The roles of microtubule-based motor proteins in mitosis: comprehensive RNAi analysis in the Drosophila S2 cell line

doi:10.1083/jcb.200303022

Published 15 September 2003. doi:10.1083/jcb.200303022

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© The Rockefeller University Press, 0021-9525/2003/9/1003 $5.00
The Journal of Cell Biology, Volume 162, Number 6, 1003-1016

Article

The roles of microtubule-based motor proteins in mitosis : comprehensive RNAi analysis in the Drosophila S2 cell line

Gohta Goshima and Ronald D. Vale

Howard Hughes Medical Institute and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94107

Address correspondence to Ronald D. Vale, Dept. of Cellular and Molecular Pharmacology/HHMI, Genentech Hall, Room N312E, University of California, San Francisco, San Francisco, CA 94107. Tel.: (415) 476-6380. Fax: (415) 476-5233. email: vale{at}cmp.ucsf.edu

Kinesins and dyneins play important roles during cell division.Using RNA interference (RNAi) to deplete individual (or combinationsof) motors followed by immunofluorescence and time-lapse microscopy,we have examined the mitotic functions of cytoplasmic dyneinand all 25 kinesins in Drosophila S2 cells. We show that fourkinesins are involved in bipolar spindle assembly, four kinesinsare involved in metaphase chromosome alignment, dynein playsa role in the metaphase-to-anaphase transition, and one kinesinis needed for cytokinesis. Functional redundancy and alternativepathways for completing mitosis were observed for many singleRNAi knockdowns, and failure to complete mitosis was observedfor only three kinesins. As an example, inhibition of two microtubule-depolymerizingkinesins initially produced monopolar spindles with abnormallylong microtubules, but cells eventually formed bipolar spindlesby an acentrosomal pole-focusing mechanism. From our phenotypicdata, we construct a model for the distinct roles of molecularmotors during mitosis in a single metazoan cell type.

Key Words: kinesin; spindle; dynein; centrosome; kinetochore

The online version of this article includes supplemental material.

Abbreviations used in this paper: DHC, dynein heavy chain; dsRNA,double-stranded RNA; MTOC, microtubule-organizing center; NEB,nuclear envelope breakdown; Pav, Pavarotti; RNAi, RNA interference.

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