Characterization of the RhoGAP proteins RGA-3 and RGA-4 and the centrosomal protein SAS-5 in the early "Caenorhabditis elegans" embryo

The Caenorhabditis elegans embryo serves as a great tool to study cell biological processes like polarization and cell divison. The first cell division is unequal and cell polarization is dependant on the Rho GTPase regulated rearrangement of the cell cortex and the following localization of PAR proteins. The embryo serves also as a great model system to study centrosome duplication, which leads to the duplication of the centrioles provided by the sperm and their assembly into mature centrosomes during S phase of the one-cell stage embryo. In this thesis, two novel Rho GTPase activation proteins (RhoGAPs), RGA-3 and RGA-4, were identified, constituting an essential part for development. Concomitant RNAi of RGA-3 and RGA-4 (rga-3/4 (RNAi)) resulted in a hyper-contractility phenotype with extensive membrane ruffling and furrowing of the zygote. These two RhoGAPs act redundantly in regulating the small GTPase RHO-1, which is essential for regulating the acto-myosin network during contractile polarization of the early C. elegans embryo. Simultaneous knock-down of rho-1 (RNAi) and rga-3/4 (RNAi) rescued the rga-3/4 ruffling phenotype demonstrating that RHO-1 is the GTPase regulated by RGA-3/4. In contrast, triple knock-down of rga-3/4 together with another small GTPase, CDC-42, which is involved in polarity maintenance in the embryo, did no rescue the rga-3/4 (RNAi) ruffling phenotype. Increased membrane ruffling was mainly observed at the anterior cortex of rga3/4 (RNAi) embryos. Consistently, RHO-1 and its effector NMY-2 were enriched in these extra furrows in rga-3/4 (RNAi) embryos. Furthermore, the known Rho GEF ECT-2 and the Rho kinase LET-502 alleviated the membrane ruffling phenotype caused by rga-3/4 (RNAi). As opposed to the third known RhoGAP of the early embryo, CYK-4, which is essential for posterior polarization and central spindle assembly, RGA-3/4 regulate anterior contractility of the early embryo. The RGAs play a role in regulating the acto-myosin network during cortical polarization, yet the initial establishment of polarity is not heavily affected in rga-3/4 (RNAi) embryos as indicated by the correct localization of the PAR proteins. However, the size of the anterior PAR-6 domain fluctuated more in rga-3/4 (RNAi) than in wild type. Over-expression of RGA-3/4 appears to be lethal in C. elegans, and no stable GFP::RGA-3 expressing line could be obtained, neither by injection nor microparticle bombardment. RGA-3/4 do not only have a role in the one-cell stage embryo, they are also necessary for germ line development. Knock-down of RGA-3/4 in the background of the let502 (sb106) mutant impaired the germ line development, a phenotype not observed for this
mutant by itself. This result indicates that both LET-502 and RGA-3/4 are required for
gonadal function.
The second part of the thesis concerned the characterization of the centrosomal
protein SAS-5. sas-5 (RNAi) resulted in a high penetrance of embryonic lethality. SAS-5 is a
centrosomal protein and essential for centrosome duplication in C. elegans. Upon sas-5
(RNAi) the first cell division appeared to be unaffected. The two centrioles provided by the
sperm were not duplicated, yet separated during S phase allowing the establishment of a
bipolar spindle in the P0 cell. The centrosome duplication defect was obvious in the
subsequent mitotic cycles and only mono-polar spindles were formed. As a consequence,
nuclear morphology was strongly affected, but did not induce apoptosis.