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Kinesins klp5+ and klp6+ are required for normal chromosome movement in mitosis

Robert R. West*, Terra Malmstrom and J. Richard McIntosh

Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347, USA



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  		Fig. 2. Chromosome segregation in wild-type and klp{Delta} cells as visualized in live cells over time. The microtubules were visualized with GFP-{alpha}-tubulin (green), and DNA stained with Hoechst 33342 (blue). (A) Wild-type progression through mitosis from metaphase (t=0) to midanaphase B (t=9.4 minutes). (B) A klp6{Delta} mitotic cell with the DNA first as a single mass asymmetrically positioned on the spindle (t=0). The DNA initiates poleward movement (t=7.6 minutes), regresses (t=8.4 minutes), then re-initiates separation (t=9.5 minutes). (C) A klp5{Delta} mitotic cell with DNA separating asymmetrically three times (t=6 minutes; t=10.2 minutes; t=13.3 minutes), finally resulting in equal, wild-type-like segregation (t=25.5 minutes).

 


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  		Fig. 1. klp{Delta} cells have aberrant DNA segregation but normal spindle structure. Live cells with microtubules labeled with GFP-{alpha}-tubulin (green) and DNA with Hoechst 33342 (blue). (A) Wild-type cell in mid-mitosis, showing two equal masses of DNA segregating on an elongating spindle. (B) klp5{Delta} cell in early-to-mid-mitosis with DNA in three masses. (C) Diploid cell with asymmetrically segregating DNA on a long spindle. (D) klp5{Delta} cells in late mitosis with symmetric, wild-type-like DNA segregation. Bar, 5 µm.

 


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  		Fig. 3. Kinetochore staining of wild-type and klp{Delta} cells. Mis12-GFP (Goshima et al., 1999Go) marked the kinetochores (green) and Hoechst 33342 stained the DNA (blue); the direction of subsequent kinetochore movement is indicated by the yellow arrows. (A-D) Wild-type cells with the expected clustered kinetochores throughout mitosis. (E-H) klp5{Delta} cells with the sister kinetochores on each chromosome separated by ~0.6 µm, and moving back and forth along the spindle. Bar, 5 µm.

 


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  		Fig. 4. Deletion of klp5+ or klp6+ extends the time in mitosis, and missegregating chromosomes appear early in mitosis. (A) Graph of the total fraction of klp5{Delta} cells in mitosis (total mitotic) and the fraction of those cells with wild-type (mitotic-NORMAL) versus abnormal (mitotic-ABNORMAL) DNA segregation. The lines show a decay in the frequency of abnormal cells with a corresponding increase in normal cells (r2=0.97). (B) The frequency of the appearance of segregating DNA in cdc25-22 and klp5{Delta} cdc25-22 strains after cell cycle arrest and release. The fraction of cells in mitosis shows a Gaussian distribution (r2=0.98) for both strains. The area under the curve for klp5{Delta} cdc25-22 is 52% greater than that for cdc25-22 cells and its mode is shifted to the right by 22%. Approximately 400 cells were counted for each time point.

 


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  		Fig. 5. Klp5p and Klp6p localize to kinetochores before anaphase. Klp5-GFP and Klp6-GFP were visualized in live cells (green) with Hoechst 33342 staining DNA (blue). (A-D) Klp5p-GFP in different cells through mitosis: prometaphase (A); metaphase (B); anaphase A (C); and anaphase B (D) (t=0-27.4 minutes). Klp6p-GFP in the same cell as it proceeds through mitosis. (E) Klp6p-GFP in the same cell as it proceeds through mitosis. Bar, 5 µm.

 

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© The Company of Biologists Ltd 2002