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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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Pav [MKLP1] functions in the formation and maintenance of the central spindle bundling during cytokinesis. (A) Cells with binuclei are abundant after Pav RNAi. Cells were fixed and stained by anti-tubulin antibodies (red) and Hoechst 33342 (green) at d 3. Bar, 10 μm. (B and C) Real-time imaging of GFP-tubulin during cytokinesis in an untreated cell (B) and Pav RNAi cells (C) on Con A–coated dish. Arrow indicates the microtubules splayed apart from the bundle. See also Videos 13–15 (available at http://www.jcb.org/cgi/content/full/jcb.200303022/DC1). Bars, 5 μm.
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fig7: Pav [MKLP1] functions in the formation and maintenance of the central spindle bundling during cytokinesis. (A) Cells with binuclei are abundant after Pav RNAi. Cells were fixed and stained by anti-tubulin antibodies (red) and Hoechst 33342 (green) at d 3. Bar, 10 μm. (B and C) Real-time imaging of GFP-tubulin during cytokinesis in an untreated cell (B) and Pav RNAi cells (C) on Con A–coated dish. Arrow indicates the microtubules splayed apart from the bundle. See also Videos 13–15 (available at http://www.jcb.org/cgi/content/full/jcb.200303022/DC1). Bars, 5 μm.

Mentions: As expected from the previous RNAi analysis in S2 cells (Somma et al., 2002) and mutant fly analysis (Adams et al., 1998), RNAi of Pavarotti (Pav; the orthologue of MKLP1/ZEN-4/CHO1) caused cytokinesis defects and showed multiple nuclei in interphase cell at very high frequencies (75%; Fig. 7 A). RNAi of the other kinesins, including Klp3A [chromokinesin] and Klp38B [Unc104], mutants of which have been shown to exhibit cytokinesis defects in testes and follicle cells, respectively (Williams et al., 1995; Ohkura et al., 1997), did not produce cytokinesis defects in S2 cells.


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)

Pav [MKLP1] functions in the formation and maintenance of the central spindle bundling during cytokinesis. (A) Cells with binuclei are abundant after Pav RNAi. Cells were fixed and stained by anti-tubulin antibodies (red) and Hoechst 33342 (green) at d 3. Bar, 10 μm. (B and C) Real-time imaging of GFP-tubulin during cytokinesis in an untreated cell (B) and Pav RNAi cells (C) on Con A–coated dish. Arrow indicates the microtubules splayed apart from the bundle. See also Videos 13–15 (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

fig7: Pav [MKLP1] functions in the formation and maintenance of the central spindle bundling during cytokinesis. (A) Cells with binuclei are abundant after Pav RNAi. Cells were fixed and stained by anti-tubulin antibodies (red) and Hoechst 33342 (green) at d 3. Bar, 10 μm. (B and C) Real-time imaging of GFP-tubulin during cytokinesis in an untreated cell (B) and Pav RNAi cells (C) on Con A–coated dish. Arrow indicates the microtubules splayed apart from the bundle. See also Videos 13–15 (available at http://www.jcb.org/cgi/content/full/jcb.200303022/DC1). Bars, 5 μm.
Mentions: As expected from the previous RNAi analysis in S2 cells (Somma et al., 2002) and mutant fly analysis (Adams et al., 1998), RNAi of Pavarotti (Pav; the orthologue of MKLP1/ZEN-4/CHO1) caused cytokinesis defects and showed multiple nuclei in interphase cell at very high frequencies (75%; Fig. 7 A). RNAi of the other kinesins, including Klp3A [chromokinesin] and Klp38B [Unc104], mutants of which have been shown to exhibit cytokinesis defects in testes and follicle cells, respectively (Williams et al., 1995; Ohkura et al., 1997), did not produce cytokinesis defects in S2 cells.

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