Actin recruitment and disassembly are driven by the activity of Rho1. (a–b′) Schematic drawing (a) and confocal microscope image (b) of a Drosophila third-instar salivary gland expressing the glue protein Sgs3-GFP (green) and the F-actin marker LifeAct-Ruby (red; A). (b′) Enlargement of boxed area in b. Large secretory cells surround an expanding lumen (L) and are filled with secretory vesicles (SV) containing the glue cargo. During secretion, vesicles fuse to the actin-rich apical membrane (AM) facing the lumen. Fused secretory vesicles (FSV) form an actomyosin coat (A) that enables contraction and expulsion of their content into the lumen. (c) A secreting gland displays active Rho1 (visualized by the sensor Ani-RBD-GFP [Munjal et al., 2015], green) and F-actin (red) enriched on vesicles that fused to the apical membrane, at various stages of contraction. (d) Time course of a single secreting vesicle. The vesicle contracts fully within 3 min, with residual actin cleared from the site of fusion after ∼1 additional minute. (d′) Kymograph monitoring active Rho1 and F-actin dynamics at the vesicle/plasma membrane interface (dashed-box area in initial panel of d). Time progresses downward as indicated by black arrow. Dynamics of actin clearing are difficult to characterize because of drastic morphological changes. (e) Addition of the ROCK inhibitor Y-27632 causes accumulation of contraction-halted vesicles, allowing unperturbed visualization of active Rho1 and actin dynamics (see also Video 1). Halted vesicles exhibit active Rho1 and actin coat enrichment, followed by Rho1 inactivation and actin coat disassembly, in a time frame comparable to that of a squeezing vesicle. (e′) Kymographs monitoring active Rho1 and F-actin dynamics at the vesicle surface (dashed-box area in initial panel of e). (e″) Graph plotting relative intensity of active Rho1 (green) and F-actin (red) in the area monitored in the kymographs (e′) over time. Intensity is normalized to the highest value within each channel. Note that both active Rho1 and actin rise sharply, but actin disassembly is gradual whereas Rho1 inactivation is sharp. (f) Dynamic profiles over time of active Rho1 and F-actin in 38 vesicles from a single contraction-halted gland. Vesicle time scales are aligned to intensity peaks. Z-score is defined as the number of standard deviations that the signal intensity differs from background levels. The duration and dynamics of active Rho1 and actin are highly similar between vesicles, despite large variations in z-score. Bars: (b, b′, and c) 20 µm; (d and e) 2 µm.