Article Figures & Data
Figures
- Figure 1.
CME of single vesicles and the fission-pore analysis. A, Simultaneous recordings of Re and Im by a cell-attached patch pipette in the double (cell-attached/whole-cell) patch configuration showed upward capacitance steps associated with exocytosis and downward capacitance steps associated with endocytosis. B, The occurrence of individual endocytic events as a function of time for each of 15 patches with at least six endocytic events. Each dot represents an individual endocytic event and the vertical dotted line indicates the onset of stimulation. C, Distribution of vesicle diameters derived from endocytic capacitance step sizes. The mean vesicle diameter is 122 ± 2.7 nm (n = 238 events). D, E, Downward steps in Im associated with single vesicle endocytosis in a control cell (D) were largely abolished in a cell treated with 240 μm anti-clathrin heavy chain antibody (E). F, Statistical analysis demonstrating that applications of anti-clathrin heavy chain antibody through the whole-cell patch pipette significantly decreased the number of endocytic events recorded within 5 min by the cell-attached patch pipette (Control: n = 48 patches; 240 μg/ml Control IgG: n = 40 patches, p > 0.05; 60 μg/ml anti-clathrin antibody: n = 75 patches, p < 0.01; 120 μg/ml anti-clathrin antibody: n = 28 patches, p < 0.01; 240 μg/ml anti-clathrin antibody: n = 30 patches, p < 0.01). **p < 0.01. G, Analysis of an individual fission-pore closure event. The traces from top to bottom show the time course of: Re, Im, and fission-pore conductance Gp. Cv is indicated in Im trace. H, The dotted box in G is shown on an expanded scale. The fission-pore kinetics were characterized by the fission-pore conductance Gp, and the duration as indicated. I–K, Application of anti-clathrin antibody at 60 μg/ml through a whole-cell patch pipette did not alter the mean Cv of the endocytic vesicles (Control: 0.87 ± 0.08 fF, n = 47 events; Anti-clathrin antibody: 0.88 ± 0.08 fF, n = 45 events, p > 0.05) (I), the fission-pore conductance Gp (Control: 173 ± 18 pS; Anti-clathrin antibody: 166 ± 21 pS, p > 0.05) (J), and the fission-pore duration (Control: 197 ± 33 ms; Anti-clathrin antibody: 188 ± 28 ms, p > 0.05) (K).
- Figure 2.
Dependence of endocytic event properties on [Ca2+]e. A, The number of endocytic events within 5 min of cell-attached recordings was reduced by ∼70% at 0.5 mm [Ca2+]e (n = 102 patches) (p < 0.01) and by ∼95% at zero [Ca2+]e (n = 46 patches) (p < 0.01), compared to 2 mm [Ca2+]e (n = 95 patches). Zero [Ca2+]e was achieved through a combination of removal of Ca2+ and addition of 5 mm EGTA. B, The number of amperometric spikes at 0.5 mm [Ca2+]e (n = 18 cells) was reduced compared to 2 mm [Ca2+]e (n = 18 cells) (p < 0.01). The experiments in A and B were performed on different cells from the same preparations. C, The endocytosis/exocytosis ratio did not differ statistically between 2 and 0.5 mm [Ca2+]e (p > 0.05). D, Representative fission-pore events at 2 mm (left) and 0.5 mm [Ca2+]e (right) in the cell-attached patch configuration. E, The mean capacitance step size of endocytic vesicles was indistinguishable between these two groups (2 mm [Ca2+]e: n = 43 events; 0.5 mm [Ca2+]e: n = 42 events) (p > 0.05). F, The fission-pore Gp was independent of [Ca2+]e (p > 0.05). G, The fission-pore duration was significantly increased (p < 0.01) at 0.5 mm [Ca2+]e. **p < 0.01.
- Figure 3.
The fission-pore duration is prolonged and its Ca2+ dependence is abolished in Syt1 KO cells. A, Representative fission-pore events at 2 mm [Ca2+]e in WT (left) and Syt1 KO cells (right) recorded in the cell-attached patch configuration. B, The number of endocytic events within 5 min of recordings was reduced in Syt1 KO cells (n = 281 patches) compared to WT cells (n = 89 patches) (p < 0.01). C, The number of amperometric spikes was decreased in Syt1 KO cells (WT: n = 21 cells; KO: n = 21 cells) (p < 0.05). The experiments in B and C were performed on different cells from the same preparations. D, The endocytosis/exocytosis ratio was also reduced in Syt1 KO cells (p < 0.01). E–G, In Syt1 KO cells, Cv of the endocytic vesicles (E) and the fission-pore conductance Gp (F) were indistinguishable between WT (n = 52 events) and KO cells (n = 43 events) (p > 0.05), but the fission-pore duration was significantly increased (p < 0.01) (G). H, The fission-pore duration was significantly dependent on [Ca2+]e in WT cells (2 mm: n = 44 events, 1 mm: n = 49 events, p < 0.01; 0.5 mm: n = 46 events, p < 0.01) but not in Syt1 KO cells (2 mm: n = 42 events, 1 mm: n = 50 events, p > 0.05; 0.5 mm: n = 39 events, p > 0.05). *p < 0.05; **p < 0.01.
- Figure 4.
The fission-pore kinetics remain unaltered on disruption in Syt1–AP-2 interaction in double (cell-attached/whole-cell) patch configuration. KR-peptide, Scr-peptide, or AA-peptide was delivered into WT cells through the whole-cell patch pipette in double (cell-attached/whole-cell) patch configuration. A, The number of endocytic events within 5 min of recordings at 2 mm [Ca2+]e was reduced by KR-peptide (25 μm: n = 129 patches, p < 0.01; 100 μm: n = 25 patches, p < 0.01) but not by Scr-peptide (25 μm: n = 97 patches, p > 0.05; 100 μm: n = 24 patches, p > 0.05) or by AA-peptide (25 μm: n = 133 patches, p > 0.05; 100 μm: n = 30 patches, p > 0.05) compared to control (n = 44 patches). B, Number of amperometric spikes indicating exocytotic events did not change in cells treated with KR-peptide (25 μm: n = 27 cells; 100 μm: n = 17 cells), Scr-peptide (25 μm: n = 21 cells; 100 μm: n = 19 cells), or AA-peptide (25 μm: n = 16 cells; 100 μm: n = 14 cells) compared to control cells (n = 27 cells) (p > 0.05). The experiments in A and B were performed in different cells from the same preparations. C, Statistical analysis showed that the endocytosis/exocytosis ratio at 2 mm [Ca2+]e was inhibited by KR-peptide (p < 0.01) but not Scr-peptide (p > 0.05) or AA-peptide (p > 0.05), indicating a role of Syt1–AP-2 interaction in regulating the endocytic capacity of CME. D, Cv of the endocytic vesicles was not changed by 25 μm KR-peptide, Scr-peptide, or AA-peptide at both 1 (Control: n = 52 events; KR-peptide: n = 47 events; Scr-peptide: n = 43 events; AA-peptide: n = 41 events) (p > 0.05) and 2 mm [Ca2+]e (Control: n = 46 events; KR-peptide: n = 44 events; Scr-peptide: n = 40 events; AA-peptide: n = 53 events) (p > 0.05). E, The fission-pore Gp was comparable among these eight groups (p > 0.05). F, Treatments with either KR-peptide, Scr-peptide, or AA-peptide produced no effects on the fission-pore duration at both 2 and 1 mm [Ca2+]e (p > 0.05), indicating that disruption in Syt1–AP-2 interaction does not affect the Ca2+ dependence of fission-pore duration. **p < 0.01.
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