Functions of the novel RhoGAP proteins RGA-3 and RGA-4 in the germ line and in the early embryo of C-elegans

We have identified two redundant GTPase activating proteins ( GAPs) - RGA- 3 and RGA- 4 - that regulate Rho GTPase function at the plasma membrane in early Caenorhabditis elegans embryos. Knockdown of both RhoGAPs resulted in extensive membrane ruffling, furrowing and pronounced pseudo- cleavages. In addition, the non- muscle myosin NMY- 2 and RHO- 1 accumulated on the cortex at sites of ruffling. RGA- 3 and RGA- 4 are GAPs for RHO- 1, but most probably not for CDC- 42, because only RHO- 1 was epistatic to the two GAPs, and the GAPs had no obvious influence on CDC- 42 function. Furthermore, knockdown of either the RHO- 1 effector, LET- 502, or the exchange factor for RHO- 1, ECT- 2, alleviated the membrane- ruffling phenotype caused by simultaneous knockdown of both RGA- 3 and RGA- 4 [ rga- 3/ 4 ( RNAi)]. GFP:: PAR- 6 and GFP:: PAR- 2 were localized at the anterior and posterior part of the early C. elegans embryo, respectively showing that rga- 3/ 4 ( RNAi) did not interfere with polarity establishment. Most importantly, upon simultaneous knockdown of RGA- 3, RGA- 4 and the third RhoGAP present in the early embryo, CYK- 4, NMY- 2 spread over the entire cortex and GFP:: PAR- 2 localization at the posterior cortex was greatly diminished. These results indicate that the functions of CYK- 4 are temporally and spatially distinct from RGA- 3 and RGA- 4 ( RGA- 3/ 4). RGA- 3/ 4 and CYK- 4 also play different roles in controlling LET- 502 activation in the germ line, because rga- 3/ 4 ( RNAi), but not cyk- 4 ( RNAi), aggravated the let- 502( sb106) phenotype. We propose that RGA- 3/ 4 and CYK- 4 control with which effector molecules RHO- 1 interacts at particular sites at the cortex in the zygote and in the germ line.