[
International C. elegans Meeting,
2001]
We have developed live and fixed assays to analyze chromosome movement and kinetochore function in one-cell stage C. elegans embryos. Unlike most eukaryotes, C. elegans has holocentric chromosomes, where the kinetochore is assembled along the length of the chromosome. Despite this dramatic structural difference, the C.elegans genome contains many genes whose homologues are implicated in the formation and function of localized kinetochores of other eukaryotes. Using strains expressing GFP-histone or GFP-tubulin we have examined chromosome segregation and spindle function in worms depleted of proteins involved in chromosome segregation by RNAi. Depletion of the C. elegans homologs of either CENP-A, (HCP-3) or CENP-C (HCP-4) results in an identical 'kinetochore-null' phenotype, characterized by complete failure of mitotic chromosome segregation as well as failure to recruit other kinetochore components and to assemble a mechanically stable spindle. Parallel analysis of embryos depleted of the C. elegans homolog of the chromosomal passenger protein INCENP (ICP-1) revealed mitotic chromosome segregation defects different from those observed in the absence of HCP-3 or HCP-4. Defects are observed before and during anaphase but the chromatin separates into two equivalently sized masses. Kinetochore components are recruited normally and mechanically stable spindles assemble that show defects later in anaphase and telophase. These detailed cytological phenotypes are also serving as high resolution 'fingerprints' for classifying new genes important for chromosome segregation that are being identified in comprehensive functional genomics screens. So far, we have one novel gene that has a 'kinetochore-null' phenotype from a RNAi based screen of the genes on chromosome III. We have also initiated an analysis of kinetochore assembly in this system. Using antibodies to kinetochore components and RNAi, we have developed a preliminary map of the dependency relationships during kinetochore assembly.
[
International Worm Meeting,
2003]
Kinetochores assemble on mitotic chromosomes to connect them to spindle microtubules and mediate their segregation. Here we use RNA interference-based genomics in C. elegans to identify a novel protein, KNL-1, that when depleted results in a kinetochore null phenotype characterized by total failure of chromosome segregation and the inability to form a stable mitotic spindle. To date, depletion of only two other kinetochore components, HCP-3 and HCP-4, thought to form the interface of the kinetochore with the underlying DNA, give this phenotype. Pair-wise RNAi-depletion and immunofluorescence experiments place KNL-1 downstream of HCP-3 and HCP-4 in kinetochore assembly. Consistent with this, KNL-1 is not required for chromosome condensation, kinetochore specification, or sister kinetochore resolution. Instead, KNL-1 directs assembly of the outer domains of the kinetochore that interact with spindle microtubules. KNL-1 is required to target five conserved outer kinetochore proteins. In extracts, KNL-1 is present in a complex with HIM-10 and the C. elegans homolog Ndc80/HEC, two widely conserved eukaryotic kinetochore proteins. Depletion of either CeNDC-80, HIM-10 or both results in less severe chromosome segregation defects, consistent with a more important role for KNL-1 in assembling the microtubule-binding interface of the kinetochore.