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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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Bipolar spindle formation defects caused by Klp61F [BimC/Eg5], Ncd [Kin C], Klp10A [Kin I], and Klp67A [Kip3] RNAi. (A) Abnormal spindle formation after RNAi of indicated four kinesins. Cells were fixed and stained by anti-tubulin antibodies (red) and Hoechst 33342 (DNA; green) at d 3 (Klp61F) or d 4 (Ncd, Klp10A and Klp67A). The majority of the mitotic cells had monopolar spindles after Klp61, Klp10A, or Klp67A RNAi, whereas reduction of Ncd induced multipolar spindle formation. In the case of Klp10A or Klp67A RNAi, long bipolar spindles were also observed. (B) γ-Tubulin staining (green) after RNAi. Spindle (red) was visualized by GFP-tubulin expression. Bipolar spindle formed in Klp10A and Klp67A RNAi cells often had only one of the two poles stained for γ-tubulin. Quantitative data and additional cell images are presented in Table II, Table III, and Figs. S2–S8. Prometa, prometaphase; Meta, metaphase; Ana, anaphase. Bars, 10 μm.
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fig3: Bipolar spindle formation defects caused by Klp61F [BimC/Eg5], Ncd [Kin C], Klp10A [Kin I], and Klp67A [Kip3] RNAi. (A) Abnormal spindle formation after RNAi of indicated four kinesins. Cells were fixed and stained by anti-tubulin antibodies (red) and Hoechst 33342 (DNA; green) at d 3 (Klp61F) or d 4 (Ncd, Klp10A and Klp67A). The majority of the mitotic cells had monopolar spindles after Klp61, Klp10A, or Klp67A RNAi, whereas reduction of Ncd induced multipolar spindle formation. In the case of Klp10A or Klp67A RNAi, long bipolar spindles were also observed. (B) γ-Tubulin staining (green) after RNAi. Spindle (red) was visualized by GFP-tubulin expression. Bipolar spindle formed in Klp10A and Klp67A RNAi cells often had only one of the two poles stained for γ-tubulin. Quantitative data and additional cell images are presented in Table II, Table III, and Figs. S2–S8. Prometa, prometaphase; Meta, metaphase; Ana, anaphase. Bars, 10 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)

Bipolar spindle formation defects caused by Klp61F [BimC/Eg5], Ncd [Kin C], Klp10A [Kin I], and Klp67A [Kip3] RNAi. (A) Abnormal spindle formation after RNAi of indicated four kinesins. Cells were fixed and stained by anti-tubulin antibodies (red) and Hoechst 33342 (DNA; green) at d 3 (Klp61F) or d 4 (Ncd, Klp10A and Klp67A). The majority of the mitotic cells had monopolar spindles after Klp61, Klp10A, or Klp67A RNAi, whereas reduction of Ncd induced multipolar spindle formation. In the case of Klp10A or Klp67A RNAi, long bipolar spindles were also observed. (B) γ-Tubulin staining (green) after RNAi. Spindle (red) was visualized by GFP-tubulin expression. Bipolar spindle formed in Klp10A and Klp67A RNAi cells often had only one of the two poles stained for γ-tubulin. Quantitative data and additional cell images are presented in Table II, Table III, and Figs. S2–S8. Prometa, prometaphase; Meta, metaphase; Ana, anaphase. Bars, 10 μm.
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fig3: Bipolar spindle formation defects caused by Klp61F [BimC/Eg5], Ncd [Kin C], Klp10A [Kin I], and Klp67A [Kip3] RNAi. (A) Abnormal spindle formation after RNAi of indicated four kinesins. Cells were fixed and stained by anti-tubulin antibodies (red) and Hoechst 33342 (DNA; green) at d 3 (Klp61F) or d 4 (Ncd, Klp10A and Klp67A). The majority of the mitotic cells had monopolar spindles after Klp61, Klp10A, or Klp67A RNAi, whereas reduction of Ncd induced multipolar spindle formation. In the case of Klp10A or Klp67A RNAi, long bipolar spindles were also observed. (B) γ-Tubulin staining (green) after RNAi. Spindle (red) was visualized by GFP-tubulin expression. Bipolar spindle formed in Klp10A and Klp67A RNAi cells often had only one of the two poles stained for γ-tubulin. Quantitative data and additional cell images are presented in Table II, Table III, and Figs. S2–S8. Prometa, prometaphase; Meta, metaphase; Ana, anaphase. Bars, 10 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.

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