The cullin gene family, of which there are five functional members and one pseudogene in C. elegans, function as ubiquitin-ligases (E3s) to facilitate ubiquitin-mediated degradation. The C. elegans cullin
cul-1 and yeast cullin CDC53 are negative and positive cell cycle regulators, respectively. We have been studying the function of the
cul-2 gene, which functions as a positive cell cycle regulator. By screening a deletion library, we have isolated a
cul-2 mutant allele,
ek1, that has a deletion of three full and one partial exons. From heterozygous parents, the
cul-2 mutant has normal somatic development through the adult stage. In the L4 stage, the
cul-2 mutant has fewer germ cells than wild type and the
cul-2 germ cell nuclei are twice as large. Quantitation of germ cell DNA reveals that the
cul-2 germ cells accumulate in G1 phase, suggesting that
cul-2 is required for progression into S phase. Due to a lack of oocytes,
cul-2 mutants lay on average only 17 eggs. The
cul-2 mutant phenotype of enlarged germ cells and lack of oocytes is partially suppressed by heterozygous mnDf100, a deficiency which removes the cyclin-dependent kinase inhibitors
cki-1 and
cki-2. This finding reinforces our previous RNAi results that CUL-2 is required for the degradation of CKI-1 and CKI-2, and suggests that CKI accumulation in
cul-2 mutants is responsible for the G1 arrest. Embryos from
cul-2 mutants arrest development with approximately 24 cells. During mitotic cycles, multiple nuclei are observed in cells; with cells having from one to five nuclei. Unlike cell cycle mutants in other organisms where multiple nuclei arise due to a failure of cytokinesis, the multiple nuclei in
cul-2 form immediately after cytokinesis and apparently result from an inability of chromosomes to coalesce into a single nucleus. The extra nuclei have nuclear pores as demonstrated by an anti-nuclear pore antibody, suggesting that their nuclear substructure may be normal. The DNA content of cells is not uniform in
cul-2 mutants, indicating that
cul-2 is required for other aspects of mitosis. Additionally, there is extreme cytoplasmic-extension activity. This activity is similar to that observed briefly in early wild type embryos after mitosis, but is much more dramatic and sustained, producing large extensions that can pinch off to form cells or cytoplasts. Loss of
cul-2 function does not appear to affect meiosis as the timing of meiosis and polar body formation is normal; however, after meiosis II, multiple pronuclei-like structures are observed. The finding that
cul-2 embryos from heterozygous parents have normal somatic development but produce arrested eggs, while
cul-2 dsRNA injection of wild type immediately produces similarly arrested eggs, suggests that
cul-2 is provided as maternal product that may perdure through the adult stage.