Functional analysis of cytoplasmic dynein heavy chain in Caenorhabditis elegans with fast-acting temperature-sensitive mutations.
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 a Caenorhabditis 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 Saccharomyces 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.
Schmidt
Diane J
DJ
Department of Biology, Indiana University, Bloomington, IN 47405, USA.
Rose
Debra J
DJ
Saxton
William M
WM
Strome
Susan
S
D000483:Alleles; D000595:Amino Acid Sequence; D000705:Anaphase; D000818:Animals; D017173:Caenorhabditis elegans; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry* / Q000235:genetics*; D002465:Cell Movement; D002467:Cell Nucleus / Q000378:metabolism; D018385:Centrosome / Q000648:ultrastructure; D048749:Cytokinesis; D003593:Cytoplasm / Q000378:metabolism*; D056445:Cytoplasmic Dyneins; D004398:Dyneins / Q000737:chemistry* / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000648:ultrastructure; D008938:Mitosis; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000737:chemistry; D004718:Saccharomycetales; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D008941:Spindle Apparatus; D013696:Temperature; D013997:Time Factors
D029742:Caenorhabditis elegans Proteins; D012330:RNA, Double-Stranded; D049452:Green Fluorescent Proteins; D056445:Cytoplasmic Dyneins; C502824:DHC-1 protein, C elegans; D004398:Dyneins