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. 2022 Jun 16;221(8):e202203017. doi: 10.1083/jcb.202203017

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© 2022 Michaud et al.

This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).

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Figure 1. — RGA-3/4 localizes to the cortex, cortical waves, the cytokinetic apparatus, and suppresses Rho activity in starfish eggs and embryos. (A) Time course of mitosis in starfish blastomeres (8 of 16 cells) expressing mNeon-RGA-3/4^R96E. mNeon-RGA-3/4^R96E first localizes to the cortex, then the equatorial cortex, then the cytokinetic apparatus. Time in min:s; scale bar = 50 µm. (B) Time course of an Ect2-loaded starfish oocyte undergoing first and second meiosis; animal pole is to the right; time in min:s; active Rho labeled with mCherry-rGBD (cyan) and mNeon-RGA-3/4^WT (orange). Waves develop coincident with polar body emission and subside between meiosis I and II. Single-channel insets of the animal pole use the deepest slice to show cortical recruitment: RGA-3/4 is cortical as the cell approaches meiosis I (MI) metaphase, while Rho is not (00:00); RGA-3/4 appears brightly in the polar body furrow (20:30); RGA-3/4 departs the cortex between MI and MII (28:00) but returns in metaphase (41:15) before waves develop. Scale bar = 50 µm. (C and C′) Waves of mNeon-RGA-3/4^WT (orange) recruitment closely follow Rho activity waves (mCherry-rGBD; cyan), and increasing dose of mNeon-RGA-3/4^WT progressively suppresses Ect2-induced excitability (Video 1); kymographs in (C′) are taken from the band denoted by ≫. All panels show post-MII oocytes at quasi-steady state. All oocytes are expressing 100 ng/μl Ect2 to induce rampant, chaotic waves. Those simultaneously loaded with 25 ng/μl mNeon-RGA-3/4 are little different from controls (not depicted); 75 or 200 ng/μl RGA-3/4 reduces wave amplitude and peak width while extending wave propagation into long runs. x scale bar = 50 µm; y scale bar = 2 min. (D) GAP-dead RGA-3/4 (mNeon-RGA-3/4^R96E; orange) coexpressed with Ect2 has no effect on excitability but recruits in the same phase as WT RGA-3/4 (see also Video 2); active Rho (mCherry-rGBD; cyan). Kymograph (right) position corresponds to ≫, stills (left) come from the time indicated by –. Kymograph x scale also applies to still images. x scale bar = 50 µm; y scale bar = 2 min. (E) Untagged RGA-3/4^WT coexpressed with high level (100 ng/μl) Ect2, labeled with GFP-rGBD (active Rho; cyan) and mCherry-UtrCH (F-actin; orange). Corresponds to Video 3. In Ect2 alone samples (left), waves are irregular, close-packed high-amplitude bursts that form broken fronts that swell and collapse; addition of modest (22 ng/μl) RGA-3/4 mRNA converts them to steadily rolling regular waves (middle); higher RGA-3/4 dose damps waves further (right), and higher still suppresses them completely (not depicted). Kymograph x scale also applies to still images; x scale bar = 50 µm; y scale bar = 2 min. Insets from stills are 2× blowups of Rho alone; scale bar = 10 µm. Insets from kymographs are 3× blowups; x scale bar = 10 µm; y scale bar = 30 s; applies to both insets.