50 fF which is close to the artifact caused by moving the patch Therefore this patch is designated inactive In the right panel robust non SG fusion gt 50 fF is observed in an active patch B The same trace is fitted to a mono exponential function rate constant k with a linear creep component c C Incubation of patches with 200 nM tetanus toxin
211. ter is bypassed using a 10 20 kHz filter will reduce the noise greatly Low pass filtering the current at 2 kHz or slower is also not recommended especially when I SQA is selected because the shape of the peak current is severely affected in this case Channel AI2 records whatever is connected to it The digitally filtered data is displayed at channel 4 It is possible to synchronize the acquisition of Capmeter with another program In this case a TTL input to PFIO is required Capmeter does not support TTL output simply because the MATLAB does not support buffered digital output Things you need to know before using 1 Please cite the paper Wang and Hilgemann 2008 2 The digital filter in Capmeter only calculates the average of data acquired in a specified time window Although the program automatically adjusts the time window so that the command sine or square waves are canceled it is still possible that the filtered current does not represent the actual current faithfully when data clipping happens 3 When PSD is selected uncheck the Sine check box results in the generation of triangular waves When SQA is selected it does not matter if the check box is 153 154 checked or not 4 For PSD the phase angle has to be adjusted again whenever you start a new acquisition even for the same cell It is also recommended to check the phase angle whenever the AO system is restarted i e changing oscillation frequency or
212. th cell parameters determined by Capmeter 6 as described above Patch amperometry The setup was connected according to Dernick et al Dernick et al 2005 with some 71 modifications In brief two Axopatch 1D amplifiers were used One of the headstages was connected to the bath for capacitance recording the other one was connected to the carbon electrode for recording the amperometric current and the patch pipette was the ground The carbon electrodes were made from 7 um carbon fibers C005711 Goodfellow corporation Oakdale PA and quartz capillaries Polymicro Technologies Phoenix AZ Flowable silicone windshield glass sealer Permatex Hartford CT was used to insulate the carbon fiber and the tip was cut to expose the carbon surface before installing Fig 3 2A The carbon electrode was installed through the infusion line of the pipette holder and connected to the amplifier using 3 M KCI and Ag AgCl wire The electrode was moved toward the patch as close as possible and a holding potential of 0 7 V was applied The amperometric signals were low pass filtered at 5 kHz by the amplifier and digitally filtered again by averaging signals acquired in a time period of 1 ms using Capmeter and then digitized at 500 Hz Recombinant proteins and antibodies The wild type and mutant E234Q GST tetanus toxin light chain fusion constructs were kindly provided by Dr Thomas C Siidhof UTSouthwestern Dallas Recombinant proteins were
213. the Workspace The Save button in Capmeter does not save snapshot data Running the program io Zoom Step Command C 100x i 44 10 1 01 ST l zoj 1 sos 12 Copyright 2006 2008 Tzu Ming Wang and Donald WV Hilgemann The most common way to launch Capmodule4 is clicking the Module button or pressing the key in Capmeters In this way Capmodule4 gets the channel carrying current device ID and command sensitivity directly from Capmeters Make sure to adjust the net gain using the and button If you get the current from the scaled 197 output of the patch clamp the net gain is af If the current is not scaled the net gain is the gain of the headstage B The value of net gain influences the stability of the tracking function and the generation of the noise Once the program is started noise is generated immediately If you do not like the noise you may click the Mute button to shot it off or change the Value property of Mute button to 1 using MATLAB GUI builder Click the Track button to keep the net current zero and the tracking potential in mV is shown in the button You may adjust the step command potential using corresponding buttons in the Step command panel and the current step size is shown in the panel To adjust the zoom simply click the desired radio button to change the Y axis setting You may take snapshots of the oscilloscope To do so click
