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.
Highlights
The spindle is a complex microtubule-based superstructure responsible for chromosome movement and segregation during mitosis and meiosis (McIntosh and Koonce, 1989; Mitchison, 1989a; Rieder, 1991; Hyman and Karsenti, 1996; Compton, 2000)
These results indicate that nuclear mitotic apparatus protein (NuMA) and HSET act through overlapping mechanisms to hold microtubule minus ends at spindle poles and permit chromosome movement to be driven by kinetochore- and polar ejection–derived forces
To determine whether chromosome movement in mitosis is affected by disruption of NuMA function, we microinjected NuMA-specific antibodies into the cytoplasm of interphase cells and monitored chromosome movements by time-lapse differential interference contrast (DIC) microscopy of those injected cells that subsequently entered mitosis
Summary
The spindle is a complex microtubule-based superstructure responsible for chromosome movement and segregation during mitosis and meiosis (McIntosh and Koonce, 1989; Mitchison, 1989a; Rieder, 1991; Hyman and Karsenti, 1996; Compton, 2000). Chromosome movement on spindles during mitosis in cultured cells has been well documented (Gorbsky, 1992; Rieder and Salmon, 1994; Inoué and Salmon, 1995; Rieder and Salmon, 1998; Khodjakov et al, 1999) and is driven by three different force-generating mechanisms (Mitchison, 1989b; Gorbsky, 1992; Rieder and Salmon, 1994; Khodjakov and Rieder, 1996; Khodjakov et al, 1999). An implicit assumption in how these force-generating mechanisms cause chromosome movement is that microtubule minus ends are firmly anchored at spindle poles. In its extreme form, this idea posits that if the microtubule minus ends were not appropriately anchored at spindle poles, the poleward forces generated by kinetochore-associated motors (coupled to microtubule depolymerization) would pull the microtubules in toward the chromosome rather than move the chromosome toward the pole.
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