We are interested in membrane traffic during cytokinesis in the early C. elegans embryo. We searched the database for embryonic lethal RNAi phenotypes that showed a defect in cytokinesis. F35B12.5 fulfilled these criteria. RNAi by injection and feeding of the 1215 bp long coding region of this gene resulted in an embryonic lethality rate of about 100%. Nomarski pictures and timelapse studies of RNAi embryos revealed a normal first cell division undistinguishable from that in wildtype embryos, whereas the second cleavage was disturbed. To determine the cellular process in which the gene product of F35B12.5 is involved, RNAi experiments were performed in the background of several GFP expressing C. elegans strains. Knock-down of F35B12.5 in
pie-1::GFP,
par-2::GFP and
par-6::GFP expressing eggs revealed that the polarization of the early embryo was normal. Furthermore, receptor-mediated endocytosis in a YP170::GFP expressing embryo was not affected by RNAi. However, investigation of the phenotype in the histone::GFP strain revealed a strong nuclear phenotype: although the histone::GFP distribution before and during the first cell division was comparable to wildtype embryos, subsequently the embryos contained big unseparated and fragmented nuclei. These results were confirmed by DAPI and FM 4-64 stainings. FM 4-64 is a lipophilic dye that marks membranes along the endocytotic pathway. F35B12.5 knock-down resulted in multicellular embryos containing multinucleated and anucleated cells. Thus, cytokinesis is not defective at each cell division. Further analysis of the F35B12.5 RNAi phenotype revealed a centrosome duplication failure as determined by
tac-1::GFP and beta-tubulin::GFP. This role in centrosome duplication is consistent with the localization of F35B12.5 at the centrosomes.