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Published online 11 June 2001. doi:10.1083/jcb.153.6.1209
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© The Rockefeller University Press, 0021-9525/2001/6/1209/ $5.00
The Journal of Cell Biology, Volume 153, Number 6, June 11, 2001 1209-1226

Original Article

Functional Analysis of Kinetochore Assembly in Caenorhabditis elegans

Karen Oegemaa,b, Arshad Desaia,b, Sonja Rybinaa, Matthew Kirkhama, and Anthony A. Hymana
a Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany
b European Molecular Biology Laboratory, 69117 Heidelberg, Germany

Correspondence to: Karen Oegema, Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany. Tel:(49) 351-210-2592 Fax:(49) 351-210-1289 E-mail:oegema{at}mpi-cbg.de.

In all eukaryotes, segregation of mitotic chromosomes requires their interaction with spindle microtubules. To dissect this interaction, we use live and fixed assays in the one-cell stage Caenorhabditis elegans embryo. We compare the consequences of depleting homologues of the centromeric histone CENP-A, the kinetochore structural component CENP-C, and the chromosomal passenger protein INCENP. Depletion of either CeCENP-A or CeCENP-C 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. The similarity 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 kinetochore assembly is the recruitment of CENP-C by CENP-A–containing chromatin. Parallel analysis of CeINCENP-depleted embryos revealed mitotic chromosome segregation defects different from those observed in the absence of CeCENP-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, kinetochore assembly and the recruitment of CeINCENP to chromosomes are independent. These results suggest distinct roles for the kinetochore and the chromosomal passengers in mitotic chromosome segregation.

Key Words: centromere, CENP, passenger, mitosis, chromosome


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