F-actin and myosin II accelerate catecholamine release from chromaffin granules

Abstract

The roles of nonmuscle myosin II and cortical actin filaments in chromaffin granule exocytosis were studied by confocal fluorescence microscopy, amperometry, and cell-attached capacitance measurements. Fluorescence imaging indicated decreased mobility of granules near the plasma membrane following inhibition of myosin II function with blebbistatin. Slower fusion pore expansion rates and longer fusion pore lifetimes were observed after inhibition of actin polymerization using cytochalasin D. Amperometric recordings revealed increased amperometric spike half-widths without change in quantal size after either myosin II inhibition or actin disruption. These results suggest that actin and myosin II facilitate release from individual chromaffin granules by accelerating dissociation of catecholamines from the intragranular matrix possibly through generation of mechanical forces.

Figure 1.

A, Confocal micrograph of a representative cell used for vesicle tracking. Vesicles were labeled with Lysotracker green for 5 min and then imaged at 25°C without stimulation. Scale bar, 20 μm. B, Trace of typical x–y coordinates followed by a single vesicle taken from control cells. C, Plots of the two dimensional MSD calculated for control and blebbistatin-treated cells. Data are presented as mean ± SEM for 94 vesicles from 9 control cells, and 92 vesicles from 10 blebbistatin-treated cells.

Figure 2.

Effects of inhibition of actin polymerization and myosin II function on cortical actin distribution. A, Confocal micrographs of chromaffin cells in control condition, and treated with 10 μm blebbistatin or 4 μm cytochalasin D. Cells were fixed after 30 min incubation with the inhibitor and stained for F-actin with Alexa 568 phalloidin. Scale bar, 20 μm. B, Schematic depicts the quantification of cortical actin. A circular region of interest inside the cell was subtracted from another region covering the entire cell. The annular width was kept equal to 1.5 μm for all cells. C, Quantified fluorescence of Alexa 568 phalloidin labeled F-actin on the cortical region. Fluorescence values were normalized to the mean value for control cells. Data are presented as mean ± SEM from a total of 20 cells per group. Triple asterisks indicate p < 0.001 (Student's unpaired t test).

Figure 3.

A, A typical recording from an untreated chromaffin cell stimulated with ionomycin. B, Spikes from each cell were normalized to their peak amplitude, aligned in time at the point of their maximum slope (occurring shortly before the spike maximum) and averaged, providing the average spike shape for this cell. The average spikes from each cell in a treatment group were again averaged in the same way to obtain the average spike shapes for control (CT, n = 19 cells, 786 spikes), blebbistatin-treated (BL, n = 18 cells, 633 spikes), and cytochalasin D-treated (CD, n = 18 cells, 1229 spikes) cells. Last, the averaged spikes were normalized to the same quantal size. The half-widths of these averaged spikes were 11.9 ms for control, 18.7 ms for blebbistatin, and 26.0 ms for cytochalasin D-treated cells.

Figure 4.

A, A single amperometric spike along with the five parameters: quantal size Q (pC), half-width (ms), peak amplitude (pA), foot signal duration (ms), and mean foot current (pA). B–G, Averaged values for half-width (B), peak amplitude (C), quantal size (D), number of exocytotic events (E), mean foot current (F), and foot signal duration (G) for control (CT, n = 19 cells, 786 spikes), blebbistatin (BL, n = 18 cells, 633 spikes), cytochalasin D (CD, n = 18 cells, 1229 spikes), ML-7 (ML7, n = 20 cells, 388 spikes), and Lat-A (LatA, n = 10 cells) expressed as percentages of control values (always taken to be 100%). Data are represented as mean ± SEM, where n is the number of cells. Differences between treatment groups were tested for statistical significance by Student's unpaired t test and are indicated by single (p < 0.05), double (p < 0.01), or triple (p < 0.001) asterisks.

Figure 5.

A, The real (blue trace) and imaginary (red trace) parts of the complex admittance are converted into fusion pore conductance G_P (green dots) and vesicle capacitance C_V (black dots). The fusion pore initial and average conductance are depicted by the dashed horizontal black lines, while the fusion pore expansion rate is the slope of the linear fit to the initial 15 ms segment of the conductance trace (solid black line). The fusion pore lifetime is the time for the conductance to reach 2 nS from its initial value. B–F, Vesicle step size (B), fusion pore initial conductance (C), fusion pore average conductance (D), fusion pore lifetime (E), and fusion pore initial expansion rate (F) for control (CT, n = 7 cells, 86 fusion pores), blebbistatin-treated (BL, n = 8 cells, 82 fusion pores), and cytochalasin D-treated (CD, n = 8 cells, 78 fusion pores) cells. Data are represented as mean ± SEM, where n is the number of cells. Statistically significant differences (p < 0.05) are indicated by single asterisks.

Figure 6.

A, B, Survival curves for foot signal duration (A) and fusion pore lifetime (B). C, D, Logarithmic plots of survival curves for foot signal duration (C) and fusion pore lifetime (D). A single exponential (dashed lines) and a power law function (dotted lines) were fitted to the data. Black: control (n = 7 cells, 86 fusion pores), green: blebbistatin (n = 8 cells, 82 fusion pores), red: cytochalasin D (n = 8 cells, 78 fusion pores).