Chromosome Movement in Mitosis Requires Microtubule Anchorage at Spindle Poles

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.

CENP-E Is Essential for Reliable Bioriented Spindle Attachment, but Chromosome Alignment Can Be Achieved via Redundant Mechanisms in Mammalian Cells

“…Studies on HeLa cells disagree as to whether depleting CENP-E from kinetochores causes spindle fragmentation Yao et al, 2000). To resolve these differences, we examined CF-PAC cells, a human cell line that stays flat during mitosis (Gordon et al, 2001). As seen in Figure 5, a significant number of anti-CENP-E-injected CF-PAC cells exhibited multiple spindle poles with chromosomes aligned to the "minor" spindle poles ( Figure 5, b-d, yellow arrows).…”

“…Furthermore, neither the injected antibodies nor an antibody directed against a different CENP-E epitope (Figures 1, c and f, and 6, b and c) detected CENP-E at the kinetochore. Moreover, depletion of CENP-E from kinetochores via antibody injection has functional consequences on aligned chromosomes as exemplified by an 87% drop in Mt-induced stretching between sister kinetochores (Table 2), with remaining tension apparently contributed by other proteins (Gordon et al, 2001). Finally, the mixed phenotype of both congressed and uncongressed chromosomes is found regardless of whether CENP-E disruption is accomplished via antibody injection, transfection of mutants , antisense RNA (Yao et al, 2000), or complete gene disruption in primary cells from mice (Putkey et al, 2000; data reported at the 40th annual meeting of the American Society for Cell Biology).…”

“…Evidently, CENP-E generates 80 -90% of the tension across the centromere independently of chromosome motion. Recent evidence suggests that 20 -30% of the Mt-induced tension across the centromere is dependent upon the spindle poles acting as an anchor for kMts (Gordon et al, 2001), but it is still unclear how these different factors effecting tension interact.…”

A Functional Relationship between NuMA and Kid Is Involved in Both Spindle Organization and Chromosome Alignment in Vertebrate Cells

“…Indirect immunofluorescence microscopy was performed as described previously (Gordon et al, 2001;Levesque and Compton, 2001). Fluorescent images were captured with a Hamamatsu Orca II cooled charge-coupled device camera mounted on an Axioplan 2 microscope (Carl Zeiss, Thornwood, NY) equipped for epifluorescence.…”

“…Methods for time-lapse videomicroscopy and chromosome velocity measurements were described previously (Gordon et al, 2001;Levesque and Compton, 2001). Chromosome velocities were obtained from the digital time-lapse record of each cell.…”