Figure 7.
Recruitment of RHGF-1 to F-actin is enhanced by myosin contractility. (A) Maximum intensity projections and middle plane images of GFP::ACT-1 and RHGF-1::tagRFP in spermathecae from control (empty vector), mel-11(RNAi), rho-1(RNAi), fln-1(RNAi), and nmy-1(RNAi) treated worms. Scale bar, 10 µm. (B) Quantification of F-actin bundle tortuosity in each of the conditions shown in A. (C) Quantification of the ratio of RHGF-1 intensity between the basal side of the cell and in the cytoplasm in each of the conditions shown in A. For both B and C: N ≥ 10. Error bars are mean ± SEM. Statistical comparisons were performed by Brown-Forsythe ANOVA-Dunnett’s T3 multiple comparisons test. Stars designate statistical significance (**** P <0.0001, *** P <0.001). (D) Tortuosity as a function of RHGF-1 enrichment at the basal side. Black line is an interpolation using the “Sigmoidal, 4PL, X is log(concentration)” function in GraphPad. Statistical assay was correlation, r = −0.76, P <0.0001. N = 50. N, number of samples analyzed. (E) Schematic model of the mechanosensitive regulation of RHO-1 in the spermatheca, combining the results on RHGF-1 from this study with our previous work on SPV-1 (Tan and Zaidel-Bar, 2015). In the empty spermatheca (left), RHGF-1 is mostly inactive in the cytoplasm, and RHO-1 is mostly inactive due to the GAP activity of SPV-1, which localizes to the apical membrane. Following ovulation, the spermatheca is filled with an oocyte (top) and its membranes and F-actin become stretched. Membrane stretching causes SPV-1 to become cytoplasmic and inactive, while F-actin stretching leads to recruitment of RHGF-1, which activates RHO-1. After fertilization (right), the level of active RHO-1 continues to increase due to a positive feedback loop involving tension-dependent RHGF-1 recruitment, RHO-1 activation, and RHO-1–induced myosin contractility. Finally, when the level of RHO-1 activation and actomyosin contractility reach the level required to squeeze the embryo out of the spermatheca (bottom), emptying of the spermatheca occurs. The loose spermatheca apical membrane folds and recruits SPV-1, which inactives RHO-1, therefore actin becomes under less tension, RHGF-1 detaches, and the cycle is completed.

Recruitment of RHGF-1 to F-actin is enhanced by myosin contractility. (A) Maximum intensity projections and middle plane images of GFP::ACT-1 and RHGF-1::tagRFP in spermathecae from control (empty vector), mel-11(RNAi), rho-1(RNAi), fln-1(RNAi), and nmy-1(RNAi) treated worms. Scale bar, 10 µm. (B) Quantification of F-actin bundle tortuosity in each of the conditions shown in A. (C) Quantification of the ratio of RHGF-1 intensity between the basal side of the cell and in the cytoplasm in each of the conditions shown in A. For both B and C: N ≥ 10. Error bars are mean ± SEM. Statistical comparisons were performed by Brown-Forsythe ANOVA-Dunnett’s T3 multiple comparisons test. Stars designate statistical significance (**** P <0.0001, *** P <0.001). (D) Tortuosity as a function of RHGF-1 enrichment at the basal side. Black line is an interpolation using the “Sigmoidal, 4PL, X is log(concentration)” function in GraphPad. Statistical assay was correlation, r = −0.76, P <0.0001. N = 50. N, number of samples analyzed. (E) Schematic model of the mechanosensitive regulation of RHO-1 in the spermatheca, combining the results on RHGF-1 from this study with our previous work on SPV-1 (Tan and Zaidel-Bar, 2015). In the empty spermatheca (left), RHGF-1 is mostly inactive in the cytoplasm, and RHO-1 is mostly inactive due to the GAP activity of SPV-1, which localizes to the apical membrane. Following ovulation, the spermatheca is filled with an oocyte (top) and its membranes and F-actin become stretched. Membrane stretching causes SPV-1 to become cytoplasmic and inactive, while F-actin stretching leads to recruitment of RHGF-1, which activates RHO-1. After fertilization (right), the level of active RHO-1 continues to increase due to a positive feedback loop involving tension-dependent RHGF-1 recruitment, RHO-1 activation, and RHO-1–induced myosin contractility. Finally, when the level of RHO-1 activation and actomyosin contractility reach the level required to squeeze the embryo out of the spermatheca (bottom), emptying of the spermatheca occurs. The loose spermatheca apical membrane folds and recruits SPV-1, which inactives RHO-1, therefore actin becomes under less tension, RHGF-1 detaches, and the cycle is completed.

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