214. the coordinates by an angle of s r as shown in B After the adjustment X reflects the actual amplitude of G and nothing contaminates PSD2 C When is properly adjusted PSD1 and PSD2 give the actual G and C respectively The mixture of the two components is a new vector with an amplitude of R and a phase angle of 0 It is important to note however by knowing R and does not mean that you can extract the actual G and C There are infinite ways to get a vector with R and and three of them are shown in D 204 PSD2 AC Figure 4 2 Online calculation of the phase angle To test whether the current Or is valid or not one can change the amount of compensated capacitance from the patch clamp and see if the change AC is solely reflected on the readout of PSD2 If is not correct changes of capacitance compensation result in signal changes both on PSD1 X X0 and PSD2 Y Yo as shown above The correct phase angle can be calculated by rotating the PSD1 PSD2 coordinate by an angle of a given that amp is tan X X0 Y Yo and Xo Yo and X Y represent values before and after changing capacitance compensation respectively 205 206 AJaATOOdsaL 70 SO2X pue 70 uISy st YOIyM STXE ZASd OU UO X pue y eUISIIO y Jo suonoofoIrd y JO wns 9y ST NOpedl TQS MoU oY UZY ATIATIOdSaI 70 UISZ pue 70 sooy SI YIM sIxe GSd 24 UO 9 4 pue Q y eulsiio y Jo suonooford y zo wns ay SI mopear
215. the data on axis2 again you simply put 0 zero in the edit box on the right side of the to button and then click Enter By clicking the Show button in the Navigate panel IQplot2 generates figures for all the axes and the data are assigned to corresponding variables in the Workspace Although you may specify the region to be displayed on axis 2 the figure for axis2 still contains all the points i e 1 end 183 184 Variables in the Workspace After the recording is stopped IQplot assigns a number of variables to the MATLAB Workspace They are introduced below DAQinfo structure containing hardware settings aiSR analogue input speed in Hz aoSR analogue output speed in Hz aoChIconvert command sensitivity for AOO in mV V starttime approximate time when the Stopped button is clicked in year month date hr min s Fig1Y it contains Y data from axis when the Show button is clicked Fig2 it contains XY pairs of axis2 data when the Show button is clicked Fig3X Fig3Y voltage and current data from axis4 when the Show button is clicked Fig4X Fig4Y voltage and charge data from axis5 when the Show button is clicked Pulselog structure containing pulse protocol and other information eventcode 0 no event 1 setting baseline 2 applying note pulse voltage values of steps in the pulse protocol mV pulseinfo first peak mV second peak mV step length AO points step increment mV number
216. the first electrophysiological recording of Ca dependent liposome fusion However when the 43 docking priming protocol was applied I could not see any capacitance jump upon Ca application not shown More importantly capacitance started to increase when Sytl Syb2 proteoliposomes were applied in the absence of Ca not shown This observation implies that either the fusion of proteoliposomes is Ca independent Mahal et al 2002 or the proteoliposomes stick onto the glass pipette faster than blank liposomes do To test it I saturated the pipette with proteoliposomes without Ca first and then move the patch to proteoliposomes with Ca to see if faster capacitance increment as Fig 2 12 A could be observed Unfortunately there was nothing happening when Sytl Syb2 liposomes were applied with Ca Fig 2 12 B indicating that the previous result could be an artifact There are too many unknowns in this novel system For example if blank liposomes could fuse to the membrane or just stick onto the pipette is unknown and I also could not exclude the possibilities that some of the proteoliposomes might fuse to the membrane in a Ca independent way Amperometry with liposomes loaded with dopamine or serotonin is probably required to answer these questions Sadly time was passing and I could not establish the amperometric recording setup before this project was suspended 44 45 B Input HA fraction Figure 2
