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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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Accumulation of metaphase by cytoplasmic dynein knockdown. (A) Mitotic index of Dhc64C [DHC] RNAi at d 4 was 1.9-fold higher than controls. (B) Percentage of metaphase and anaphase in mitotic cells. Metaphase cells were accumulated, whereas anaphase was less frequently observed in RNAi samples. (C) Metaphase cells with congressed chromosomes. Cells treated with dsRNA for Dhc64C [DHC] were fixed and stained by γ-tubulin (red) and Hoechst 33342 (green) at d 4. Spindle-like structure was visualized by the overexposure of anti- γ-tubulin signals. Majority of the metaphase cells (64%; n = 22) had two punctate signals of γ-tubulin like untreated cells, and no apparent morphological defects in the spindle were detected (not depicted). Bar, 10 μm.
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fig6: Accumulation of metaphase by cytoplasmic dynein knockdown. (A) Mitotic index of Dhc64C [DHC] RNAi at d 4 was 1.9-fold higher than controls. (B) Percentage of metaphase and anaphase in mitotic cells. Metaphase cells were accumulated, whereas anaphase was less frequently observed in RNAi samples. (C) Metaphase cells with congressed chromosomes. Cells treated with dsRNA for Dhc64C [DHC] were fixed and stained by γ-tubulin (red) and Hoechst 33342 (green) at d 4. Spindle-like structure was visualized by the overexposure of anti- γ-tubulin signals. Majority of the metaphase cells (64%; n = 22) had two punctate signals of γ-tubulin like untreated cells, and no apparent morphological defects in the spindle were detected (not depicted). Bar, 10 μm.

Mentions: Genetic analyses and antibody inhibition experiments in Drosophila suggested that cytoplasmic DHC (Dhc64C) is involved in spindle formation (Robinson et al., 1999; Sharp et al., 2000b), chromosome movement in prometaphase and anaphase A (Sharp et al., 2000a), and transport of Rod, a spindle checkpoint protein (Wojcik et al., 2001). After Dhc64C [DHC] RNAi, we did not observe defects in spindle morphology, γ-tubulin localization, or chromosome segregation, but detected a 1.5-fold accumulation of cells with fully congressed metaphase chromosomes and a higher mitotic index (5.6%; Fig. 6). The congressed chromosome mass was more compact than normal metaphase, most likely reflecting the prolonged metaphase state. This phenotype indicates that the metaphase-to-anaphase transition is significantly delayed by the absence of Dhc64C [DHC], but that chromosome movements and spindle integrity are not affected. Identical anaphase delay phenotypes were observed for both 4 and 7 d of dsRNA treatment (unpublished data).


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)

Accumulation of metaphase by cytoplasmic dynein knockdown. (A) Mitotic index of Dhc64C [DHC] RNAi at d 4 was 1.9-fold higher than controls. (B) Percentage of metaphase and anaphase in mitotic cells. Metaphase cells were accumulated, whereas anaphase was less frequently observed in RNAi samples. (C) Metaphase cells with congressed chromosomes. Cells treated with dsRNA for Dhc64C [DHC] were fixed and stained by γ-tubulin (red) and Hoechst 33342 (green) at d 4. Spindle-like structure was visualized by the overexposure of anti- γ-tubulin signals. Majority of the metaphase cells (64%; n = 22) had two punctate signals of γ-tubulin like untreated cells, and no apparent morphological defects in the spindle were detected (not depicted). Bar, 10 μm.
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Related In: Results  -  Collection

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fig6: Accumulation of metaphase by cytoplasmic dynein knockdown. (A) Mitotic index of Dhc64C [DHC] RNAi at d 4 was 1.9-fold higher than controls. (B) Percentage of metaphase and anaphase in mitotic cells. Metaphase cells were accumulated, whereas anaphase was less frequently observed in RNAi samples. (C) Metaphase cells with congressed chromosomes. Cells treated with dsRNA for Dhc64C [DHC] were fixed and stained by γ-tubulin (red) and Hoechst 33342 (green) at d 4. Spindle-like structure was visualized by the overexposure of anti- γ-tubulin signals. Majority of the metaphase cells (64%; n = 22) had two punctate signals of γ-tubulin like untreated cells, and no apparent morphological defects in the spindle were detected (not depicted). Bar, 10 μm.
Mentions: Genetic analyses and antibody inhibition experiments in Drosophila suggested that cytoplasmic DHC (Dhc64C) is involved in spindle formation (Robinson et al., 1999; Sharp et al., 2000b), chromosome movement in prometaphase and anaphase A (Sharp et al., 2000a), and transport of Rod, a spindle checkpoint protein (Wojcik et al., 2001). After Dhc64C [DHC] RNAi, we did not observe defects in spindle morphology, γ-tubulin localization, or chromosome segregation, but detected a 1.5-fold accumulation of cells with fully congressed metaphase chromosomes and a higher mitotic index (5.6%; Fig. 6). The congressed chromosome mass was more compact than normal metaphase, most likely reflecting the prolonged metaphase state. This phenotype indicates that the metaphase-to-anaphase transition is significantly delayed by the absence of Dhc64C [DHC], but that chromosome movements and spindle integrity are not affected. Identical anaphase delay phenotypes were observed for both 4 and 7 d of dsRNA treatment (unpublished data).

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
Related in: MedlinePlus