Figure 5.

Inhibition of myosin II with blebbistatin interferes with actin ring formation and constriction, and prevents dextran-positive endosomal fission. A–D, Bovine adrenal chromaffin cells transfected with Lifeact-GFP were stimulated with nicotine (100 μm) in the presence or absence of blebbistatin (10 min preincubation at 10 μm) and imaged by time-lapse confocal microscopy at the level of the footprint. A, Representative confocal image of the cortical actin network of a chromaffin cell before (prestimulation) and 8 min after nicotine stimulation (poststimulation) with or without blebbistatin. Note that blebbistatin prevents the formation of actin rings. Scale bar, 5 μm. Analysis of the number of rings formed per cell (B; six control cells; five blebbistatin-treated cells) and their duration (C; n = 52 rings from eight control cells; n = 14 rings from four blebbistatin-treated cells) and their rate of constriction (D; n = 10 rings from three control cells; n = 6 rings from two blebbistatin-treated cells). Note that blebbistatin significantly blocks the formation of rings and prevents their constriction. As a result, the few rings that form fail to constrict. E, Chromaffin cells transfected with Lifeact-GFP were incubated with 70 kDa dextran-TMRh and were stimulated with nicotine (100 μm) in the presence or absence of blebbistatin (10 min preincubation at 10 μm) and imaged by time-lapse confocal microscopy at the level of the footprint. Scale bar, 5 μm. F, Analysis of the number of dextran-positive structures following stimulation (n = 6 control cells and n = 21 blebbistatin-treated cells). G, Dextran-positive structures formed following stimulation in the presence of blebbistatin were photobleached using 100% laser (20 iterations) and were time lapse imaged at 0.5 Hz. All dextran-positive structures fully recovered their fluorescence, indicating persisting continuity with the extracellular dextran (n = 11 Bleached-Recovered dextran-positive structures from three cells; n = 3 Immediate Recovery dextran-positive structures from three cells). H, Plasma membrane portions of the cell were used as a control for recovery. GPI-positive rings that were also positive for Lifeact-RFP actin ring GPI/AR− rings were able to recover to the same extent as GPI/AR+ rings following blebbistatin pretreatment (p > 0.05, ns; n = 6 cells). Error bars are mean ± SEM.