Michaud A et al. (2022), A versatile cortical pattern-forming circuit ba...

XB-ART-59169

J Cell Biol 2022 Aug 01;2218:. doi: 10.1083/jcb.202203017.

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A versatile cortical pattern-forming circuit based on Rho, F-actin, Ect2, and RGA-3/4.

Michaud A , Leda M , Swider ZT , Kim S , He J , Landino J , Valley JR , Huisken J , Goryachev AB , von Dassow G , Bement WM .

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Many cells can generate complementary traveling waves of actin filaments (F-actin) and cytoskeletal regulators. This phenomenon, termed cortical excitability, results from coupled positive and negative feedback loops of cytoskeletal regulators. The nature of these feedback loops, however, remains poorly understood. We assessed the role of the Rho GAP RGA-3/4 in the cortical excitability that accompanies cytokinesis in both frog and starfish. RGA-3/4 localizes to the cytokinetic apparatus, "chases" Rho waves in an F-actin-dependent manner, and when coexpressed with the Rho GEF Ect2, is sufficient to convert the normally quiescent, immature Xenopus oocyte cortex into a dramatically excited state. Experiments and modeling show that changing the ratio of RGA-3/4 to Ect2 produces cortical behaviors ranging from pulses to complex waves of Rho activity. We conclude that RGA-3/4, Ect2, Rho, and F-actin form the core of a versatile circuit that drives a diverse range of cortical behaviors, and we demonstrate that the immature oocyte is a powerful model for characterizing these dynamics.

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Species referenced: Xenopus laevis
Genes referenced: arhgap35 cyb5r1 ect2 rho
GO keywords: cytoskeleton [+]

