Embryonic morphogenesis involves properly coordinated cell migrations, which are dependent on rearrangement of the actin cytoskeleton. In C. elegans, actin dynamics are modulated by axonal guidance signals functioning through the WAVE/SCAR complex, which is regulated by the CED-10 GTPase. Previous work in our lab, identified UNC-40/DCC, SAX-3/Robo, and VAB-1/Eph as axonal guidance signals regulating the actin cytoskeleton through CED-10 and the WAVE/SCAR complex. Exactly how these axonal guidance proteins function to regulate WAVE is still unclear. To gain insight into the mechanisms that govern the regulation of WAVE/SCAR, we performed an RNAi screen to identify genes that enhance unc-40 embryonic lethality. From this screen we identified HUM-7, the C. elegans homolog of vertebrate Myosin IX gene. Loss of HUM-7 alone, resulted in a low percentage of dead embryos, some with phenotypes similar to that observed in wve-1 mutants. Loss of HUM-7 enhanced the lethality of unc-40, but suppressed sax-3 lethality, suggesting that HUM-7 likely functions in a pathway parallel to UNC-40 and similar to SAX-3. Similar to vertebrate Myosin IX, HUM-7 has a myosin domain in its head and a RhoGAP domain in its C-terminus. Based on the presence of the GAP domain and the wve-1-like embryonic phenotypes, we predicted that HUM-7 likely functions as a GAP for the CED-10 GTPase in the WAVE/SCAR pathway. To test our prediction, we examined genetic and RNAi doubles of hum-7 and hypomorphic alleles of three C. elegans GTPases, RHO-1, CDC-42 and CED-10 or their regulators. Based on our genetic observations, HUM-7 appears to function as a GAP for RHO-1 and CDC-42 but not CED-10 during embryogenesis. Protein-protein interaction assays supported these findings since the GAP domain of HUM-7 bound to GTP-loaded RHO-1 and CDC-42, but not CED-10. To test if HUM-7 truly functions to regulate RHO-1 we compared it to RGA-2, a GAP known to function with RHO-1 during elongation. Loss of RGA-2 leads to elongation phenotypes similar to what we observe in a subset of hum-7 embryos. Like rga-2, loss of HUM-7 enhances loss of MEL-11/myosin phosphatase and rescues let-502/rho-1 kinase, suggesting that HUM-7 is a negative regulator of RHO-1 activity. Based on live imaging analyses, loss of HUM-7 results in increased F-actin levels in the C. elegans embryo, and increased actin dynamics at the leading edge of the migrating cells. These results support a role for HUM-7 in two key events occurring during morphogenesis, the initiation of cell migrations and elongation. Taken together, our results suggest that tight regulation of RHO-1 activity is necessary for modulating correct F-actin levels and dynamics during two key steps of embryonic development.
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