217. the log of the decaying current transient solid line 107 gt Ww Ca ionophore gt sale h E g T 3 aay o m 3 75m 200m 762um l 3 H i oO 2 i sats a g z silicone sealer carpoh fiber 3M KCI quartz tubing hot melt gue O oO 3 a F 407 200 so nd i er i a ae 5 Time s C D lt x 100uM free Ca ATP GTP S _ be 18 JE axa T ATP GTP ra a 8i 3 S Jg T t 18 5 R 3 O Z E L S D 50 2 2 L lt x L 4 4 4 4 4 4 4 4 4 i i zi i 10 Time 8 5 Time s Figure 3 2 Amperometric and capacitance measurement in RBL cells A Schematic illustration of the intra patch pipette carbon electrode Carbon electrodes were prepared to allow facile insertion and manipulation in the patch pipette holder employed B In the cell attached configuration 2 uM of calcium ionophore A23187 triggers profuse exocytosis Two distinct vesicle pools are observed One is the secretory granule SG pool with large amplitude capacitance steps and amperometric spikes upon stimulation The other pool at the end of the trace contains vesicles of much smaller size that do not release serotonin non SG pool C Amperometric recording of SG fusion in a 20 um diameter excised patch D Expandsion of C In most cases SGs are lost from membrane patches during the excision procedure Occasionally when SGs are preserved fusion gives rise to capacitance steps of
218. tical significance was determined by Student s t test All error bars in figures represent S E M 74 3 4 Results Distinct vesicle populations in RBL cells To study serotonin secretion in RBL cells carbon electrodes were prepared as described in Methods and mounted in a quartz tube with a Ag AgCl electrode that could be inserted into the patch pipette Fig 3 2A to detect the released serotonin In the cell attached configuration application of 2 uM calcium ionophore A23187 induced massive membrane fusion that was detected as an increase of membrane capacitance Fig 3 2B Two types of vesicle populations were observed one is the secretory granule SG pool accompanied with big capacitance steps and amperometric spikes the other one observed here at the end of the trace contains vesicles of much smaller size that are evidently not filled with serotonin non SG pool The capacitance steps of SG fusion were in the range of tens of fF indicating that the diameters of the SGs were in the submicro to micrometer range consistent with values reported by most Spudich and Braunstein 1995 but not all investigators Smith et al 2003 Empirically we found that treating the cells with latrunculin A facilitated both the formation of giant excised patches and the preservation of non SGs on the patches Nevertheless our success rate to preserve SGs in the RBL giant patches was prohibitive for routine studies perhaps because the SGs are n
219. tion at 15 krpm for 15 min with a JA20 rotor followed by additional centrifugation at 35 krpm for 30 min with a Ti70 rotor Beckman Coulter The supernatant was collected into a 50 ml centrifuge tube and 2 4 ml of Ni NTA beads and additional 1 mM of PMSF were added into the crude protein lysate After overnight binding at 4 C with gentle agitation the beads were washed sequentially with 12 ml of 5 10 20 and 30 mM imidazole prepared in solution containing Triton X 100 50 NaH PO 300 NaCl 2 B mercaptoethanol 1 PMSF in mM and supplied with 0 1 Triton X 100 and then washed with 40 and 50 mM imidazole prepared in the same solution except that 0 1 Triton X 100 was substituted by 1 octyl B D glucopyranoside OG To elute the protein 2 5 ml of 100 150 200 250 and 500 mM imidazole prepared in the same OG solution were added sequentially and the eluates were collected in 1 5 ml microtubes Quality of each fraction was examined using Coomassie blue staining after electrophoresis Molar concentration of Sytl was determined using dot blot with serially diluted samples standard C2AB of Sytl with known concentation and monoclonal antibody CL41 1 Synaptic Systems 28 Purification of Synaptobrevin 2 from E coli The protocol for purifying Syb2 was adopted from protocol described previously Chen et al 2006 The GST glutathione S transferase Syb2 fusion construct was transformed into E coli strain BL21 A single colony
