Functional Analysis of Kinetochore Assembly in Caenorhabditis elegans

doi:10.1083/jcb.153.6.1209

Published online 11 June 2001. doi:10.1083/jcb.153.6.1209

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© The Rockefeller University Press, 0021-9525/2001//1209 $5.00
The Journal of Cell Biology, Volume 153, Number 6, , 2001 1209-1226

Original Article

Functional Analysis of Kinetochore Assembly in Caenorhabditis elegans

Karen Oegema^a^,b, Arshad Desai^a^,b, Sonja Rybina^a, Matthew Kirkham^a, and Anthony A. Hyman^a

^a Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany
^b European Molecular Biology Laboratory, 69117 Heidelberg, Germany
Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.(49) 351-210-1289(49) 351-210-2592

oegema{at}mpi-cbg.de

In all eukaryotes, segregation of mitotic chromosomes requirestheir interaction with spindle microtubules. To dissect thisinteraction, we use live and fixed assays in the one-cell stageCaenorhabditis elegans embryo. We compare the consequences ofdepleting homologues of the centromeric histone CENP-A, thekinetochore structural component CENP-C, and the chromosomalpassenger protein INCENP. Depletion of either CeCENP-A or CeCENP-Cresults in an identical "kinetochore null" phenotype, characterizedby complete failure of mitotic chromosome segregation as wellas failure to recruit other kinetochore components and to assemblea mechanically stable spindle. The similarity of their depletionphenotypes, combined with a requirement for CeCENP-A to localizeCeCENP-C but not vice versa, suggest that a key step in kinetochoreassembly is the recruitment of CENP-C by CENP-A–containingchromatin. Parallel analysis of CeINCENP-depleted embryos revealedmitotic chromosome segregation defects different from thoseobserved in the absence of CeCENP-A/C. Defects are observedbefore and during anaphase, but the chromatin separates intotwo equivalently sized masses. Mechanically stable spindlesassemble that show defects later in anaphase and telophase.Furthermore, kinetochore assembly and the recruitment of CeINCENPto chromosomes are independent. These results suggest distinctroles for the kinetochore and the chromosomal passengers inmitotic chromosome segregation.

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

The online version of this article contains supplementary material.

K. Oegema and A. Desai contributed equally to this work.

Abbreviations used in this paper: DIC, differential interferencecontrast; GFP, green fluorescent protein; MT, microtubule; NEBD,nuclear envelope breakdown; RNAi, RNA-mediated interference.

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