Functional analysis of kinetochore assembly in Caenorhabditis elegans

In all eukaryotes, segregation of mitotic chromosomes requires their intera ction with spindle microtubules. To dissect this interaction, we use live a nd fixed assays in the one-cell stage Caenorhabditis elegans embryo. We com pare the consequences of depleting homologues of the centromeric histone CE NP-A, the kinetochore structural component CENP-C, and the chromosomal pass enger protein INCENP. Depletion of either CeCENP-A or CeCENP-C results in a n identical "kinetochore null" phenotype, characterized by complete failure of mitotic chromosome segregation as well as failure to recruit other kine tochore components and to assemble a mechanically stable spindle. The simil arity of their depletion phenotypes, combined with a requirement for CeCENP -A. to localize CeCENP-C but not vice versa, suggest that a key step in kin etochore assembly is the recruitment of CENP-C by CENP-A-containing chromat in. Parallel analysis of CeINCENP-depleted embryos revealed mitotic chromos ome segregation defects different from those observed in the absence of CeC ENP-A/C. Defects are observed before and during anaphase, but the chromatin separates into two equivalently sized masses. Mechanically stable spindles assemble that show defects later in anaphase and telophase. Furthermore, k inetochore assembly and the recruitment of CeINCENP to chromosomes are inde pendent. These results suggest distinct roles for the kinetochore and the c hromosomal passengers in mitotic chromosome segregation.