220. tivity for AOO in mV V handles aiSR acquisition speed for AI in Hz handles aoSR acquisition speed for AO in Hz handles aidata n by 3 matrix containing all the processed data handles aodata column array containing output signals for AOO handles datasample current sampled at 20 Hz using MATLAB function peekdata handles starttime the time when the Stopped button is clicked handles TRIGGERDELAY the default trigger delay value Negative value is used so that the pre trigger signals are acquired handles triggerdelay it is the trigger delay value used for the current record 4 6 Dynamically linked subroutines CapEngine4 mexw32 CapEngine4 is the core processing unit of Capmeter6v3 It performs all the required calculations to generate digitized data These functions include 1 assigning relative time to every data point 2 performing background digital filtering for Ch3 AI1 and Ch4 AI2 3 performing PSD 4 performing I SQA and 5 performing Q SQA The syntax for using PSD in MATLAB is time Ch3 Ch4 Ch1 Y Ch2 X CapEngine4 1 time AI1 A12 fck3 fck4 filterpt aiSamplesPerTrigger PSDref PSD90 The first input argument is 1 which tells CapEngine4 to use PSD 2 for I SQA and 3 for Q SQA Input arguments fck3 and fck4 determine whether Ch3 and Ch4 are digitally filtered or not and argument filterpt is the number of points used for averaging Argument aiSamplesPerTrigger tells CapEngine4 the length of each
221. ty and ion exchange chromatographic purification the quality of the purified Syb2 was quite good Fig 2 5 The concentration of the most concentrated fraction was 450 ug ml Although the quality was good and the quantity was enough for reconstitution it is possible to further optimize the expression and purification procedures For example incubating the cells at 37 C after IPTG induction may increase 38 the total expressed Syb2 tremendously personal communication Chen et al 2006 In addition the protocol for chromatographic purification may also need to be adjusted since there was still a lot of Syb2 in the flow through Fig 2 5 Quality of the liposomes Syb2 reconstitution with detergent mediated direct incorporation method has been well documented Chen et al 2006 however reconstitution of integral Sytl using this method has never been published as of July 2004 To test the right condition for Sytl reconstitution different OG concentration were tested OG concentration of 0 67 was used for Syb2 proteoliposome preparation However as shown in Fig 2 6 columns 5 7 it is clear that Sytl tends to form aggregation at this concentration and the best OG concentration for Sytl reconstitution is 0 77 if total lipid concentration is 5 mM To check if the reconstituted proteins really get integrated into liposomes 2 M urea was used to remove proteins sticking on the liposomes Again reconstitution condition for Syb
222. u may not adjust the amplitude 1 2 mV if command sensitivity is 20 mV V above the original setting You can lower the amplitude to whatever value you want but remember there is still a trigger signal that is 1 mV larger than the initial peak amplitude not peak to peak 158 Using SQA Although the coding for SQA is quite difficult and has spent me a huge amount of time using SQA is quite easy The first step is to select the algorithm and the second step is to click the Stopped button to start recording That s it If the net gain a is 1 then 10 pF 1 for Cm 1 nS for Gm and 1 MQ for Ra The hints will show up when you move the mouse cursor onto the algorithm pop up menu for few seconds Note that if the SQA letters become red Foregroundcolor that means the fitting routines fail to extract the information and NaN is assigned to all cell parameters If the entire pop up menu become red Backgroundcolor that means the peak current might have overload the board gt 10 V and the command amplitude should be decreased I will recommend you to restart the recording in this circumstance if possible For your information there is an Auto check box beneath the Sine check box It is checked automatically when you select SQA Right click on the check box and the context menu shows you two options 1 frequency and 2 fitting range The fitting range option is selected by default that means the fitting routine uses a tau
