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Mitosis and Kinesin

 

Research summary of comprehensive RNAi analyses of Drosophila microtubule-base motor proteins

 

Members of the kinesin superfamily of proteins and cytoplasmic dynein have been shown to play major roles as molecular motors in various cargo transports along microtubules and in mitosis. While the roles of microtubule motors in mitosis have been comprehensively analyzed in the budding yeast, which has six kinesin genes belonging to five subfamilies and one cytoplasmic dynein heavy chain (DHC) gene, there have been no comprehensive examination of motor function in a single cell type of multicellular organisms, many of which have more than 20 kinesin genes in their genome.

The Drosophila S2 cell system is excellent for functional analysis of mitotic genes, since they are very sensitive to dsRNA-mediated gene silencing. We have also reported previously that S2 cells spread on concanavalin A (ConA)-coated surfaces and execute normal mitosis (Rogers et al., 2002). This preparation provides outstanding imaging of the mitotic spindle and enables real-time observation of mitotic events by light microscopy (Movie 1, below).

We catalogued the Drosophila microtubule-based motor gene family by BLAST homology, and prepared dsRNAs targeting all 25 kinesins and one cytoplasmic DHC for inhibition. Using 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 S2 cells. We found that RNAi of 8 kinesins and cytoplasmic dynein causes mitotic defects, including monopolar spindle formation (Klp61F [BimC/Eg5], Ncd [KinC], Klp10A [KinI], Klp67A [Kip3]), chromosome misalignment (CENP-meta [CENP-E], Nod [Kid], Klp3A [chromokinesin], Klp67A), anaphase delay (Dhc64C [cDHC]), and cytokinesis failure (Pav [MKLP1]) (Figure 1). Some of the phenotypes are unexpected from the previous studies. 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 depolymerising kinesins (Klp10A and Klp67A) initially produced monopolar spindles with abnormally long microtubules, but cells eventually formed bipolar spindles by an acentrosomal pole-focusing mechanism (Figure 2 and Movie 2, below). This study represents the first comprehensive analysis of microtubule-based motor function during mitosis in a single metazoan cell type, and we constructed a model for the distinct roles of molecular motors during mitosis in this cell type (Figure 3, below) (Goshima and Vale, 2003).

 

Fig. 1

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Fig. 2

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Fig. 3

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Movie 1 - GFP-tubulin in mitosis movie 1 (wt). Time-lapse observation of GFP-tubulin in wild-type S2 cells during bipolar spindle formation. Images are taken every 10 sec using spinning-disk confocal microscopy.

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Movie 1a - S2 Anaphase

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Movie 2 - GFP-tubulin in mitosis movie (klp10A RNAi). Time-lapse observation of GFP-tubulin in S2 cells after Klp10A [Kin I] RNAi. Monopolar spindle is initially formed, but is converted to bipolar spindle through acentrosomal pole formation. Images are taken every 10 sec using spinning-disk confocal microscopy.

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Movie 3 - EB1-GFP in mitosis. Time-lapse observation of EB1-GFP in mitosis. EB1-GFP is accumulated at the growing tips of microtubules. Images were taken every 1 sec using spinning-disk confocal microscopy.

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updated 4/9/07


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