Coordinated oscillations of active Rho1, F-actin, and RhoGAP71E. (a) Coordinated oscillatory behavior of the active Rho1 sensor (green) and LifeAct-Ruby (red) on a single contraction-halted vesicle from a Y-27632–treated salivary gland. (a′) Kymograph monitoring active Rho1 and F-actin dynamics at the vesicle surface (dashed-box area in initial panel of a). (a″) Graph plotting relative intensity of active Rho1 (green) and F-actin (red) in the area monitored in the kymograph (a′) over time. Intensity is normalized to the highest value within each channel. (b) Histogram showing the time in minutes between peaks of actin accumulation in oscillating vesicles (n = 43 in four glands). Oscillations most frequently reach their maximum ∼6 min after the previous peak, similar to the time scale of a single actin cycle. Of vesicles that were tracked for at least 15.5 min (defined by 90th percentile of oscillation peaks +3 s), 52.9% displayed at least one oscillation in addition to their first peak (n = 34 in four glands). (c) Coordinated oscillatory behavior of RhoGAP71E-GFP (green) and LifeAct-Ruby (red) on a single contraction-halted vesicle from a Y-27632–treated salivary gland. Coordinated dynamics of actin and RhoGAP71E was observed in 78% of oscillating vesicles (n = 18 in three glands). (c′) Kymograph monitoring RhoGAP71E-GFP and F-actin dynamics at the vesicle surface (dashed-box area in initial panel of c). (c″) Graph plotting relative intensity of RhoGAP71E-GFP (green) and F-actin (red) in the area monitored in the kymograph (c′) over time. Intensity is normalized to the highest value within each channel. Note that RhoGAP71E-GFP and actin appear and disappear in a similar temporal pattern, consistent with a role for F-actin in RhoGAP71E recruitment. Bars, 2 µm.