Control of local Rho GTPase crosstalk by Abr

Abstract

Background: The Rho GTPases-Rho, Rac, and Cdc42-regulate the dynamics of F-actin (filamentous actin) and myosin-2 with considerable subcellular precision. Consistent with this ability, active Rho and Cdc42 occupy mutually exclusive zones during single-cell wound repair and asymmetric cytokinesis, suggesting the existence of mechanisms for local crosstalk, but how local Rho GTPase crosstalk is controlled is unknown.

Results: Using a candidate screen approach for Rho GTPase activators (guanine nucleotide exchange factors; GEFs) and Rho GTPase inactivators (GTPase-activating proteins; GAPs), we find that Abr, a protein with both GEF and GAP activity, regulates Rho and Cdc42 during single-cell wound repair. Abr is targeted to the Rho activity zone via active Rho. Within the Rho zone, Abr promotes local Rho activation via its GEF domain and controls local crosstalk via its GAP domain, which limits Cdc42 activity within the Rho zone. Depletion of Abr attenuates Rho activity and wound repair.

Conclusions: Abr is the first identified Rho GTPase regulator of single-cell wound healing. Its novel mode of targeting by interaction with active Rho allows Abr to rapidly amplify local increases in Rho activity using its GEF domain while its ability to inactivate Cdc42 using its GAP domain results in sharp segregation of the Rho and Cdc42 zones. Similar mechanisms of local Rho GTPase activation and segregation enforcement may be employed in other processes that exhibit local Rho GTPase crosstalk.

Figure 1. A screen of GEFs and GAPs during wound healing identifies the GEF-GAP Abr, which colocalizes with active Rho

(A) Schematic of candidate regulator screen. (B) Oocytes expressing mRFP-wGBD (red) and endogenous Abr (green) as detected by antibody staining (top panel) but not in oocytes stained with secondary antibody alone (bottom panel). (C) Oocyes expressing Abr-3XeGFP and mRFP-wGBD mRNA. (D) Oocyes expressing 3XmCherry-Abr and eGFP-rGBD mRNA. (E) Oocytes expressing eGFP-farnesyl and 3XmCherry-Abr mRNA. Z-view is shown before and after wounding.

Figure 2. Abr requires its substrate-binding domains and active Rho for localization

(A) Schematic showing Abr domain structure and 3XeGFP-tagged constructs used for localization studies. (B) Oocytes expressing Abr-3XeGFP, AbrΔDH-3XeGFP, or AbrΔGAP-3XeGFP. The localization of each at 60 s post-wounding is shown. (C) Kymographs from cells expressing 3XmCherry-Abr and eGFP-rGBD. W=wound. (D) A vertical line was drawn in (C) through the region where Abr and active Rho are recruited starting after wounding through 46 s post-wounding. The intensity of each was plotted over time. (E) Cells expressing mRFP-wGBD, Abr-3XeGFP and C3 exoenzyme where indicated. (F) PM Z-views of cells expressing eGFP-farnesyl and 3XmCherry-Abr alone or with either CA Cdc42, CA Rho, or GEF-H1 as shown. Cells were incubated in latrunculin A (lat) where indicated.

Figure 3. Abr inhibits the Cdc42 zone and broadens the Rho zone

(A) Cells expressing mRFP-wGBD and eGFP-rGBD alone (top panel) or 100 μg/ml Abr mRNA (bottom panel). (B) The Cdc42 zone is shown with increasing concentrations of Abr mRNA. (C) The intensity of the Cdc42 zone was quantified at 60 s post-wounding with increasing concentrations of Abr mRNA (n=9; **p<0.01, ***p<0.005; Tukey’s multiple comparison test). (D) Rho zone width was quantified at 90 s post-wounding with increasing concentrations of Abr mRNA (n=6; *p<0.05, **p<0.01;Tukey’s multiple comparison test).

Figure 4. GAP-dead Abr prevents Cdc42 inhibition and promotes zone overlap

(A) Domain structure of GAP-dead mutant, Abr RN/AA, and Abr RN/AA-3XeGFP. (B) Abr RN/AA 3X-eGFP was injected along with mRFP-wGBD; 60 s post-wounding. (C) Cdc42 zone intensity was quantified in controls and Abr RN/AA-expressing cells (n=12; p=0.6941;Unpaired T-test). (D) Cdc42 and Rho zone width were quantified in control and Abr RN/AA-expressing cells (for Rho: n=12; ***p< 0.0001; for Cdc42: n=12, *p< 0.05; Unpaired T-test). (E) Cells injected with mRFP-wGBD and eGFP-rGBD alone (top panel) and with 500 μg/ml Abr RN/AA (bottom panel); 90 s post-wounding.

Figure 5. GEF-dead Abr inhibits the Rho zone but not the Cdc42 zone

(A) Domain structure of GEF-dead mutant, Abr SR/AA, and Abr SR/AA-3XeGFP. (B) Oocytes expressing Abr SR/AA-3XeGFP and mRFP-wGBD; 60 s post-wounding (scale bar=20 μm). (C) Rho zone intensity was quantified in control cells and those expressing either WT Abr or Abr SR/AA (n=10; *p<0.05; Tukey’s multiple comparison test). (D) Cdc42 zone intensity was quantified in control cells and those expressing either WT Abr or Abr SR/AA (n=10; ***p<0.0001; Tukey’s multiple comparison test). (E) Cells expressing eGFP-rGBD and mRFP-wGBD alone (top panel) or with 500 μg/ml Abr SR/AA (bottom panel).

Figure 6. Abr localizes with active Rho in embryos and Abr depletion inhibits Rho and perturbs wound healing

(A) Embryos expressing eGFP-rGBD and 3XmCherry-Abr (scale bar=20 μm). (B) Embryos expressing Abr-3XeGFP and mCherry-UtrCH to label F-actin were wounded near a cell border. Top panel=merge, cell border labeled by arrowheads. Bottom panel=Abr, Abr accumulation at the cell border indicated by an arrow (scale bar=20 μm). (C) Embryos expressing 3XmCherry-wGBD and eGFP-rGBD alone (top panel), or with WT Abr (bottom panel). Stress folds indicated by arrows (scale bar=20 μm). (D) Embryos were either uninjected, injected with 2 mM control MO, or 1 mM Abr MO and 1 mM Abr homologue MO (AbrMO 1+2) and homogenized 18 h post-fertilization. Abr and tubulin were detected by immunoblotting. (E) Embryos expressing eGFP-rGBD, mCherry-wGBD and Wee1 with either control MO or Abr MO 1+2 and imaged 18 h post-fertilization. Top panel shows control MO phenotype. Second and third panels represent wounds from Abr MO 1+2 -injected embryos. (F) Rho and Cdc42 zone intensity from cells in (E) was quantified after 48 s (Rho: n=25, ***p<0.0001; Cdc42; n=17, p=0.2907; Unpaired T-test). (G) Rho zone width was quantified from cells in (E) (n=17; ***p<0.001; Unpaired T-test).

Figure 7. Model for Abr at wounds

Abr is recruited to the Rho zone, where it interacts specifically with active Rho through its DH and GAP domains. Once recruited, Abr positively regulates Rho activity via the GEF domain. Simultaneously, the Abr speeds Cdc42 inactivation in the Rho zone through its GAP activity, maintaining zone segregation.