To study the effect of NCS-1 in exocytosis and Ca2+ response, I performed amperometry recording and Ca2+ imaging on the untransfected cell and a cell expressing GFP; NCS-1; NCS-1G2A; NCS-1E120Q; NCS-1R102Q. The untransfected cells are normal chromaffin cells. I used the group as a comparison for transfection effect.
The GFP group was tagged by green fluorescence as a control group which had no effect on function. The data mentioned below were presented as the mean ± SEM and analyzed by Kruskal-Wallis ANOVA with Mann-Whitney post hoc test (*: p < 0.05 when compared with the GFP group).
3.1 The traces of amperometry
Amperometry is an electrochemical technique using carbon fiber electrode to oxidize released chemicals during exocytosis. To determine release contents, chromaffin cells which have vigorous secretory function were used for the catecholamine oxidation. Fig.1 shows the experimental set up and the carbon fiber electrode. I stimulated cells for 3 s with high K+ starting at the 1s and record the spikes of exocytosis in one sweep for 30 s. The protocol repeated 3 times with an interval of 2 min. Fig. 2 A-F show the representative raw traces in one sweep of different treatments. Consider the occurrence time, numbers and size of the spike,
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these groups present different characteristics. In general, the GFP group is similar to the NCS-1 group, and the other 3 mutant groups are 3 types respectively. The details of statistics were mentioned in the following sections. Fig.3 shows the spike analysis.
The amplitude (pA) is the maximal current change of a spike; the rise time (ms) is 10
% - 90 % of the period which a spike climbed from the baseline to the peak; the decay time (ms) is the period which a spike fell from the peak to the 37% of peak height; the area (fC) under single spike is the quantity of electric charge; the half width (ms) is the period at the 50% of peak height. The amplitude and area are determined as the contents of neurotransmitters; the rise time, decay time and half width are related to the fusion kinetics of vesicles.
3.2 Cells overexpressing NCS-1 maintains the exocytosis; cells overexpressing
NCS-1
G2Aor NCS-1
R102Qdown-regulate the exocytosis; cells overexpressing
NCS-1
E120Qhave an elevated exocytosis level.
To find out the exocytosis level, I analyzed the data of amplitude, area and spike numbers. Fig. 4 A and B show the averaged amplitude and area in 3 sweeps per spike per cell in different groups. The amplitude (pA): untransfected, 59.5 ± 6.3*; GFP, 75.0
± 6.7; NCS-1, 88.8 ± 9.1; NCS-1G2A, 48.0 ± 5.2*; NCS-1E120Q, 69.8 ± 6.8; NCS-1R102Q, 36.1 ± 2.1*. The area (fC): untransfected, 845.1 ± 78.4; GFP, 929.3 ± 162.7; NCS-1,
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1137.4 ± 167.7; NCS-1G2A, 671.3 ± 105.5; NCS-1E120Q, 1035.8 ± 88.6*; NCS-1R102Q, 462.8 ± 79.6*. Compared with the GFP group, cells overexpressing NCS-1 have no significant change; NCS-1G2A and NCS-1R102Q have smaller amplitude or area, which represent the inactive release; cells overexpressing NCS-1E120Q have larger area, which represent the dynamic release. Fig. 4 C and D shows the average of spike numbers and total area in 3 sweeps per cell in different groups. The spike numbers:
untransfected, 11.3 ± 2.6*; GFP, 24.6 ± 4.2; NCS-1, 29.0 ± 10.9; NCS-1G2A, 9.1± 3.7*;
NCS-1E120Q, 32.1 ± 7.1; NCS-1R102Q, 7.7 ± 2.6*. The total area (fC): untransfected, 10790.9 ± 2796.5*; GFP, 20084.6 ± 3902.2; NCS-1, 30591.3 ± 10612.5; NCS-1G2A, 6041.9 ± 2676.8*; NCS-1E120Q, 35578.7 ± 8734.5; NCS-1R102Q, 5587.7 ± 2807.0*.
The spike numbers represent the exocytosis events; the total area represents the release contents of a cell. Cells overexpressing NCS-1G2A or NCS-1R102Q have fewer spikes and total area; that suggest the inhibition of exocytosis. The sample size of spike numbers: untransfected, 519; GFP, 788; NCS-1, 465; NCS-1G2A, 118;
NCS-1E120Q, 642; NCS-1R102Q, 100. The sample size of cell numbers: untransfected, 46; GFP, 32; NCS-1, 16; NCS-1G2A, 13; NCS-1E120Q, 20; NCS-1R102Q, 13.