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	Figure 4. Coexpression of Ect2 and RGA-3/4 induces high-level excitability in immature frog oocytes. (A–D) Still frames (top) and kymographs (bottom) from representative oocytes expressing probe for active Rho (GFP-rGBD). Kymographs generated from 1-px line drawn at position ≫. Kymograph x scale also applies to still images; x scale bar = 50 µm, y scale bar = 5 min; see also Video 8. (A) Control oocyte expressing only active Rho probe shows no waves. (B) Oocyte expressing untagged, nonimportable Ect2 (Ect2ΔNLS) shows isolated, low-amplitude Rho waves. (C) Oocyte expressing Ect2ΔNLS and RGA-3/4WT shows high-amplitude waves with multiple spiral cores and continuous waves across cortex. (D) Oocyte expressing RGA-3/4WT alone shows no waves. (E) Quantification of relative wave amplitude across conditions in A–D. Each dot represents a single oocyte; group mean ± SD. 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; control, n = 8; Ect2ΔNLS, n = 6; Ect2 ΔNLS + RGA-3/4WT, n = 10; RGA-3/4WT, n = 7; six experiments. (F) Light-sheet imaging of immature oocyte expressing Ect2ΔNLS and RGA-3/4WT shows cortical waves present over entire animal cortex; scale bar = 100 µm; see also Video 9. (G) All oocytes express probe for active Rho (GFP-rGBD) and Ect2ΔNLS. Expression of p190RhoGAP (panel 2) or RGA-3/4 R80E (panel 3) do not support high-level cortical excitability; kymograph x scale also applies to still images; x scale bar = 25 µm, y scale bar = 2 min. (H) Quantification of relative wave amplitude across experimental groups described in G. Each dot represents a single oocyte; group mean ± SD. One-way ANOVA with Tukey post hoc test for multiple comparisons; **, P < 0.0001; Ect2ΔNLS, n = 27; p190RhoGAP, n = 9; RGA-3/4R80E, n = 16; RGA-3/4WT, n = 21; five experiments.
	Figure 5. RGA-3/4 recruitment to waves trails Rho activation and slightly leads peak of F-actin recruitment. All oocytes expressing untagged Ect2ΔNLS and RGA-3/4WT to generate cortical waves. (A) Frog oocyte expressing probe for active Rho (cyan; GFP-rGBD) and tagged RGA-3/4WT (orange; RGA-3/4WT-3xGFP). Kymographs (bottom) generated from 1-px line drawn at position ≫. Kymograph x scale also applies to still images; x scale bar = 20 µm; y scale bar = 5 min. (B) Representative intensity profile of active Rho and RGA-3/4WT. (C) Cross-correlational analysis of cell in A showing 18-s delay between Rho activation and RGA-3/4WT recruitment. (D) Frog oocyte expressing probe for F-actin (cyan; mCherry-UtrCH) and tagged RGA-3/4 (orange; RGA-3/4WT-3xGFP). Kymographs (bottom) generated from 1-px line drawn at position ≫. Kymograph x scale also applies to still images; x scale bar = 20 µm; y scale bar = 5 min. (E) Representative intensity profile of F-actin and RGA-3/4WT. (F) Cross-correlational analysis of cell in D showing 8-s delay between peak RGA-3/4WT recruitment and peak F-actin signal.
	Figure 6. Recruitment of cytokinetic participants to immature oocyte waves. All oocytes express untagged Ect2ΔNLS and RGA-3/4WT to generate cortical waves. (A) Frog oocyte expressing probe for active Rho (cyan; mCherry-rGBD) and tagged anillin (orange; Anillin-3xGFP). Kymographs (bottom) generated from 1-px line drawn at position ≫. Kymograph x scale also applies to still images; x scale bar = 40 µm; y scale bar = 1 min. (B) Representative intensity profile of active Rho and anillin. (C) Cross-correlational analysis of cell in A showing 18-s delay between Rho activation and anillin recruitment. (D) Waving frog oocyte expressing probes for active Rho (cyan; mCherry-rGBD) and myosin (orange; Sf9-mNeon). Kymographs (bottom) generated from 1-px line drawn at position ≫. Kymograph x scale also applies to still images; x scale bar = 20 µm; y scale bar = 2 min. (E) Representative intensity profile of Rho and myosin dynamics for cell in D. (F) Cross-correlational analysis of cell in D showing 57-s delay between Rho activation and Myosin recruitment. (G) Still frames of oocytes expressing probe for active Rho (cyan; mCherry-rGBD) and tagged Xenopus Dias 1, 2, or 3 (orange; Dia1-3xGFP, Dia2-3xGFP, Dia3-3xGFP). Only Dia3 is recruited robustly to cortical waves. Scale bar = 25 µm. (H) Representative intensity profiles of active Rho (cyan) with Dias 1, 2, and 3 (orange).
	Figure S3. Variation in Rho activity patterns in oocytes expressing Ect2 alone and quantification for oocytes coexpressing Ect2 and RGA. (A) All oocytes express probe for active Rho (GFP-rGBD). Panel 1, rGBD only; panels 2–5, examples of phenotypes from Ect2ΔNLS overexpression: static patches of Rho activity but no traveling waves (panel 2, arrowhead); tiny cluster of waves (panel 3, arrowhead) and diffuse wave patterns (panel 4, arrowhead) in same oocyte; wave patches (panel 5, arrowhead), surrounded by dormant cortex (panel 5, asterisk); scale bars = 50 µm. (B) Example still-frame difference subtraction of oocyte from (Fig. 4 C), showing segmentation process for measuring end-to-end lengths of cortical waves; scale bar = 50 µm. (C) One-way ANOVA with Tukey post hoc test for multiple comparisons, comparing end-to-end lengths across experimental groups. Each dot represents a single oocyte; group mean ± SD; data distribution was assumed to be normal but was not formally tested. Cells coexpressing Ect2ΔNLS and RGA-3/4WT are significantly different from all other groups; controls, n = 8; RGA-3/4WT, n = 7; Ect2ΔNLS, n = 11; Ect2ΔNLS + RGA-3/4WT, n = 12; seven experiments; ****, P < 0.0001. (D) Plot of percentage of cells displaying cortical waves across each experimental condition; controls, n = 8; RGA-3/4WT, n = 7; Ect2ΔNLS, n = 36; Ect2ΔNLS + RGA-3/4WT, n = 37; 13 experiments.

References [+] :

Barnhart, Adhesion-Dependent Wave Generation in Crawling Cells. 2017, Pubmed