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In animal cells, cytokinesis is powered by a contractile ring of actin filaments (F-actin) and myosin-2. Formation of the contractile ring is dependent on the small GTPase RhoA, which is activated in a precise zone at the cell equator. It has long been assumed that cytokinesis and other Rho-dependent processes are controlled in a sequential manner, whereby Rho activation by guanine nucleotide exchange factors (GEFs) initiates a particular event, and Rho inactivation by GTPase activating proteins (GAPs) terminates that event. MgcRacGAP is a conserved cytokinesis regulator thought to be required only at the end of cytokinesis. Here we show that GAP activity of MgcRacGAP is necessary early during cytokinesis for the formation and maintenance of the Rho activity zone. Disruption of GAP activity by point mutation results in poorly focused Rho activity zones, whereas complete removal of the GAP domain results in unfocused zones that show lateral instability and/or rapid side-to-side oscillations. We propose that the GAP domain of MgcRacGAP has two unexpected roles throughout cytokinesis: first, it transiently anchors active Rho, and second, it promotes local Rho inactivation, resulting in the constant flux of Rho through the GTPase cycle.
Figure 2. Testing the GTPase flux modela–d, Embryos were injected with GFP-rGBD to monitor active Rho along with WT Mgc (b), Mgc R384A (c), or Mgc ΔGAP (d) and imaged during cytokinesis. Frames taken from time-lapse movies are shown. A single blastomere is outlined in red in the first frame, and the Rho activity zone is marked with an arrow in the second frame. Scale bars, 40 μm. e, Average Rho zone width / longitudinal diameter for multiple cells. Mean ± SE. Control, n = 12 embryos; WT Mgc, n = 9; R384A, n = 10; ΔGAP, n = 12. *p < 0.05; **p < 0.01; ***p < 0.005. f–i, Gene replacement experiments where embryos were injected with Mgc MO.2 to knock down endogenous MgcRacGAP along with mRNAs for WT Mgc (g), Mgc R384A (h), or Mgc ΔGAP (i). The embryos were also injected with GFP-rGBD to monitor active Rho. A single blastomere is outlined in red in the first frame, and the Rho activity zone is marked with an arrow in the second frame. Scale bars, 40 μm. j, Effects of constitutively-active Rho (CA-Rho) on Rho zone dynamics. Frames taken from time-lapse movies showing Rho activity dynamics during cytokinesis in control cells and cells expressing CA-Rho are shown. Cytokinetic Rho zones (arrows) are broader and brighter in cells expressing CA-Rho and are much slower to furrow. Moreover, the amount of Rho activity outside the equtorial regions is much higher in those cells expressing CA-Rho than the controls. Note that these phenotypes closely resemble those obtained with the R384A GAP-DEAD mutant. Scale bars, 40 μm.
Figure 3. Downstream consequences of abnormal GTPase fluxaâd, Embryos were injected with GFP-UtrCH to monitor F-actin along with WT Mgc (b), Mgc R384A (c), or Mgc ÎGAP (d) and imaged during cytokinesis. Frames taken from time-lapse movies are shown. A single blastomere is outlined in red in the first frame, and the Rho activity zone is marked with an arrow in the second frame. Scale bars, 40 μm.
Figure 4. Rho activity zones in MgcRacGAP ÎGAP-expressing cells oscillatea, z-stack cross-section kymographs of the region where the Rho activity zone is forming for control, WT Mgc-, Mgc R384A-, or Mgc ÎGAP-expressing cells. Appearance of the Rho activity zone is marked with an arrowhead, and the furrow is marked with an arrow. Asterisks indicate places where the Rho activity zone splits. b, Kymographs made along the lines shown in the first frame of c and d. Yellow dots indicate F-actin oscillations. câd, Control (c) or Mgc ÎGAP-expressing cells (d) were co-injected with EMTB-3XGFP to monitor microtubules (green) and mChe-UtrCH to monitor F-actin (red) and imaged during cytokinesis. Frames from time-lapse movies are shown. Yellow dots in d are fiduciary marks to help visualize the F-actin oscillations. Scale bars, 20 μm. e, Model showing that equatorial accumulation of Ect2 and MgcRacGAP leads to focused activation of Rho, which in turn leads to focused activation of Rho effectors. Our results are consistent with the GTPase flux model, suggesting that Ect2 locally activates Rho, while MgcRacGAP counterbalances Ect2 by locally inactivating Rho, keeping Rho in a constant state of flux through the GTPase cycle and maintaining a focused Rho activity zone at the cell equator.
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