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

Goshima G, Vale RD - J. Cell Biol. (2003)

Bottom Line: Functional redundancy and alternative pathways for completing mitosis were observed for many single RNAi knockdowns, and failure to complete mitosis was observed for only three kinesins.As an example, inhibition of two microtubule-depolymerizing kinesins initially produced monopolar spindles with abnormally long microtubules, but cells eventually formed bipolar spindles by an acentrosomal pole-focusing mechanism.From our phenotypic data, we construct a model for the distinct roles of molecular motors during mitosis in a single metazoan cell type.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94107, USA.

ABSTRACT
Kinesins and dyneins play important roles during cell division. Using RNA interference (RNAi) to deplete individual (or combinations of) motors followed by immunofluorescence and time-lapse microscopy, we have examined the mitotic functions of cytoplasmic dynein and all 25 kinesins in Drosophila S2 cells. We show that four kinesins are involved in bipolar spindle assembly, four kinesins are involved in metaphase chromosome alignment, dynein plays a role in the metaphase-to-anaphase transition, and one kinesin is needed for cytokinesis. Functional redundancy and alternative pathways for completing mitosis were observed for many single RNAi knockdowns, and failure to complete mitosis was observed for only three kinesins. As an example, inhibition of two microtubule-depolymerizing kinesins initially produced monopolar spindles with abnormally long microtubules, but cells eventually formed bipolar spindles by an acentrosomal pole-focusing mechanism. From our phenotypic data, we construct a model for the distinct roles of molecular motors during mitosis in a single metazoan cell type.

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Real-time imaging of GFP-tubulin during the abnormal spindle formation in RNAi cells. Images were taken every 10 s at single optical section using spinning-disk confocal microscopy. (A) A Klp61F [BimC/Eg5] RNAi cell. A monopolar spindle is formed through MTOC fusion. (B) A Ncd [Kin C] RNAi cell. Multiple spindles are formed during prometaphase. (C) Klp10A [Kin I] RNAi cells. Monopolar spindle is initially formed, but is converted to bipolar spindle through acentrosomal pole fusion. (D) Klp67A [Kip3] RNAi cells. A monopolar spindle is maintained for 34 min (top panels), or is eventually converted to a monastral bipolar spindle (bottom panels). See also Videos 5–12 (available at http://www.jcb.org/cgi/content/full/jcb.200303022/DC1. Bars, 5 μm.
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fig4: Real-time imaging of GFP-tubulin during the abnormal spindle formation in RNAi cells. Images were taken every 10 s at single optical section using spinning-disk confocal microscopy. (A) A Klp61F [BimC/Eg5] RNAi cell. A monopolar spindle is formed through MTOC fusion. (B) A Ncd [Kin C] RNAi cell. Multiple spindles are formed during prometaphase. (C) Klp10A [Kin I] RNAi cells. Monopolar spindle is initially formed, but is converted to bipolar spindle through acentrosomal pole fusion. (D) Klp67A [Kip3] RNAi cells. A monopolar spindle is maintained for 34 min (top panels), or is eventually converted to a monastral bipolar spindle (bottom panels). See also Videos 5–12 (available at http://www.jcb.org/cgi/content/full/jcb.200303022/DC1. Bars, 5 μm.

Mentions: RNAi of four kinesins (Klp61F [BimC/Eg5], Klp10A [Kin I], Klp67A [Kip3], and Ncd [Kin C]) produced distinct types of abnormal spindles (Fig. 3; Figs. S2–S6, available at http://www.jcb.org/cgi/content/full/jcb.200303022/DC1). In Klp61F [BimC/Eg5] RNAi cells, the mitotic index was fourfold higher than control cells, and virtually all mitotic cells (97%; n = 102) had monopolar spindles with a single γ-tubulin staining foci at the MTOC, and either a single chromosome mass (72%; Fig. 3 A, top) or scattered chromosomes (25%, Fig. 3 A, bottom; also see Table II and Table III, and Fig. S2). This phenotype is consistent with the fly Klp61F [BimC/Eg5] mutant (Heck et al., 1993). From time-lapse imaging of GFP-tubulin in Klp61F [BimC/Eg5] RNAi cells, it was apparent that the two or more MTOCs in prophase fused into one monopolar spindle after NEB (Fig. 4 A; Video 5). The monopolar spindle was stable for >30 min (Video 6).


