Figure S2.
IT-RHO-1 responds to ECT-2 GEF and RGA-3/4 GAP depletion. (A) To generate IT-RHO-1 the superfolder GFP (sGFP) was inserted into a conserved external exposed loop of RHO-1. Three-dimensional structure of C. elegans RHO-1 (UniProt ID Q22038) and IT-RHO-1 with sfGFP (green) predicted by AlphaFold https://galaxyproject.org/citing-galaxy/ (Galaxy Community, 2022). The position of the effector binding site and the external loop are highlighted. The local confidence of the AlphaFold prediction is indicated by the scores of the predicted local distance difference test (pLDDT). (B) Mean number of progeny per worm of wild type (rho-1/rho-1) and homozygote (it-rho-1/it-rho-1) hermaphrodites. Error bars are standard deviation (SD) and n = number of progenies analyzed. (C) Immunoblot of wild-type (rho-1/rho-1) or heterozygote (rho-1/it-rho1) adult hermaphrodites treated with anti-RHO-1 (left) or anti-GFP antibodies (right). Please note that with the anti-RHO-1 antibody a non-specific band is present at the same height as the IT-RHO-1 (black star). After image acquisition, the membranes were washed with buffer and probed with anti-Actin antibodies to ensure similar loading. The intensity of the different bands is indicated in % (mean of three worm extracts). (D) Immunoblot of control and rho-1(RNAi) worms probed with anti-RHO-1 and anti-Actin antibodies. The intensity of the RHO-1 bands is indicated in % (mean of 3 worm extracts). The membrane was cut and the upper part was treated with the anti-RHO-1 and the lower part with the anti-Actin antibodies. (E) Cytokinesis success determined by live-cell imaging on central planes images for one-cell embryos derived from wild type (rho-1/rho-1), heterozygote (rho-1/it-rho-1) or rho-1(RNAi) treated hermaphrodite animals and n = number of embryos analyzed. (F) Time in seconds (s) from NEBD to the completion of cytokinesis in wild type (rho-1/rho-1) and IT-RHO-1 (rho-1/it-rho-1) expressing embryos quantified on central plane images from live-cell imaging. Error bars are SD and n = number of embryos. (G) Confocal single z-plane cortical images and magnifications of the equatorial region (right) of IT-RHO-1 expressing embryos for the indicated RNAi conditions and time points after NEBD during cytokinesis. Scale bars are 5 µm. (H) Normalized cortical fluorescence intensity of IT-RHO-1 from the anterior (0%) to the posterior (100%) pole for indicated RNAi conditions and n = number of embryos analyzed. (I) Mean normalized fluorescence intensity of IT-RHO-1 at the cell equator for indicated RNAi conditions at 180 s after NEBD. Error bars are SEM. P values in comparison to uninjected controls were calculated using a two-tailed Student’s t test are **P < 0.01 and ***P < 0.001. For all, time in seconds (s) after NEBD is indicated. Source data are available for this figure: SourceData FS2.

IT-RHO-1 responds to ECT-2 GEF and RGA-3/4 GAP depletion. (A) To generate IT-RHO-1 the superfolder GFP (sGFP) was inserted into a conserved external exposed loop of RHO-1. Three-dimensional structure of C. elegans RHO-1 (UniProt ID Q22038) and IT-RHO-1 with sfGFP (green) predicted by AlphaFold https://galaxyproject.org/citing-galaxy/ (Galaxy Community, 2022). The position of the effector binding site and the external loop are highlighted. The local confidence of the AlphaFold prediction is indicated by the scores of the predicted local distance difference test (pLDDT). (B) Mean number of progeny per worm of wild type (rho-1/rho-1) and homozygote (it-rho-1/it-rho-1) hermaphrodites. Error bars are standard deviation (SD) and n = number of progenies analyzed. (C) Immunoblot of wild-type (rho-1/rho-1) or heterozygote (rho-1/it-rho1) adult hermaphrodites treated with anti-RHO-1 (left) or anti-GFP antibodies (right). Please note that with the anti-RHO-1 antibody a non-specific band is present at the same height as the IT-RHO-1 (black star). After image acquisition, the membranes were washed with buffer and probed with anti-Actin antibodies to ensure similar loading. The intensity of the different bands is indicated in % (mean of three worm extracts). (D) Immunoblot of control and rho-1(RNAi) worms probed with anti-RHO-1 and anti-Actin antibodies. The intensity of the RHO-1 bands is indicated in % (mean of 3 worm extracts). The membrane was cut and the upper part was treated with the anti-RHO-1 and the lower part with the anti-Actin antibodies. (E) Cytokinesis success determined by live-cell imaging on central planes images for one-cell embryos derived from wild type (rho-1/rho-1), heterozygote (rho-1/it-rho-1) or rho-1(RNAi) treated hermaphrodite animals and n = number of embryos analyzed. (F) Time in seconds (s) from NEBD to the completion of cytokinesis in wild type (rho-1/rho-1) and IT-RHO-1 (rho-1/it-rho-1) expressing embryos quantified on central plane images from live-cell imaging. Error bars are SD and n = number of embryos. (G) Confocal single z-plane cortical images and magnifications of the equatorial region (right) of IT-RHO-1 expressing embryos for the indicated RNAi conditions and time points after NEBD during cytokinesis. Scale bars are 5 µm. (H) Normalized cortical fluorescence intensity of IT-RHO-1 from the anterior (0%) to the posterior (100%) pole for indicated RNAi conditions and n = number of embryos analyzed. (I) Mean normalized fluorescence intensity of IT-RHO-1 at the cell equator for indicated RNAi conditions at 180 s after NEBD. Error bars are SEM. P values in comparison to uninjected controls were calculated using a two-tailed Student’s t test are **P < 0.01 and ***P < 0.001. For all, time in seconds (s) after NEBD is indicated. Source data are available for this figure: SourceData FS2.

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