3.3 Cells overexpressing NCS-1
E120Qhave long vesicle fusion time.
To detect the fusion kinetics of vesicles, I analyzed the rise time, decay time and
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half width. Fig. 5 A-C shows the averaged rise time, decay time and half width in 3 sweeps per spike per cell in different groups. The rise time (ms) : untransfected, 17.4
± 1.4; GFP, 16.4 ± 2.3; NCS-1, 15.5 ± 1.1 ; NCS-1G2A, 21.5 ± 4.1 ; NCS-1E120Q, 19.3 ± 1.4; NCS-1R102Q, 13.0 ± 1.8. The decay time (ms) : untransfected, 17.5 ± 2.0*; GFP, 15.5 ± 4.5; NCS-1, 14.5 ± 2.7; NCS-1G2A, 15.6 ± 5.2; NCS-1E120Q, 17.1 ± 2.2*;
NCS-1R102Q, 12.1 ± 2.0. The half width (ms): untransfected, 17.3 ± 1.9; GFP, 15.2 ± 4.0; NCS-1, 14.2 ± 2.2; NCS-1G2A, 16.5 ± 4.2; NCS-1E120Q, 17.6 ± 2.1; NCS-1R102Q, 11.9 ± 1.5. The rise time means the fusion time before maximal release, these experiment groups have no significant when compare with the GFP group. The decay time means the fusion time after maximal release. Cells overexpressing NCS-1E120Q have longer decay time. The half width is the combined changes of rise time and decay time, or it is related to the size of a spike. These experiment groups have no significant change when compare with the GFP group. The above results indicate the fusion mode change in cells overexpressing NCS-1E120Q. The sample size of spike numbers: untransfected, 519; GFP, 788; NCS-1, 465; NCS-1G2A, 118; NCS-1E120Q, 642; NCS-1R102Q, 100. The sample size of cell numbers: untransfected, 46; GFP, 32;
NCS-1, 16; NCS-1G2A, 13; NCS-1E120Q, 20; NCS-1R102Q, 13.
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3.4 Cells overexpressing NCS-1
G2Aor NCS-1
R102Qhave relative infrequent
exocytosis in the 3
rdsweep.
To separate the contribution to exocytosis in different sweeps, I presented spike numbers and total area by different sweeps. Fig. 6 A shows the average of spike numbers per sweep per cell in different groups. The 1st sweep: untransfected, 4.3 ± 1.0 ; GFP, 7.6 ± 1.8; NCS-1, 9.6 ± 4.4; NCS-1G2A, 4.5 ± 1.9; NCS-1E120Q, 12.2 ± 3.3;
NCS-1R102Q, 3.8 ± 1.6. The 2nd sweep: untransfected, 3.7 ± 1.0*; GFP, 8.8 ± 2.2;
NCS-1, 8.1 ± 4.7; NCS-1G2A, 2.5 ± 0.9; NCS-1E120Q, 11.2 ± 2.5; NCS-1R102Q, 2.8 ± 1.0.
The 3rd sweep: untransfected, 3.3 ± 0.8*; GFP, 8.2 ± 1.6; NCS-1, 11.3 ± 3.7;
NCS-1G2A, 2.1 ± 1.0*; NCS-1E120Q, 8.8 ± 2.3; NCS-1R102Q, 1.1 ± 0.5*. Fig. 6 B shows the average of total area (fC) per sweep per cell in different groups. The 1st sweep:
untransfected, 3739.4 ± 1134.2; GFP, 4407.5 ± 960.5; NCS-1, 8915.2 ± 3640.2;
NCS-1G2A, 2784.9 ±1305.4; NCS-1E120Q, 10248.9 ± 2975.4; NCS-1R102Q, 2908.6 ± 1894.0. The 2nd sweep: untransfected, 3690.0 ± 1006.5*; GFP, 8107.0 ± 2207.2;
NCS-1, 9240.1 ± 4909.8; NCS-1G2A, 1554.2 ± 614.6; NCS-1E120Q, 13435.4 ± 3128.5;
NCS-1R102Q, 2078.6 ± 933.5. The 3rd sweep: untransfected, 3361.5 ± 924.6*; GFP, 7570.0 ± 1604.3; NCS-1, 12436.1 ± 4836.9; NCS-1G2A, 1702.8 ± 860.9*; NCS-1E120Q, 11894.4 ± 4029.7; NCS-1R102Q, 600.6 ± 314.9*. These experiment groups have no significant change when compare with the GFP group in the 1st sweep and the 2nd
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sweep. Cells overexpressing NCS-1G2A or NCS-1R102Q have relative fewer spikes and total area in the 3rd sweep. The sample size of spike numbers: untransfected, 519; GFP, 788; NCS-1, 465; NCS-1G2A, 118; NCS-1E120Q, 642; NCS-1R102Q, 100. The sample size of cell numbers: untransfected, 46; GFP, 32; NCS-1, 16; NCS-1G2A, 13;
NCS-1E120Q, 20; NCS-1R102Q, 13.
