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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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CENP-E localizes normally to kinetochores in cells lacking organized spindle poles. CFPAC-1 cells that were uninjected (A) or microinjected with antibodies to both NuMA and HSET (B) were fixed and processed for immunofluorescence microscopy using antibodies to the kinetochore-associated motor protein CENP-E and the DNA-specific dye DAPI, as indicated. Bar: 20 μm.
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Figure 6: CENP-E localizes normally to kinetochores in cells lacking organized spindle poles. CFPAC-1 cells that were uninjected (A) or microinjected with antibodies to both NuMA and HSET (B) were fixed and processed for immunofluorescence microscopy using antibodies to the kinetochore-associated motor protein CENP-E and the DNA-specific dye DAPI, as indicated. Bar: 20 μm.

Mentions: That microtubule minus ends were loosely organized at poles after perturbation of NuMA and tightly focused at poles after perturbation of HSET raised the possibility that these proteins play redundant roles in spindle pole function. To test this idea, we injected cells with antibodies to both NuMA and HSET and monitored chromosome dynamics by time-lapse DIC microscopy (Fig. 3). Chromosomes in cells injected with antibodies to both NuMA and HSET appeared to experience Brownian motion, but failed to undergo detectable directed movement (Fig. 3 A). Instead, the chromosomes remained loosely arranged near the cell center. In some cases (e.g., Fig. 3), chromosomes coalesced into a metaphase-like arrangement, but in other cases the chromosomes formed a loose group in the cell center and did not align efficiently (e.g., see Fig. 6 B). Spindles in cells injected with both antibodies were bi-oriented but lacked organized poles, and centrosomes were dissociated from the bulk of the microtubules connected to the chromosomes (Fig. 3 B). The injected antibody was concentrated around the centrosomes as well as in aggregates in the cytoplasm, consistent with the sequestration of NuMA and HSET away from their normal sites of localization (Fig. 3 C). Many of the chromosomes appeared to have K fibers associated with each sister kinetochore, and the K fibers extend normally toward opposite sides of the cell, but K fibers of different chromosomes did not focus at poles. These cells did not enter anaphase, similar to when cells were injected with NuMA-specific antibodies alone.


Chromosome movement in mitosis requires microtubule anchorage at spindle poles.

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

CENP-E localizes normally to kinetochores in cells lacking organized spindle poles. CFPAC-1 cells that were uninjected (A) or microinjected with antibodies to both NuMA and HSET (B) were fixed and processed for immunofluorescence microscopy using antibodies to the kinetochore-associated motor protein CENP-E and the DNA-specific dye DAPI, as indicated. Bar: 20 μm.
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Related In: Results  -  Collection

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

Figure 6: CENP-E localizes normally to kinetochores in cells lacking organized spindle poles. CFPAC-1 cells that were uninjected (A) or microinjected with antibodies to both NuMA and HSET (B) were fixed and processed for immunofluorescence microscopy using antibodies to the kinetochore-associated motor protein CENP-E and the DNA-specific dye DAPI, as indicated. Bar: 20 μm.
Mentions: That microtubule minus ends were loosely organized at poles after perturbation of NuMA and tightly focused at poles after perturbation of HSET raised the possibility that these proteins play redundant roles in spindle pole function. To test this idea, we injected cells with antibodies to both NuMA and HSET and monitored chromosome dynamics by time-lapse DIC microscopy (Fig. 3). Chromosomes in cells injected with antibodies to both NuMA and HSET appeared to experience Brownian motion, but failed to undergo detectable directed movement (Fig. 3 A). Instead, the chromosomes remained loosely arranged near the cell center. In some cases (e.g., Fig. 3), chromosomes coalesced into a metaphase-like arrangement, but in other cases the chromosomes formed a loose group in the cell center and did not align efficiently (e.g., see Fig. 6 B). Spindles in cells injected with both antibodies were bi-oriented but lacked organized poles, and centrosomes were dissociated from the bulk of the microtubules connected to the chromosomes (Fig. 3 B). The injected antibody was concentrated around the centrosomes as well as in aggregates in the cytoplasm, consistent with the sequestration of NuMA and HSET away from their normal sites of localization (Fig. 3 C). Many of the chromosomes appeared to have K fibers associated with each sister kinetochore, and the K fibers extend normally toward opposite sides of the cell, but K fibers of different chromosomes did not focus at poles. These cells did not enter anaphase, similar to when cells were injected with NuMA-specific antibodies alone.

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