Figure 2.
Figure 2. RhoGAP71E is required for Rho1 inactivation. A candidate-based RNAi screen identified RhoGAP71E as a regulator of Rho1 inactivation in the salivary gland. (a) Contraction-halted glands expressing the sensor for active Rho1 (green) and LifeAct-Ruby (red) along with RhoGAP71E RNAi (VDRC109797) show sustained active Rho1 and actin coat. (a′) Kymographs 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 kymographs (a′) over time. Intensity is normalized to the highest value within each channel. Note that both active Rho1 and actin rise sharply and are sustained on the vesicle for >20 min. (b) Dynamic profiles over time of active Rho1 and F-actin in 29 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. Note that although Rho1 and actin rise sharply, they are sustained above initial levels over many minutes (compare time scale to Fig. 1 f). (c) Glands expressing RhoGAP71E-GFP, an endogenously expressed GFP-tagged version of RhoGAP71E (green) and LifeAct-Ruby (red). RhoGAP71E-GFP appears to be enriched in most actin-coated vesicles. (d) A single secreting vesicle in such a gland expressing RhoGAP71E-GFP (green) and LifeAct-Ruby (red), with TMR-dextran (red) injected into the lumen to mark fused vesicles. Yellow arrowhead marks appearance of dextran within the vesicle, preceding formation of the bright actin coat on the vesicle surface. RhoGAP71E-GFP and actin appear to be simultaneously enriched on vesicles within 30 s after vesicle fusion. (e) Glands expressing RhoGAP71E-GFP (green) and LifeAct-Ruby (red). Addition of the F-actin polymerization inhibitor LatA to the medium caused loss of both actin coat formation and RhoGAP71E recruitment to vesicles. Time corresponds to time elapsed since LatA addition. (f) A single secreting vesicle in such a LatA-treated gland expressing RhoGAP71E-GFP (green) and LifeAct-Ruby (red), injected with TMR-dextran (red) into the lumen. Yellow arrowhead marks appearance of dextran and onset of fusion. Although vesicles still fuse 17 min after LatA addition, actin and RhoGAP71E-GFP are not recruited to newly fused vesicles, demonstrating that RhoGAP71E recruitment requires F-actin. (g) LatA-treated glands expressing the active Rho1 sensor (green) and LifeAct-Ruby (red). Unlike RhoGAP71E-GFP, active Rho1 is enriched on a large subset of vesicles, despite the failed recruitment of actin. (h) A single secreting vesicle in such a LatA-treated gland expressing active Rho1 (green) and LifeAct-Ruby (red), and injected with TMR-dextran (red) into the lumen. Active Rho1 is recruited and sustained on the fused vesicle, with a progressively punctate pattern forming over time. In addition, compound vesicle fusion leads to formation of abnormally large vesicles. Under these conditions, active Rho1 is sustained for at least 8 min in ∼39% of vesicles (n = 85 in four glands; see also Fig. S3), implying lack of Rho1 inactivation in the absence of actin and RhoGAP71E. Bars: (a, d, f, and h) 2 µm; (c, e, and g) 20 µm.

RhoGAP71E is required for Rho1 inactivation. A candidate-based RNAi screen identified RhoGAP71E as a regulator of Rho1 inactivation in the salivary gland. (a) Contraction-halted glands expressing the sensor for active Rho1 (green) and LifeAct-Ruby (red) along with RhoGAP71E RNAi (VDRC109797) show sustained active Rho1 and actin coat. (a′) Kymographs 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 kymographs (a′) over time. Intensity is normalized to the highest value within each channel. Note that both active Rho1 and actin rise sharply and are sustained on the vesicle for >20 min. (b) Dynamic profiles over time of active Rho1 and F-actin in 29 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. Note that although Rho1 and actin rise sharply, they are sustained above initial levels over many minutes (compare time scale to Fig. 1 f). (c) Glands expressing RhoGAP71E-GFP, an endogenously expressed GFP-tagged version of RhoGAP71E (green) and LifeAct-Ruby (red). RhoGAP71E-GFP appears to be enriched in most actin-coated vesicles. (d) A single secreting vesicle in such a gland expressing RhoGAP71E-GFP (green) and LifeAct-Ruby (red), with TMR-dextran (red) injected into the lumen to mark fused vesicles. Yellow arrowhead marks appearance of dextran within the vesicle, preceding formation of the bright actin coat on the vesicle surface. RhoGAP71E-GFP and actin appear to be simultaneously enriched on vesicles within 30 s after vesicle fusion. (e) Glands expressing RhoGAP71E-GFP (green) and LifeAct-Ruby (red). Addition of the F-actin polymerization inhibitor LatA to the medium caused loss of both actin coat formation and RhoGAP71E recruitment to vesicles. Time corresponds to time elapsed since LatA addition. (f) A single secreting vesicle in such a LatA-treated gland expressing RhoGAP71E-GFP (green) and LifeAct-Ruby (red), injected with TMR-dextran (red) into the lumen. Yellow arrowhead marks appearance of dextran and onset of fusion. Although vesicles still fuse 17 min after LatA addition, actin and RhoGAP71E-GFP are not recruited to newly fused vesicles, demonstrating that RhoGAP71E recruitment requires F-actin. (g) LatA-treated glands expressing the active Rho1 sensor (green) and LifeAct-Ruby (red). Unlike RhoGAP71E-GFP, active Rho1 is enriched on a large subset of vesicles, despite the failed recruitment of actin. (h) A single secreting vesicle in such a LatA-treated gland expressing active Rho1 (green) and LifeAct-Ruby (red), and injected with TMR-dextran (red) into the lumen. Active Rho1 is recruited and sustained on the fused vesicle, with a progressively punctate pattern forming over time. In addition, compound vesicle fusion leads to formation of abnormally large vesicles. Under these conditions, active Rho1 is sustained for at least 8 min in ∼39% of vesicles (n = 85 in four glands; see also Fig. S3), implying lack of Rho1 inactivation in the absence of actin and RhoGAP71E. Bars: (a, d, f, and h) 2 µm; (c, e, and g) 20 µm.

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