In the ubiquitin proteasome system, E3 ubiquitin ligases serve to ubiquitylate target substrates and thereby target them for the proteasome dependent degradation. During C. elegans early embryogenesis, the CUL-2 and CUL-3 E3 ligase complexes play essential roles by regulating the degradation of developmental factors. However, the targets of these E3 ligases and their developmental significance are not entirely known. Here we found a new role for the CUL-3 E3 ligase during cytokinesis. Interestingly, C. elegans zygote P0 cell is known to undergo an asymmetric cytokinesis, with the contractile ring on one side closing faster than on the opposite side, such that the midbody is eventually off-centered. We found that cul-3(RNAi) resulted in a loss of this asymmetry during cytokinesis. The CUL-3 binding partner and substrate adaptor MEL-26 also is required for this asymmetric cytokinesis. Using a GFP::CUL-3 fusion we generated by CRISPR/Cas9 genome editing, and an anti-MEL-26 antibody, we found that both CUL-3 and MEL-26 localized to the cleavage furrow during cytokinesis, suggesting that this E3 ligase complex functions at the contractile ring. The CUL-3/MEL-26 complex may be selectively transported to the cleavage furrow, as RNAi knockdown of the early endosomal component RAB-5 specifically diminished CUL-3/MEL-26 localization at the cleavage furrow. How does this E3 ligase on the contractile ring regulate asymmetric cytokinesis? Surprisingly, we found that ubiquitylation at the contractile ring was strongly asymmetric, and this ubiquitylation was CUL-3 dependent. A previous study has reported the asymmetric accumulation of contractile ring components, including the non-muscle myosin NMY-2, to the side where the ring closes faster during asymmetric cytokinesis. We found that in cul-3(RNAi) embryos, NMY-2 localization became symmetric, suggesting that CUL-3 dependent ubiquitylation is required for the formation of an asymmetric contractile ring. Simultaneous staining of ubiquitin and NMY-2 showed that poly-ubiquitin exclusively localized to the fast closing side while NMY-2 localized to both sides with enrichment on the fast side. Moreover, poly-ubiquitin only localized to the inner contractile ring while NMY-2 also localized to the outer ring. In summary, we have found that the CUL-3/MEL-26 E3 ubiquitin ligase regulates the asymmetric ubiquitination of an unknown protein at the inner contractile ring on the fast closing side during this asymmetric cytokinesis. To our knowledge, this is the first example of an asymmetric ubiquitination within a cell. Moreover, this ubiquitylation is required for the asymmetric localization of NMY-2 to the contractile ring and for the asymmetry of this cytokinesis. .
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