Every dividing cell must replicate its DNA and then segregate the DNA to each daughter cell. Errors in chromosome segregation can result in cells with abnormal chromosome content, a hallmark of cancer cells. Cellular mechanisms termed "checkpoints" detect DNA damage, monitor progression of DNA replication and mitosis and couple the S-and M-phases of the cell cycle. We are interested in the
lin-5 gene because its mutant phenotype suggests that
lin-5 is essential for chromosome segregation and for coupling DNA replication and mitosis (Albertson et al., 1978; Horvitz and Sulston, 1980, 1981). We have cloned the
lin-5 gene and have shown that
lin-5 encodes a novel protein. LIN-5 protein is present at the meiotic spindle and localizes to the centrosomes throughout mitosis. These results are consistent with an essential role for
lin-5 in mitosis. In
lin-5(0) animals, postembryonic cells enter mitosis at the normal time, the nuclear envelope breaks down, the chromatin condenses and a bipolar spindle is formed. However in general, metaphase plate formation is incomplete, anaphase is not initiated and cytokinesis is absent. Despite defects in chromosome segregation, the cells exit mitosis without significant delay, replicate their DNA again and enter another abortive mitosis. Additionally, in
lin-5 mutants centrosome duplication continues after abortive mitoses, consistent with an uncoupling of cell-cycle events. To further understand the role of
lin-5 in mitosis we are addressing the following questions: 1) how does
lin-5 act at the centrosome to promote mitosis? and 2) is
lin-5 required to delay cells in mitosis in the presence of mitotic defects? A further characterization of the
lin-5 mutant phenotype has given us some insight into the role of
lin-5 and screens will allow us to identify genes that act with
lin-5 in both of these processes. As in other eukaryotes, defects in chromosome segregation are expected to activate a mitotic checkpoint in C. elegans. To test the role of
lin-5 in a mitotic checkpoint, we used the
lin-5(
ev571ts) allele. We predicted if
lin-5 has a role in chromosome segregation as well as mitotic checkpoint control that partial inactivation at a semipermissive temperature would result in a partial delay in exit from mitosis. Cell cycle timing studies showed that at the semipermissive temperature defects in chromosome segregation delayed exit from mitosis, lengthening the time in mitosis on average 2.5-fold as compared to wild type. In contrast, the
lin-5(0) allele showed only slight variation from wild type with respect to time in mitosis. These results suggest that
lin-5 is required in mitosis and generates a signal that delays cells in mitosis in the presence of mitotic defects. To further understand the role of
lin-5 in chromosome segregation, we have initiated a screen for enhancers of the partial loss-of-function allele
ev571 and obtained 24 candidate enhancers. The initial characterization of these enhancers is in progress. A two hybrid screen is also in progress in order to identify LIN-5 interacting proteins (see abstract by Marian Walhout and Marc Vidal). Sequence from the C. elegans Genome Project has revealed a gene with 43% overall identity to
lin-5. In order to determine if this gene has a similar mutant phenotype as
lin-5, I made use of RNAi and obtained mutant F1 progeny with low penetrance. I am now injecting RNA from this gene into
lin-5(
ev571ts) animals to determine if disruption of both of these homologous genes produces a stronger mutant phenotype, suggesting that these genes might function in similar mitotic or meitoic processes. We are trying to understand the molecular mechanisms that maintain chromosome stability during cell division. The localization of LIN-5 protein to the centrosome and its role in chromosome segregation emphasizes the importance of centrosomes in this process.