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. 2012 Mar 14;32(11):3778–3785. doi: 10.1523/JNEUROSCI.3540-11.2012

Figure 1.

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).