223. ucker W C Weber T and Chapman E R 2004 Reconstitution of ca2 regulated membrane fusion by synaptotagmin and snares Science 304 435 438 Verhage M Maia A S Plomp J J Brussaard A B Heeroma J H et al 2000 Synaptic assembly of the brain in the absence of neurotransmitter secretion Science 287 864 869 Vitale N Mawet J Camonis J Regazzi R Bader M et al 2005 The small gtpase rala controls exocytosis of large dense core secretory granules by interacting with arf6 dependent phospholipase d1 J Biol Chem 280 29921 29928 Wang P Chicka M C Bhalla A Richards D A and Chapman E R 2005 Synaptotagmin vii is targeted to secretory organelles in pc12 cells where it functions as a high affinity calcium sensor Mol Cell Biol 25 8693 8702 Wang T M and Hilgemann D W 2008 Ca dependent non secretory vesicle fusion in a secretory cell J Gen Physiol In press Weber T Zemelman B V McNew J A Westermann B Gmachl M et al 1998 Snarepins minimal machinery for membrane fusion Cell 92 759 772 225 Whiteheart S W Rossnagel K Buhrow S A Brunner M Jaenicke R et al 1994 N ethylmaleimide sensitive fusion protein a trimeric atpase whose hydrolysis of atp is required for membrane fusion J Cell Biol 126 945 954 Williams R M Shear J B Zipfel W R Maiti S and Webb W W 1999 Mucosal mast cell secretion processes imaged using three photon microscopy of 5
224. use kit to prepare plasmid DNA but I don t Unless the DNA is going to be used for transfection I use DNA prepared by traditional protocol for all other purposes including DNA sequencing Miniprep kit is expensive and the eluted DNA concentration is low To save some money and time for concentrating DNA I still use the traditional way a lot The protocol is modified from the standard one and summarized below Single colony of E coli was inoculated into each tube containing 2 ml LB 1 tryptone 0 5 yeast extract 1 NaCl medium with appropriate antibiotic and cultured at 37 C overnight with vigorously shaking The overnight culture was poured into a 1 5 ml microtube and centrifuged at 12000 g for 30 s The supernatant was removed and the pellet was resuspended in 100 ul Solution I 50 mM glucose 25 mM Tris Cl 10 mM EDTA pH 8 0 An amount of 200 ul freshly prepared Solution II 0 2 N NaOH 1 SDS was added into the suspension and the microtube was inverted gently for several times to lyse the bacteria Subsequently 150 ul ice cold Solution HI 3 M potassium acetate 11 5 glacial acetic acid was added and the mixture was inverted gently for several times and placed on ice for 3 5 minutes optional After centrifugation at 12000 g for 5 minutes the supernatant containing plasmid DNA was transferred into a fresh microtube Phenol chloroform extraction is optional If desired an equal volume of 22 phenol chloroform isoamyl alco
225. w it sounds crazy If I move my chair during the recording not always but sometimes it does give some artifacts in the record So I recommend you not to move anything including your body during the recording Besides tips mentioned above you can increase the oscillation frequency and amplitude of the sine waves to reduce noise in capacitance records And as a last resort you can apply running mean median filter to suppress the noise However keep in mind that the kinetics of the trace is slower than original after filtering Tips for making carbon electrodes The standard ways for making carbon electrodes are well documented Chow and von R den Ludolf 1995 Dernick et al 2005 Mundroff and Wightman 2002 and the electrodes are even commercially available However these types of electrodes do not fit my patch clamp headstage and it is also difficult for me to make a delicate and specialized pipette holder like that So I decided to design one that is easy to make and easy to use Fig 3 2A Before describing the procedures for making electrodes I would like to thank Dr Marc Llaguno whose specialty is carbon nano tube He is the one who suggested me to insulate the electrodes using windshield glass sealer Steps for making carbon electrodes are listed below l Glue two quartz tubings together Cut a 2 cm piece of 75 um i d quartz tubing and a 8 cm piece of 200 um i d quartz tubing Insert about ha
