Localization of RGA-3/4 to the germinal vesicle, meiotic cytokinetic apparatus, nucleus, and mitotic cytokinetic apparatus in starfish. (A) Localization of GAP-dead RGA-3/4 (mNeon-RGA-3/4^R96E; cyan) and F-actin (mCherry-UtrCH; orange) to the germinal vesicle and cortex of the immature starfish oocyte, respectively. Scale bar = 50 µm. (B–B″) Time course of mNeon-RGA-3/4^R96E localization during second meiosis. Time in min:s; scale bar = 50 µm. Insets (B′ and B″) enlarge nascent cytokinetic apparatus 2.5× as indicated by boxes; scale bar = 10 µm. (C) mNeon-RGA-3/4^R96E recruitment to the equatorial cortex during cytokinesis and corresponding kymographs. Note low-amplitude wavelets throughout furrow ingression. x scale bar = 50 µm; y scale bar = 2 min. (D) mNeon-RGA-3/4^R96E localization in cleaving blastomeres of 32-cell starfish embryo; *, cells in interphase; o, cells in early anaphase (note cortical accumulation of mNeon-RGA-3/4^R96E compared with interphase cells); ∞, cells that have commenced cytokinesis; arrowheads, reforming nuclei. Time in min:s. Scale bar = 50 µm. (E) Cross-correlational analysis of a starfish cell expressing mNeon-RGA-3/4^WT and mCherry-rGBD showing a 15-s delay between peak Rho activity and RGA-3/4^WT recruitment. Corresponds to experiments shown in Fig. 1 C (75 ng/μl). (E′) Representative intensity profile of active Rho and RGA-3/4^R96E. (F–H) Quantification of period (F), temporal width (G), and relative amplitude (H) for experiments shown in Fig. 1 E. Each dot represents a single oocyte; group mean ± 95% confidence interval; 0 ng/μl, n = 16; 22 ng/μl, n = 16; 66 ng/μl, n = 15; two experiments. One-way ANOVA with Tukey post hoc test for multiple comparisons; data distribution was assumed to be normal but was not formally tested; **, P < 0.01; ****, P < 0.0001.