Starfish RGA-3/4 behaves like an actin-dependent Rho inhibitor. (A) Stills and corresponding kymograph from a post-meiosis II (MII) oocyte coexpressing Ect2 (100 ng/μl) and mNeon-RGA-3/4^WT (cyan; 75 ng/μl) as well as mCherry-UtrCH (orange) to detect F-actin. RGA-3/4 and F-actin overlap almost perfectly. RGA-3/4 image and kymograph is from maximum-intensity projection of three z-slices per time point minus 0.9 times the minimum value over a 20–time point sliding window to reduce background autofluorescence. Kymograph position corresponds to ≫. Kymograph x scale also applies to still images; x scale bar = 50 µm; y scale bar = 2 min. Inset from kymograph is a 3× blowup; x scale bar = 10 µm; y scale bar = 30 s. (B) Cross-correlational analysis of a representative starfish cell expressing mNeon-RGA-3/4WT and mCherry-UtrCH showing a 4.8-s delay between peak RGA recruitment peak actin signal. (B′) Representative intensity profile of RGA-3/4^WT and F-actin. (C) Rho activity (GFP-rGBD; cyan) versus F-actin (mCherry-UtrCH; orange) in a post-MII Ect2-loaded oocyte flooded with 200 nm latrunculin B at time 0 (corresponds to Video 4). Before treatment, this cell experienced steady high-amplitude rolling waves; within minutes of treatment, wave amplitude was noticeably enhanced. Black arrow to right of kymograph indicates latrunculin B addition at time 0. Kymograph x scale also applies to still images. x scale bar = 50 µm; y scale bar = 2 min. (D) Same experiment as in C but with mNeon-RGA-3/4^WT (orange) instead of UtrCH (corresponds to Video 5). Dose of Ect2 and RGA-3/4 titrated to induce steady, rolling waves before treatment. After treatment, Rho amplitude is noticeably enhanced, and waves are more closely packed. RGA-3/4 continues to occupy a phase immediately following Rho. (D′) Another oocyte from the same batch, in which the pretreatment behavior is somewhat higher on the excitability spectrum. Treatment likewise enhances wave amplitude, packs waves more tightly, and breaks wave fronts into irregular bursts. RGA-3/4 images background-subtracted as in A. Kymograph x scale in D′ applies to all still images in D–D′. x scale bar = 50 µm; y scale bar = 2 min. (E) GAP-dead RGA-3/4 continues to track F-actin throughout latrunculin treatment. Similar treatment to C and D but with mNeon-RGA-3/4^R96E (left; cyan) and mCherry-UtrCH (right; orange). Times are min:s relative to flooding with 200 nm latrunculin B. RGA-3/4 images are not background-subtracted. Top two panels are from one oocyte, the bottom one from another oocyte in the same treatment batch. Scale bar = 50 µm. Insets from kymographs are 3× blowups; x scale bar = 10 µm; y scale bar = 30 s; applies to all insets. (F–F″) Cleaving embryonic cells coexpressing mNeon-RGA-3/4^R96E and mCherry-UtrCH, the latter underlabeled to avoid interfering with actin-dependent events; a micropipette filled with 0.5% LM agarose + 4 µm latrunculin B is parked next to the cell on the right and moved into position (white asterisk) at time 0; scale bar = 50 µm. Furrow stalls within minutes; F-actin band breaks into pulsed contractions (kymograph, inset [F′], generated from position ≫ ≪, 2.5× blowup, x scale bar = 5 µm, y scale bar = 2 min; compare to Fig. S1 C); RGA-3/4 continues to nearly match F-actin (2.5× blowup, inset [F″], scale bar = 5 µm). Scale bar = 50 µm.