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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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Mitosis of untreated S2. (A) Untreated S2 cells expressing GFP-tubulin (green) were fixed and stained with γ-tubulin antibody (red) and Hoechst 33342 (blue). Bar, 5 μm. (B) Time-lapse observation of GFP-tubulin in untreated cells, which have variable numbers of prophase MTOCs (Table II). Bipolar spindle was formed directly from two MTOCs (top) or in an extreme case, from eight MTOCs joined through fusion process (middle). The bottom cells failed to complete MTOC fusion, and a tripolar spindle was formed. Images were taken every 10 s (top; single optical section) or 20 s (middle and bottom; Z-projection, 0.66 μm × 10 and 0.58 μm × 10, respectively) using a spinning-disk confocal microscopy. See also Videos 1–3 (available at http://www.jcb.org/cgi/content/full/jcb.200303022/DC1). Bar, 5 μm. (C) Time-lapse phase-contrast images of a GFP-tubulin cell from anaphase to cytokinesis. Phase images were taken every 30 s using wide-field microscopy. The image of GFP-tubulin was obtained at the last frame (1980 s). See also Video 4. Bar, 10 μm.
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fig1: Mitosis of untreated S2. (A) Untreated S2 cells expressing GFP-tubulin (green) were fixed and stained with γ-tubulin antibody (red) and Hoechst 33342 (blue). Bar, 5 μm. (B) Time-lapse observation of GFP-tubulin in untreated cells, which have variable numbers of prophase MTOCs (Table II). Bipolar spindle was formed directly from two MTOCs (top) or in an extreme case, from eight MTOCs joined through fusion process (middle). The bottom cells failed to complete MTOC fusion, and a tripolar spindle was formed. Images were taken every 10 s (top; single optical section) or 20 s (middle and bottom; Z-projection, 0.66 μm × 10 and 0.58 μm × 10, respectively) using a spinning-disk confocal microscopy. See also Videos 1–3 (available at http://www.jcb.org/cgi/content/full/jcb.200303022/DC1). Bar, 5 μm. (C) Time-lapse phase-contrast images of a GFP-tubulin cell from anaphase to cytokinesis. Phase images were taken every 30 s using wide-field microscopy. The image of GFP-tubulin was obtained at the last frame (1980 s). See also Video 4. Bar, 10 μm.

Mentions: To visualize poles of the mitotic spindle, we stained with γ-tubulin antibody in cells stably expressing GFP-tagged tubulin (Rogers et al., 2002). Mitotic γ-tubulin foci always colocalized with microtubule-organizing centers (termed here γ-tubulin–MTOCs) from which astral microtubules emanate and may reflect the presence of centriole-containing centrosomes. In prophase before NEB, we found that the number of γ-tubulin–MTOCs was quite variable (Fig. 1; Fig. S1 B). Half of the cells contained three or more foci of γ-tubulin staining (Table II). Such abnormal γ-tubulin–MTOC numbers in prophase have been observed in many mammalian tumor cell lines (Marx, 2001), and may reflect the long-term propagation and immortalization of the S2 cell line. After NEB, the proportion of cells with three or more γ-tubulin–MTOCs decreased to 30%, suggesting that the MTOC fusion took place during spindle formation. To gain more insight on this abnormal spindle formation process, we performed time-lapse observation of GFP-tubulin in living cells using spinning-disk confocal microscopy. Cells that had two MTOCs in prophase (Fig. 1 B, top row; Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200303022/DC1) successfully formed bipolar spindle within 5 min after NEB. Cells with more than three MTOCs in prophase formed either bipolar spindles through the fusion of MTOCs (Fig. 1 B, middle row; Video 2) or multipolar spindles (Fig. 1 B, bottom row; Video 3). Even in the latter case, MTOC fusion partially took place, as four MTOCs initially observed in prophase fused into three. Although phototoxicity prevented continuous live observation from prophase through to late mitotic stages, fixed-cell images suggest that chromosomes can be segregated equally to daughter cells by multipolar spindles (Fig. S1 B, bottom right cell).


