Rhythmic opening and closing of vesicles during constitutive exo- and endocytosis in chromaffin cells

Abstract

Constitutive exo- and endocytic events are expected to increase and diminish the cell surface area in small spontaneous steps. Indeed, cell-attached patch-clamp measurements in resting chromaffin cells revealed spontaneous upward and downward steps in the electrical capacitance of the plasma membrane. The most frequent step size indicated cell surface changes of <0.04 microm(2), corresponding to vesicles of <110 nm diameter. Often downward steps followed upward steps within seconds, and vice versa, as if vesicles transiently opened and closed their lumen to the external space. Transient openings and closings sometimes alternated rhythmically for tens of seconds. The kinase inhibitor staurosporine dramatically increased the occurrence of such rhythmic episodes by making vesicle closure incomplete and by inhibiting fission. Staurosporine also promoted transient closures of large endocytic vesicles possibly representing remnants of secretory granules. We suggest that staurosporine blocks a late step in the endocytosis of both small and large vesicles, and that endocytosis involves a reaction cascade that can act as a chemical oscillator.

Shape bistability of a membrane neck: A toggle switch to control vesicle content release

“…Our measurements of the cell membrane neck conductance reveal stepwise repetitive changes of the conductance, similar to those obtained on the BLM tubule. In retrospect, we found a qualitatively similar behavior in the conductance between fusing or fissioning compartments in data on various cell systems (17,(19)(20)(21)33). Overall, the seemingly common ability of a cell membrane connection to switch between two states with high and low permeability substantiates the hypothesis of shape bistability of cellular necks.…”

“…Fig. 4A shows that in IC-21 cells, both upward and downward capacitance changes were often reversible; i.e., the capacitance ''flickered'' (17,(19)(20)(21)33). The occurrence of such oscillations was Ϸ15% of all detected fusion͞fission events (50 of 346 total).…”

“…The permeability of cell membrane necks (also referred to as ''fission pores'') has been studied only in a few systems (17)(18)(19)(20). One of the most intriguing findings is the pore ''flicker,'' fluctuations of the pore conductance between high and low level (17,(19)(20)(21). IC-21 cells, capable of endocytosis and phagocytosis (22,23), can form relatively large endocytic granules, close in size to the smallest end-ring in the BLM system.…”

Regulation of transferrin-induced endocytosis by wild-type and C282Y-mutant HFE in transfected HeLa cells

“…In cell-attached configuration, an 8-kHz sine wave of 280 mV peak to peak was applied to the patch membrane, and the data points were sampled at 350 Hz. Phase settings for cell-attached measurements were done as recently described (17). In whole cell recordings, we adjusted the phase angle by short, repetitive resistance changes between the bath electrode and the EPC-7 head stage until no deflections were seen in the capacitance trace.…”

“…Fast capacitance steps in cell-attached measurements were detected by using an automated algorithm, as previously described (17). Only solitary steps with amplitudes between 0.15 and 2 femtofarads (fF) were included in our data set when the capacitance noise within 150-ms sections before and after the step was Ͻ50 attofarads (aF).…”