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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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Microtubule attachment to kinetochores in microinjected cells. CFPAC-1 cells that were uninjected (A) or microinjected with antibodies to both NuMA and HSET (B) were processed for transmission electron microscopy. Arrows in A and B point to centrosomes. The electron-dense material detected in B near the centrosomes is most likely the antibody-induced aggregates of NuMA and HSET, which are prominently detected by immunofluorescence microscopy. (C) High magnification views to highlight kinetochore-microtubule interactions in the chromosomes identified as 1–4 in B. The kinetochore regions of chromosome 2 were located in the adjacent serial section from that shown in B. Bars: (A and B) 3 μm; (C) 100 nm.
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Figure 5: Microtubule attachment to kinetochores in microinjected cells. CFPAC-1 cells that were uninjected (A) or microinjected with antibodies to both NuMA and HSET (B) were processed for transmission electron microscopy. Arrows in A and B point to centrosomes. The electron-dense material detected in B near the centrosomes is most likely the antibody-induced aggregates of NuMA and HSET, which are prominently detected by immunofluorescence microscopy. (C) High magnification views to highlight kinetochore-microtubule interactions in the chromosomes identified as 1–4 in B. The kinetochore regions of chromosome 2 were located in the adjacent serial section from that shown in B. Bars: (A and B) 3 μm; (C) 100 nm.

Mentions: To verify that microtubules were attached to kinetochores in cells injected with antibodies to both NuMA and HSET, we examined injected cells by transmission electron microscopy (Fig. 5). Uninjected control cells showed organized spindles with tightly focused spindle poles (Fig. 5 A). In contrast, cells injected with antibodies to both NuMA and HSET lacked organized spindle poles (Fig. 5 B), consistent with images obtained by fluorescence microscopy (Fig. 3 B). At high magnification, many of the chromosomes had identifiable kinetochores and multiple microtubules were observed attached to both kinetochores, consistent with the formation of bi-oriented K fibers (Fig. 5 C). These data demonstrate that the lack of directed chromosome movement in the absence of organized spindle poles does not arise from a lack of microtubule binding to kinetochores or formation of K fibers, although this limited analysis does not permit us to determine whether each kinetochore has obtained a full complement of microtubules.


Chromosome movement in mitosis requires microtubule anchorage at spindle poles.

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

Microtubule attachment to kinetochores in microinjected cells. CFPAC-1 cells that were uninjected (A) or microinjected with antibodies to both NuMA and HSET (B) were processed for transmission electron microscopy. Arrows in A and B point to centrosomes. The electron-dense material detected in B near the centrosomes is most likely the antibody-induced aggregates of NuMA and HSET, which are prominently detected by immunofluorescence microscopy. (C) High magnification views to highlight kinetochore-microtubule interactions in the chromosomes identified as 1–4 in B. The kinetochore regions of chromosome 2 were located in the adjacent serial section from that shown in B. Bars: (A and B) 3 μm; (C) 100 nm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Microtubule attachment to kinetochores in microinjected cells. CFPAC-1 cells that were uninjected (A) or microinjected with antibodies to both NuMA and HSET (B) were processed for transmission electron microscopy. Arrows in A and B point to centrosomes. The electron-dense material detected in B near the centrosomes is most likely the antibody-induced aggregates of NuMA and HSET, which are prominently detected by immunofluorescence microscopy. (C) High magnification views to highlight kinetochore-microtubule interactions in the chromosomes identified as 1–4 in B. The kinetochore regions of chromosome 2 were located in the adjacent serial section from that shown in B. Bars: (A and B) 3 μm; (C) 100 nm.
Mentions: To verify that microtubules were attached to kinetochores in cells injected with antibodies to both NuMA and HSET, we examined injected cells by transmission electron microscopy (Fig. 5). Uninjected control cells showed organized spindles with tightly focused spindle poles (Fig. 5 A). In contrast, cells injected with antibodies to both NuMA and HSET lacked organized spindle poles (Fig. 5 B), consistent with images obtained by fluorescence microscopy (Fig. 3 B). At high magnification, many of the chromosomes had identifiable kinetochores and multiple microtubules were observed attached to both kinetochores, consistent with the formation of bi-oriented K fibers (Fig. 5 C). These data demonstrate that the lack of directed chromosome movement in the absence of organized spindle poles does not arise from a lack of microtubule binding to kinetochores or formation of K fibers, although this limited analysis does not permit us to determine whether each kinetochore has obtained a full complement of microtubules.

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