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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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Chromosome congression defects after CENP-meta [CENP-E], Klp3A [chromokinesin], and Nod [Kid] RNAi. Cells were treated with dsRNA targeting for indicated genes and were fixed and stained by anti-tubulin antibodies (red) and Hoechst 33342 (green) at d 4. Bar in the left column represents 10 μm. Magnified images of the chromosomes are shown in the right column (Bar, 5 μm). Control nontreated cell is shown in A. Misaligned chromosomes were frequently detected after CENP-meta (B) and Klp3A (C) RNAi, whereas stretched chromatin was observed after Nod RNAi (D). More severe misalignment phenotypes appeared when RNAi of a kinetochore kinesin and two chromokinesins were combined in E (see also Table IV). Additional images are presented in Fig. S9 and Fig. S10.
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fig5: Chromosome congression defects after CENP-meta [CENP-E], Klp3A [chromokinesin], and Nod [Kid] RNAi. Cells were treated with dsRNA targeting for indicated genes and were fixed and stained by anti-tubulin antibodies (red) and Hoechst 33342 (green) at d 4. Bar in the left column represents 10 μm. Magnified images of the chromosomes are shown in the right column (Bar, 5 μm). Control nontreated cell is shown in A. Misaligned chromosomes were frequently detected after CENP-meta (B) and Klp3A (C) RNAi, whereas stretched chromatin was observed after Nod RNAi (D). More severe misalignment phenotypes appeared when RNAi of a kinetochore kinesin and two chromokinesins were combined in E (see also Table IV). Additional images are presented in Fig. S9 and Fig. S10.

Mentions: In addition to Klp67A [Kip3] RNAi, depletion of three kinesins, CENP-meta [CENP-E], Klp3A [chromokinesin], and Nod [Kid], caused chromosome misalignment at the metaphase plate. RNAi of Klp3A [chromokinesin] (Williams et al., 1995) and the kinetochore-localized CENP-meta [CENP-E] kinesin (Yucel et al., 2000) exhibited indistinguishable phenotypes: an increased frequency of prometaphase-like cells with bipolar spindles and misaligned chromosomes that were detached from the metaphase plate (Fig. 5, B and C; Table IV; Fig S9, available at http://www.jcb.org/cgi/content/full/jcb.200303022/DC1). The mitotic indices (3.5%) were only slightly higher than the control, and spindle morphology looked normal. Similarly, RNAi of Nod, a DNA-binding kinesin reported to be essential for chromosome alignment in meiosis I (Theurkauf and Hawley, 1992), had no effect on spindle morphogenesis but showed higher frequencies of prometaphase-like cells (Fig. 5 D and Fig. S9 T). In this case, complete separation of chromosomes from the metaphase plate was rare, but most chromosome arms were extended along the spindle axis toward the poles.


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)

Chromosome congression defects after CENP-meta [CENP-E], Klp3A [chromokinesin], and Nod [Kid] RNAi. Cells were treated with dsRNA targeting for indicated genes and were fixed and stained by anti-tubulin antibodies (red) and Hoechst 33342 (green) at d 4. Bar in the left column represents 10 μm. Magnified images of the chromosomes are shown in the right column (Bar, 5 μm). Control nontreated cell is shown in A. Misaligned chromosomes were frequently detected after CENP-meta (B) and Klp3A (C) RNAi, whereas stretched chromatin was observed after Nod RNAi (D). More severe misalignment phenotypes appeared when RNAi of a kinetochore kinesin and two chromokinesins were combined in E (see also Table IV). Additional images are presented in Fig. S9 and Fig. S10.
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getmorefigures.php?uid=PMC2172859&req=5

fig5: Chromosome congression defects after CENP-meta [CENP-E], Klp3A [chromokinesin], and Nod [Kid] RNAi. Cells were treated with dsRNA targeting for indicated genes and were fixed and stained by anti-tubulin antibodies (red) and Hoechst 33342 (green) at d 4. Bar in the left column represents 10 μm. Magnified images of the chromosomes are shown in the right column (Bar, 5 μm). Control nontreated cell is shown in A. Misaligned chromosomes were frequently detected after CENP-meta (B) and Klp3A (C) RNAi, whereas stretched chromatin was observed after Nod RNAi (D). More severe misalignment phenotypes appeared when RNAi of a kinetochore kinesin and two chromokinesins were combined in E (see also Table IV). Additional images are presented in Fig. S9 and Fig. S10.
Mentions: In addition to Klp67A [Kip3] RNAi, depletion of three kinesins, CENP-meta [CENP-E], Klp3A [chromokinesin], and Nod [Kid], caused chromosome misalignment at the metaphase plate. RNAi of Klp3A [chromokinesin] (Williams et al., 1995) and the kinetochore-localized CENP-meta [CENP-E] kinesin (Yucel et al., 2000) exhibited indistinguishable phenotypes: an increased frequency of prometaphase-like cells with bipolar spindles and misaligned chromosomes that were detached from the metaphase plate (Fig. 5, B and C; Table IV; Fig S9, available at http://www.jcb.org/cgi/content/full/jcb.200303022/DC1). The mitotic indices (3.5%) were only slightly higher than the control, and spindle morphology looked normal. Similarly, RNAi of Nod, a DNA-binding kinesin reported to be essential for chromosome alignment in meiosis I (Theurkauf and Hawley, 1992), had no effect on spindle morphogenesis but showed higher frequencies of prometaphase-like cells (Fig. 5 D and Fig. S9 T). In this case, complete separation of chromosomes from the metaphase plate was rare, but most chromosome arms were extended along the spindle axis toward the poles.

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