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

Abstract

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.

Cortical domain correction repositions the polarity boundary to match the cytokinesis furrow in C. elegans embryos

“…1D). Similar PAR domain size correction can be observed in other mutants with enlarged posterior domains, such as rga-3/4(RNAi) embryos (Schmutz et al, 2007;Schonegg et al, 2007), in two-cell embryos with spindle orientation defects (Gomes et al, 2001) and in subsequent divisions of the germline lineage in wild-type C. elegans embryos (see Figs S1 and S2 in the supplementary material; see Movie 2 in the supplementary material). C. elegans embryos appear to match the PAR domain boundary to the site of cell division.…”

Elaborating polarity: PAR proteins and the cytoskeleton

“…These challenges have made it difficult to determine whether CYK-4 corresponds to the centrosome-dependent polarization cue, or if CYK-4 and the centrosome cue function in parallel. The functionally redundant RhoGAP proteins RGA-3 and RGA-4 (hereafter RGA-3/4) attenuate myosin activity to limit the extent of actomyosin contraction (Schmutz et al, 2007;Schonegg et al, 2007). In rga-3/4(RNAi) embryos, the myosin network is more dense and filamentous and contracts too far anteriorly, creating a smaller anterior PAR domain.…”

Widely Conserved Signaling Pathways in the Establishment of Cell Polarity

“…However, CYK-4 is also a component of a complex involved in cytokinesis, and in this situation, at least, it is specific for Rac inactivation rather than Rho-1 (Canman et al 2008). Two other RhoGAPs are present in C. elegans but seem to have a distinct function from CYK-4 because they control the ratio of the anterior/posterior domain rather than its formation (Schmutz et al 2007;Schonegg et al 2007). Rho-1 GTP normally activates a downstream kinase that phosphorylates myosin light chain, increasing contractility, but the accumulation of the GAP depletes Rho-GTP and relaxes actomyosin at the posterior cortex.…”