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)

Mitosis of untreated S2. (A) Untreated S2 cells expressing GFP-tubulin (green) were fixed and stained with γ-tubulin antibody (red) and Hoechst 33342 (blue). Bar, 5 μm. (B) Time-lapse observation of GFP-tubulin in untreated cells, which have variable numbers of prophase MTOCs (Table II). Bipolar spindle was formed directly from two MTOCs (top) or in an extreme case, from eight MTOCs joined through fusion process (middle). The bottom cells failed to complete MTOC fusion, and a tripolar spindle was formed. Images were taken every 10 s (top; single optical section) or 20 s (middle and bottom; Z-projection, 0.66 μm × 10 and 0.58 μm × 10, respectively) using a spinning-disk confocal microscopy. See also Videos 1–3 (available at http://www.jcb.org/cgi/content/full/jcb.200303022/DC1). Bar, 5 μm. (C) Time-lapse phase-contrast images of a GFP-tubulin cell from anaphase to cytokinesis. Phase images were taken every 30 s using wide-field microscopy. The image of GFP-tubulin was obtained at the last frame (1980 s). See also Video 4. Bar, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172859&req=5

fig1: Mitosis of untreated S2. (A) Untreated S2 cells expressing GFP-tubulin (green) were fixed and stained with γ-tubulin antibody (red) and Hoechst 33342 (blue). Bar, 5 μm. (B) Time-lapse observation of GFP-tubulin in untreated cells, which have variable numbers of prophase MTOCs (Table II). Bipolar spindle was formed directly from two MTOCs (top) or in an extreme case, from eight MTOCs joined through fusion process (middle). The bottom cells failed to complete MTOC fusion, and a tripolar spindle was formed. Images were taken every 10 s (top; single optical section) or 20 s (middle and bottom; Z-projection, 0.66 μm × 10 and 0.58 μm × 10, respectively) using a spinning-disk confocal microscopy. See also Videos 1–3 (available at http://www.jcb.org/cgi/content/full/jcb.200303022/DC1). Bar, 5 μm. (C) Time-lapse phase-contrast images of a GFP-tubulin cell from anaphase to cytokinesis. Phase images were taken every 30 s using wide-field microscopy. The image of GFP-tubulin was obtained at the last frame (1980 s). See also Video 4. Bar, 10 μm.
Mentions: To visualize poles of the mitotic spindle, we stained with γ-tubulin antibody in cells stably expressing GFP-tagged tubulin (Rogers et al., 2002). Mitotic γ-tubulin foci always colocalized with microtubule-organizing centers (termed here γ-tubulin–MTOCs) from which astral microtubules emanate and may reflect the presence of centriole-containing centrosomes. In prophase before NEB, we found that the number of γ-tubulin–MTOCs was quite variable (Fig. 1; Fig. S1 B). Half of the cells contained three or more foci of γ-tubulin staining (Table II). Such abnormal γ-tubulin–MTOC numbers in prophase have been observed in many mammalian tumor cell lines (Marx, 2001), and may reflect the long-term propagation and immortalization of the S2 cell line. After NEB, the proportion of cells with three or more γ-tubulin–MTOCs decreased to 30%, suggesting that the MTOC fusion took place during spindle formation. To gain more insight on this abnormal spindle formation process, we performed time-lapse observation of GFP-tubulin in living cells using spinning-disk confocal microscopy. Cells that had two MTOCs in prophase (Fig. 1 B, top row; Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200303022/DC1) successfully formed bipolar spindle within 5 min after NEB. Cells with more than three MTOCs in prophase formed either bipolar spindles through the fusion of MTOCs (Fig. 1 B, middle row; Video 2) or multipolar spindles (Fig. 1 B, bottom row; Video 3). Even in the latter case, MTOC fusion partially took place, as four MTOCs initially observed in prophase fused into three. Although phototoxicity prevented continuous live observation from prophase through to late mitotic stages, fixed-cell images suggest that chromosomes can be segregated equally to daughter cells by multipolar spindles (Fig. S1 B, bottom right cell).

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