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Chromosome movement in mitosis requires microtubule anchorage at spindle poles.

Gordon MB, Howard L, Compton DA - J. Cell Biol. (2001)

Bottom Line: Perturbation of NuMA alone disrupts spindle pole organization and delays anaphase onset, but does not alter the velocity of oscillatory chromosome movement in prometaphase.Perturbation of HSET alone increases the duration of prometaphase, but does not alter the velocity of chromosome movement in prometaphase or anaphase.These results demonstrate that anchorage of microtubule minus ends at spindle poles mediated by overlapping mechanisms involving both NuMA and HSET is essential for chromosome movement during mitosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.

ABSTRACT
Anchorage of microtubule minus ends at spindle poles has been proposed to bear the load of poleward forces exerted by kinetochore-associated motors so that chromosomes move toward the poles rather than the poles toward the chromosomes. To test this hypothesis, we monitored chromosome movement during mitosis after perturbation of nuclear mitotic apparatus protein (NuMA) and the human homologue of the KIN C motor family (HSET), two noncentrosomal proteins involved in spindle pole organization in animal cells. Perturbation of NuMA alone disrupts spindle pole organization and delays anaphase onset, but does not alter the velocity of oscillatory chromosome movement in prometaphase. Perturbation of HSET alone increases the duration of prometaphase, but does not alter the velocity of chromosome movement in prometaphase or anaphase. In contrast, simultaneous perturbation of both HSET and NuMA severely suppresses directed chromosome movement in prometaphase. Chromosomes coalesce near the center of these cells on bi-oriented spindles that lack organized poles. Immunofluorescence and electron microscopy verify microtubule attachment to sister kinetochores, but this attachment fails to generate proper tension across sister kinetochores. These results demonstrate that anchorage of microtubule minus ends at spindle poles mediated by overlapping mechanisms involving both NuMA and HSET is essential for chromosome movement during mitosis.

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Chromosome movement in a CFPAC-1 cell microinjected with antibodies to NuMA. (A) Selected DIC images from the video record of a mitotic cell that has been microinjected with an antibody to NuMA. Times are indicated in hours:minutes:seconds. The arrow highlights a chromosome that makes a pronounced movement toward the cell equator. (B and C) The cell featured in A (B) and a second, unrelated injected mitotic CFPAC-1 cell (C) were fixed and processed for immunofluorescence microscopy using antibodies specific for tubulin, the injected NuMA antibody, and the DNA-specific dye DAPI, as indicated. Arrows indicate centrosomes and arrowheads indicate sites of microtubule convergence (see Video 1 in online supplement). Bar: 20 μm.
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Figure 1: Chromosome movement in a CFPAC-1 cell microinjected with antibodies to NuMA. (A) Selected DIC images from the video record of a mitotic cell that has been microinjected with an antibody to NuMA. Times are indicated in hours:minutes:seconds. The arrow highlights a chromosome that makes a pronounced movement toward the cell equator. (B and C) The cell featured in A (B) and a second, unrelated injected mitotic CFPAC-1 cell (C) were fixed and processed for immunofluorescence microscopy using antibodies specific for tubulin, the injected NuMA antibody, and the DNA-specific dye DAPI, as indicated. Arrows indicate centrosomes and arrowheads indicate sites of microtubule convergence (see Video 1 in online supplement). Bar: 20 μm.

Mentions: Quicktime videos to accompany Fig. 1Fig. 2Fig. 3Fig. 4 are available at http//:www.jcb.org/cgi/content/full/152/3/425/DC1.


Chromosome movement in mitosis requires microtubule anchorage at spindle poles.

Gordon MB, Howard L, Compton DA - J. Cell Biol. (2001)

Chromosome movement in a CFPAC-1 cell microinjected with antibodies to NuMA. (A) Selected DIC images from the video record of a mitotic cell that has been microinjected with an antibody to NuMA. Times are indicated in hours:minutes:seconds. The arrow highlights a chromosome that makes a pronounced movement toward the cell equator. (B and C) The cell featured in A (B) and a second, unrelated injected mitotic CFPAC-1 cell (C) were fixed and processed for immunofluorescence microscopy using antibodies specific for tubulin, the injected NuMA antibody, and the DNA-specific dye DAPI, as indicated. Arrows indicate centrosomes and arrowheads indicate sites of microtubule convergence (see Video 1 in online supplement). Bar: 20 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196006&req=5

Figure 1: Chromosome movement in a CFPAC-1 cell microinjected with antibodies to NuMA. (A) Selected DIC images from the video record of a mitotic cell that has been microinjected with an antibody to NuMA. Times are indicated in hours:minutes:seconds. The arrow highlights a chromosome that makes a pronounced movement toward the cell equator. (B and C) The cell featured in A (B) and a second, unrelated injected mitotic CFPAC-1 cell (C) were fixed and processed for immunofluorescence microscopy using antibodies specific for tubulin, the injected NuMA antibody, and the DNA-specific dye DAPI, as indicated. Arrows indicate centrosomes and arrowheads indicate sites of microtubule convergence (see Video 1 in online supplement). Bar: 20 μm.
Mentions: Quicktime videos to accompany Fig. 1Fig. 2Fig. 3Fig. 4 are available at http//:www.jcb.org/cgi/content/full/152/3/425/DC1.

Bottom Line: Perturbation of NuMA alone disrupts spindle pole organization and delays anaphase onset, but does not alter the velocity of oscillatory chromosome movement in prometaphase.Perturbation of HSET alone increases the duration of prometaphase, but does not alter the velocity of chromosome movement in prometaphase or anaphase.These results demonstrate that anchorage of microtubule minus ends at spindle poles mediated by overlapping mechanisms involving both NuMA and HSET is essential for chromosome movement during mitosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.

ABSTRACT
Anchorage of microtubule minus ends at spindle poles has been proposed to bear the load of poleward forces exerted by kinetochore-associated motors so that chromosomes move toward the poles rather than the poles toward the chromosomes. To test this hypothesis, we monitored chromosome movement during mitosis after perturbation of nuclear mitotic apparatus protein (NuMA) and the human homologue of the KIN C motor family (HSET), two noncentrosomal proteins involved in spindle pole organization in animal cells. Perturbation of NuMA alone disrupts spindle pole organization and delays anaphase onset, but does not alter the velocity of oscillatory chromosome movement in prometaphase. Perturbation of HSET alone increases the duration of prometaphase, but does not alter the velocity of chromosome movement in prometaphase or anaphase. In contrast, simultaneous perturbation of both HSET and NuMA severely suppresses directed chromosome movement in prometaphase. Chromosomes coalesce near the center of these cells on bi-oriented spindles that lack organized poles. Immunofluorescence and electron microscopy verify microtubule attachment to sister kinetochores, but this attachment fails to generate proper tension across sister kinetochores. These results demonstrate that anchorage of microtubule minus ends at spindle poles mediated by overlapping mechanisms involving both NuMA and HSET is essential for chromosome movement during mitosis.

Show MeSH
Related in: MedlinePlus