226. ween 20 N N N N tetramethyl ethylenediamin tetanus toxin angle total internal reflection fluorescence microscopy tris hydroxymethyl aminomethane target membrane SNARE voltage signal amplitude command voltage steady state potential vesicle SNARE wortmannin xxii Chapter 1 General introduction 1 1 Proteins involved in membrane fusion SNAREs and SNARE cycle Membrane fusion is a fundamental biophysical phenomenon that is utilized and tightly regulated in a living organism Neurotransmitters are released upon vesicle fusion and by means of the tightly controlled fusion machinery the release of neurotransmitters is spatially restricted to the active zone of the presynaptic terminal and temporally controlled upon stimuli Fusion between the vesicle and plasma membranes requires the interaction between these two lipid bilayers In general it is wildly accepted that the process at least in part is mediated by the SNARE soluble N ethylmaleimide sensitive factor attachment protein receptor proteins which are evolutionarily conserved among species and are utilized in both exocytic and endocytic pathways Fig 1 1 Jahn et al 2003 Synaptobrevin Syb is a SNARE protein that is expressed on the vesicle v SNARE and syntaxin Stx and SNAP25 synaptosomal associated protein of 25 kDa are distributed on the target membrane t SNAREs These three components can form a four helix bundle Fig 1 2 termed the SNARE complex or
227. within a two hour period 53 a Trypsin ug ml In 4 8 12 16 16 TX 52 3 35 3 28 7 21 3 V761 a Syt1 lumen portion B Trypsin ug ml P In 2 4 8 12 16 16 TX T va j 7 52 3 n l l en 35 3 pm p 28 7 Ee 21 30 V216 a Syt1 cytoplasmic tail C Syt1 Syb2 _TX Syb2 TX P L chymo_ P L tma A Gia hae 52 3 35 3 acy 28 7 a ae aean 21 30 ma DOM P939 a Syb2 Figure 2 9 Orientation tests of Syt1 Syb2 liposomes The orientations of incorporated proteins were tested by enzymatic digestion followed by western blot with antibodies against cytoplasmic or lumen portion of the proteins A With antibody against Sytl lumen portion V761 the smear on western film indicated correct orientated Sytl boxed region B antibody against Sytl cytoplasmic portion V216 was employed Signals remained on the film indicated inversely incorporated Syt1 boxed region C The orientations of Syb2 in Syt1 Syb2 prepared in 0 77 OG and Syb2 only prepared in 0 67 OG proteoliposomes were also tested P pellet In input TX Triton X 100 L Syb2 liposome only chymo chymotrypsin BNG2 StxlA BPR Munc18 1 BMG SNAP25A Figure 2 10 The triple marker transient expression system A Ideally cells co expressing Stxl1A SNAP25A and Munc18 1 have green nucleus green plasma membrane and red cytoplasm B amp C Epifluorescence images of cells co transfected with all three expression vectors are shown The major problem was that
228. xpeak np SPC for i 0 i lt SPC i dataB i dataA indexpeak correct the polarity np i signB assign data and dataBtime i dataTime indexpeak np i dataTime indexpeak np set initial time to 0 Cfind dataB 0 Cmax dataB SPC SPC amp ftemp amp fc if fc 0 lastmax ftemp fc 1 fc l is the last one else lastmax 10 if lastmax gt SPC 12 lastmax SPC 12 D int floor double SPC lastmax 3 w D spl lastmax sp2 spl D sp3 sp2 D if sp3 w gt SPC w SPC sp3 for i 0 i lt w i sl dataB sp1 i s2 dataB sp2 i s3 dataB sp3 i sl w s2 w s3 w get asymptote if 2 s2 s3 sl 0 asymp else quality 0 if quality s2 s2 s1 s3 2 s2 s3 s1 procedures for peak and time constant estimation To correct the polarity of the curve the routine multiplies the curve with the sign of its average variable signB For asymptote estimation the averages of three equally spaced sections are used as introduced in Eq 3 2 Rather than starting from the first point to the last one a variable called astmax is introduced As shown in Fig 4 5B dots in the presence of a filter function the peak is usually delayed and rounded If the entire data range were used for asymptote estimation apparently the value of section A would be und

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