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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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Reduction of kinesins and dynein after RNAi. (A) Reduction of seven microtubule motor proteins after dsRNA addition. Immunoblotting of KHC, Ncd, Klp3A, Pav, Cos2, Klp61F, and Dhc64C for cultures subjected to RNAi for 3 (Pav and Klp61F), 4 (others), or 7 (Dhc64C) days. All lanes contained an equal load of total proteins (not depicted). Quantitative analyses (not depicted) indicated >90% of reduction of each protein. (B) Specificity of RNAi. dsRNA against KHC or Ncd did not affect the levels of the other five kinesins. (C) Triple RNAi (KHC/Ncd/Klp3A, right lane) yielded efficient protein reduction of all three kinesins.
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fig2: Reduction of kinesins and dynein after RNAi. (A) Reduction of seven microtubule motor proteins after dsRNA addition. Immunoblotting of KHC, Ncd, Klp3A, Pav, Cos2, Klp61F, and Dhc64C for cultures subjected to RNAi for 3 (Pav and Klp61F), 4 (others), or 7 (Dhc64C) days. All lanes contained an equal load of total proteins (not depicted). Quantitative analyses (not depicted) indicated >90% of reduction of each protein. (B) Specificity of RNAi. dsRNA against KHC or Ncd did not affect the levels of the other five kinesins. (C) Triple RNAi (KHC/Ncd/Klp3A, right lane) yielded efficient protein reduction of all three kinesins.

Mentions: +, protein reduction after RNAi confirmed using specific antibodies (see Fig. 2); -, not tested because antibodies were unavailable.


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)

Reduction of kinesins and dynein after RNAi. (A) Reduction of seven microtubule motor proteins after dsRNA addition. Immunoblotting of KHC, Ncd, Klp3A, Pav, Cos2, Klp61F, and Dhc64C for cultures subjected to RNAi for 3 (Pav and Klp61F), 4 (others), or 7 (Dhc64C) days. All lanes contained an equal load of total proteins (not depicted). Quantitative analyses (not depicted) indicated >90% of reduction of each protein. (B) Specificity of RNAi. dsRNA against KHC or Ncd did not affect the levels of the other five kinesins. (C) Triple RNAi (KHC/Ncd/Klp3A, right lane) yielded efficient protein reduction of all three kinesins.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172859&req=5

fig2: Reduction of kinesins and dynein after RNAi. (A) Reduction of seven microtubule motor proteins after dsRNA addition. Immunoblotting of KHC, Ncd, Klp3A, Pav, Cos2, Klp61F, and Dhc64C for cultures subjected to RNAi for 3 (Pav and Klp61F), 4 (others), or 7 (Dhc64C) days. All lanes contained an equal load of total proteins (not depicted). Quantitative analyses (not depicted) indicated >90% of reduction of each protein. (B) Specificity of RNAi. dsRNA against KHC or Ncd did not affect the levels of the other five kinesins. (C) Triple RNAi (KHC/Ncd/Klp3A, right lane) yielded efficient protein reduction of all three kinesins.
Mentions: +, protein reduction after RNAi confirmed using specific antibodies (see Fig. 2); -, not tested because antibodies were unavailable.

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