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

Goshima G, Vale RD - J. Cell Biol. (2003)

Real-time imaging of GFP-tubulin during the abnormal spindle formation in RNAi cells. Images were taken every 10 s at single optical section using spinning-disk confocal microscopy. (A) A Klp61F [BimC/Eg5] RNAi cell. A monopolar spindle is formed through MTOC fusion. (B) A Ncd [Kin C] RNAi cell. Multiple spindles are formed during prometaphase. (C) Klp10A [Kin I] RNAi cells. Monopolar spindle is initially formed, but is converted to bipolar spindle through acentrosomal pole fusion. (D) Klp67A [Kip3] RNAi cells. A monopolar spindle is maintained for 34 min (top panels), or is eventually converted to a monastral bipolar spindle (bottom panels). See also Videos 5–12 (available at http://www.jcb.org/cgi/content/full/jcb.200303022/DC1. Bars, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172859&req=5

fig4: Real-time imaging of GFP-tubulin during the abnormal spindle formation in RNAi cells. Images were taken every 10 s at single optical section using spinning-disk confocal microscopy. (A) A Klp61F [BimC/Eg5] RNAi cell. A monopolar spindle is formed through MTOC fusion. (B) A Ncd [Kin C] RNAi cell. Multiple spindles are formed during prometaphase. (C) Klp10A [Kin I] RNAi cells. Monopolar spindle is initially formed, but is converted to bipolar spindle through acentrosomal pole fusion. (D) Klp67A [Kip3] RNAi cells. A monopolar spindle is maintained for 34 min (top panels), or is eventually converted to a monastral bipolar spindle (bottom panels). See also Videos 5–12 (available at http://www.jcb.org/cgi/content/full/jcb.200303022/DC1. Bars, 5 μm.
Mentions: RNAi of four kinesins (Klp61F [BimC/Eg5], Klp10A [Kin I], Klp67A [Kip3], and Ncd [Kin C]) produced distinct types of abnormal spindles (Fig. 3; Figs. S2–S6, available at http://www.jcb.org/cgi/content/full/jcb.200303022/DC1). In Klp61F [BimC/Eg5] RNAi cells, the mitotic index was fourfold higher than control cells, and virtually all mitotic cells (97%; n = 102) had monopolar spindles with a single γ-tubulin staining foci at the MTOC, and either a single chromosome mass (72%; Fig. 3 A, top) or scattered chromosomes (25%, Fig. 3 A, bottom; also see Table II and Table III, and Fig. S2). This phenotype is consistent with the fly Klp61F [BimC/Eg5] mutant (Heck et al., 1993). From time-lapse imaging of GFP-tubulin in Klp61F [BimC/Eg5] RNAi cells, it was apparent that the two or more MTOCs in prophase fused into one monopolar spindle after NEB (Fig. 4 A; Video 5). The monopolar spindle was stable for >30 min (Video 6).

Bottom Line: Functional redundancy and alternative pathways for completing mitosis were observed for many single RNAi knockdowns, and failure to complete mitosis was observed for only three kinesins.As an example, inhibition of two microtubule-depolymerizing kinesins initially produced monopolar spindles with abnormally long microtubules, but cells eventually formed bipolar spindles by an acentrosomal pole-focusing mechanism.From our phenotypic data, we construct a model for the distinct roles of molecular motors during mitosis in a single metazoan cell type.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94107, USA.

ABSTRACT
Kinesins and dyneins play important roles during cell division. Using RNA interference (RNAi) to deplete individual (or combinations of) motors followed by immunofluorescence and time-lapse microscopy, we have examined the mitotic functions of cytoplasmic dynein and all 25 kinesins in Drosophila S2 cells. We show that four kinesins are involved in bipolar spindle assembly, four kinesins are involved in metaphase chromosome alignment, dynein plays a role in the metaphase-to-anaphase transition, and one kinesin is needed for cytokinesis. Functional redundancy and alternative pathways for completing mitosis were observed for many single RNAi knockdowns, and failure to complete mitosis was observed for only three kinesins. As an example, inhibition of two microtubule-depolymerizing kinesins initially produced monopolar spindles with abnormally long microtubules, but cells eventually formed bipolar spindles by an acentrosomal pole-focusing mechanism. From our phenotypic data, we construct a model for the distinct roles of molecular motors during mitosis in a single metazoan cell type.

Show MeSH