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MBC in Press, published online ahead of print December 22, 2004
Mol. Biol. Cell 10.1091/mbc.E04-06-0523

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Submitted on June 26, 2004
Revised on November 17, 2004
Accepted on December 2, 2004

Functional Analysis of Cytoplasmic Dynein Heavy Chain in Caenorhabditis elegans with Fast-acting Temperature-sensitive Mutations

Diane J. Schmidt, Debra J. Rose, William M. Saxton, and Susan Strome*

Department of Biology, Indiana University, Bloomington, IN 47405

Monitoring Editor: Martin Chalfie

Cytoplasmic dynein, a minus-end-directed microtubule motor, has been implicated in many cellular and developmental processes. Identification of specific cellular processes that rely directly on dynein would be facilitated by a means to induce specific and rapid inhibition of its function. We have identified conditional variants of C. elegans dynein heavy chain (DHC-1) that lose function within a minute of a modest temperature upshift. Mutant embryos generated at elevated temperature show defects in centrosome separation, pronuclear migration, rotation of the centrosome/nucleus complex, bipolar spindle assembly, anaphase chromosome segregation, and cytokinesis. Our analyses of mutant embryos generated at permissive temperature and then upshifted quickly just before events of interest indicate that DHC-1 is required specifically for rotation of the centrosome/nucleus complex, for chromosome congression to a well-ordered metaphase plate, and for timely initiation of anaphase. Our results do not support the view that DHC-1 is required for anaphase B separation of spindle poles and chromosomes. A P-loop mutation identified in two independent dominant temperature-sensitive alleles of dhc-1, when engineered into the DHC1 gene of S. cerevisiae, conferred a dominant temperature-sensitive dynein loss-of-function phenotype. This suggests that temperature-sensitive mutations can be created for time-resolved function analyses of dyneins and perhaps other P-loop proteins in a variety of model systems.

*Corresponding author. E-mail: sstrome{at}bio.indiana.edu




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