Distinct effect of actin cytoskeleton disassembly on exo- and endocytic events in a membrane patch of rat melanotrophs

Abstract

We used the cell-attached mode of patch-clamp technique to measure discrete attofarad steps in membrane capacitance (C(m)), reporting area changes in the plasma membrane due to unitary exocytic and endocytic events. To investigate the role of the actin cytoskeleton in elementary exocytic and endocytic events, neuroendocrine rat melanotrophs were treated with Clostridium spiroforme toxin (CST), which specifically depolymerises F-actin. The average amplitude of exocytic events was not significantly different in control and in CST-treated cells. However, the amplitude of endocytic events was significantly smaller in CST-treated cells as compared to controls. The frequency of exocytic events increased by 2-fold in CST-treated cells relative to controls. In control cells the average frequency of exocytic events (upsilon;(exo)) was lower than the frequency of endocytic events (upsilon;(endo)) with a ratio upsilon;(exo)/upsilon;(endo) < 1. In the toxin treated cells, the predominant process was exocytosis with a ratio (upsilon;(exo)/upsilon;(endo) > 1). To study the coupling between the two processes, the slopes of regression lines relating upsilon;(exo) and upsilon;(endo) in a given patch of membrane were studied. The slopes of regression lines were similar, whereas the line intercepts with the y-axis were significantly different. The increased frequency of unitary exocytic events in CST-treated cells is consistent with the view, that the actin cytoskeleton acts as a barrier for exocytosis. While the disassembly of the actin cytoskeleton diminishes the size of unitary endocytic events, suggesting an important role of the actin cytoskeleton in determining the size of endocytic vesicles, the coupling between exocytosis and endocytosis in a given patch of membrane was independent of the state of the actin cytoskeleton.

Figure 1. Phalloidin-stained actin cytoskeleton in melanotroph cell

Confocal micrographs of phalloidin-stained actin cytoskeleton in a control (left) and a cell pretreated with CST.

Figure 2. Representative recordings of unitary exocytic and endocytic events

Representative recordings of exocytic (A, C) and endocytic (B, D) events in control cells (left column) and in CST-treated cells (right column). * denotes a calibration pulse used to determine the correct phase separation of the lock-in amplifier. Note that there is no projection of this pulse between the top and bottom traces in panel C. C_m stands for the imaginary part of admittance (proportional to membrane capacitance) and G_a to the real part of admittance of a cell-attached recording.

Figure 3. The amplitude of exocytic and endocytic events

The average amplitude of exocytic (left) and endocytic (right) events in control and CST-treated cells. Numbers adjacent to columns represent a number of events. * Statistically significant (P < 0.002). Bars show s.e.m.

Figure 4. The frequency of exocytic and endocytic events

A, the average frequency of exocytic events (left) and endocytic events (right) in control and CST-treated cells. B, the ratio between frequencies of exocytic and endocytic events in control and CST-treated cells. Numbers adjacent to columns represent numbers of cells examined. * Statistically significant difference (P < 0.02).

Figure 5. Relationship between the frequencies of exocytic and endocytic events

The relationship between the frequency of exocytic and the frequency of endocytic events in control (○) and in CST-treated (•) cells. Regression lines (obtained using SPSS SigmaPlot software) were drawn according to the equations depicted on the figure. Note that the intercepts, but not the slopes of lines, are significantly different (P < 0.01). Moreover, the slope of the CST-treated cells is significantly different from the slope coefficient vaalue 2 (P < 0.01).

Figure 6. Images of cytosolic [Ca²⁺] in control and CST-treated melanotrophs

A, representative ratio fura-2 image of cells showing cytosolic [Ca²⁺]_i in control conditions, and B, 1 h after the addition of the CST. Note that the average [Ca²⁺]_i has not changed significantly after the CST treatment in comparison to a rise in ionomycin-induced rise in [Ca²⁺]_i (C). Panel on the right indicates the colour-coded concentration of [Ca²⁺].

Figure 7. Two models of coupling between exocytosis and endocytosis

A, the same membrane added to the plasma membrane by exocytosis is retrieved by endocytosis (‘kiss-and-run’). B, exocytosed membrane appears to be loosely coupled to endocytosis that occurs in a different membrane microdomain. * denotes an inhibitory action of CST on the size of endocytic vesicles, whereas + denotes an increase and - a decrease in the frequency of events by the CST-treatment.