Spindle dynamics and the role of gamma-tubulin in early Caenorhabditis elegans embryos.
gamma-Tubulin is a ubiquitous and highly conserved component of centrosomes in eukaryotic cells. Genetic and biochemical studies have demonstrated that gamma-tubulin functions as part of a complex to nucleate microtubule polymerization from centrosomes. We show that, as in other organisms, Caenorhabditis elegans gamma-tubulin is concentrated in centrosomes. To study centrosome dynamics in embryos, we generated transgenic worms that express GFP::gamma-tubulin or GFP::beta-tubulin in the maternal germ line and early embryos. Multiphoton microscopy of embryos produced by these worms revealed the time course of daughter centrosome appearance and growth and the differential behavior of centrosomes destined for germ line and somatic blastomeres. To study the role of gamma-tubulin in nucleation and organization of spindle microtubules, we used RNA interference (RNAi) to deplete C. elegans embryos of gamma-tubulin. gamma-Tubulin (RNAi) embryos failed in chromosome segregation, but surprisingly, they contained extensive microtubule arrays. Moderately affected embryos contained bipolar spindles with dense and long astral microtubule arrays but with poorly organized kinetochore and interpolar microtubules. Severely affected embryos contained collapsed spindles with numerous long astral microtubules. Our results suggest that gamma-tubulin is not absolutely required for microtubule nucleation in C. elegans but is required for the normal organization and function of kinetochore and interpolar microtubules.
Strome
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Department of Biology, Indiana University, Bloomington, Indiana 47405-3700, USA. sstrome@bio.indiana.edu
Powers
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Dunn
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Reese
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Malone
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White
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Seydoux
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Saxton
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D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D017173:Caenorhabditis elegans / Q000196:embryology* / Q000502:physiology*; D018385:Centrosome / Q000378:metabolism; D002875:Chromosomes / Q000378:metabolism / Q000502:physiology; D049452:Green Fluorescent Proteins; D006657:Histones / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000378:metabolism; D008969:Molecular Sequence Data; D017785:Photons; D010957:Plasmids / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014404:Tubulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D006657:Histones; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D014404:Tubulin; D049452:Green Fluorescent Proteins; D012313:RNA