3.5 Cells overexpressing NCS-1
G2Ahave a relative uniform distribution on the
exocytosis events
To know the occurring timing of spikes, I draw a spikes accumulation figure. The sample size of spike numbers: untransfected, 519; GFP, 788; NCS-1, 465; NCS-1G2A, 118; NCS-1E120Q, 642; NCS-1R102Q, 100. Fig.7. shows that in untransfected group, GFP group, NCS-1group, NCS-1E120Q group and NCS-1R102Q group, there are about 80 % spikes occurred during 0 s-12 s in the recoding which last for 30 s; the curves are exponential distribution. But the curve of NCS-1G2A group is near linear; it has a relative uniform distribution of spikes.
3.6 The traces of calcium imaging
To measure the intracellular Ca2+ concentration ([Ca2+]i) changes, the cell was loaded Ca2+ sensitive dye, Fura-2, and repetitively stimulated with High K+ for 3 s
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with an interval of 2 min. The cells were record the fluorescent emission of the excitation at both 340 nm and 380 nm and the measurements were created a 340 / 380 ratio. Fig. 8 A - F show the representative traces of fluorescence intensity ratio from the different treatments for 3 stimulations during 380 s. In this thesis, I focused on the fluorescence intensity changes which were measured from baseline to the peak height;
generally the NCS-1G2A group has smallest changes. The details of statistics were mentioned in the following section.
3.7 Cells overexpressing NCS-1
G2Areduces calcium response
Fig. 9 shows the average of fluorescence intensity change (ΔF) per stimulation per cell in different groups. The 1st stimulation: untransfected, 2.4 ± 0.2; GFP, 1.6 ± 0.4; NCS-1, 1.4 ± 0.2; NCS-1G2A, 0.6 ± 0.2*; NCS-1E120Q, 1.6 ± 0.3; NCS-1R102Q, 1.6
± 0.3. The 2nd stimulation: untransfected, 1.8 ± 0.2; GFP, 1.5 ± 0.3; NCS-1, 1.1 ± 0.2;
NCS-1G2A, 0.4 ± 0.2*; NCS-1E120Q, 1.3 ± 0.2; NCS-1R102Q, 1.2 ± 0.3. The 3rd stimulation: untransfected, 1.6 ± 0.2; GFP, 1.3 ± 0.3; NCS-1, 0.9 ± 0.2; NCS-1G2A, 0.3
± 0.1*; NCS-1E120Q, 1.2 ± 0.3 ; NCS-1R102Q, 0.91 ± 0.2. The sample size of cell numbers: untransfected, 19; GFP, 9; NCS-1, 23; NCS-1G2A, 11; NCS-1E120Q, 10;
NCS-1R102Q, 13. As can be seen in the graph, all of these groups have the similar fluorescence intensity changes except the NCS-1G2A group. Cells expressing
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NCS-1G2A have significant low calcium response every stimulation.
3.8 The relationship between Ca
2+response and exocytosis
Fig. 10 A and B plots the changes in the Ca2+ against the total spike numbers and summarized area, respectively, during the first stimulation. The arrangement of the 6 points is not linear, and there are a main group includes GFP, NCS-1and NCS-1E120Q. The relationship between exocytosis and Ca2+ response is indirect in the regulation of NCS-1 and the other mutants.
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