The Kinesin-Related Protein, Hset, Opposes the Activity of Eg5 and Cross-Links Microtubules in the Mammalian Mitotic Spindle

Abstract

We have prepared antibodies specific for HSET, the human homologue of the KAR3 family of minus end-directed motors. Immuno-EM with these antibodies indicates that HSET frequently localizes between microtubules within the mammalian metaphase spindle consistent with a microtubule cross-linking function. Microinjection experiments show that HSET activity is essential for meiotic spindle organization in murine oocytes and taxol-induced aster assembly in cultured cells. However, inhibition of HSET did not affect mitotic spindle architecture or function in cultured cells, indicating that centrosomes mask the role of HSET during mitosis. We also show that (acentrosomal) microtubule asters fail to assemble in vitro without HSET activity, but simultaneous inhibition of HSET and Eg5, a plus end-directed motor, redresses the balance of forces acting on microtubules and restores aster organization. In vivo, centrosomes fail to separate and monopolar spindles assemble without Eg5 activity. Simultaneous inhibition of HSET and Eg5 restores centrosome separation and, in some cases, bipolar spindle formation. Thus, through microtubule cross-linking and oppositely oriented motor activity, HSET and Eg5 participate in spindle assembly and promote spindle bipolarity, although the activity of HSET is not essential for spindle assembly and function in cultured cells because of centrosomes.

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

“…Second, centrosomes remained associated with the spindle, microtubule minus ends were focused at the poles, and spindles were bipolar indicating that inhibition of Kid function restored spindle pole organization to cells lacking NuMA function (compare Figures 1C to D and 3C to D). We hypothesized that spindle poles were organized in the absence of Kid and NuMA function by the activity of the kinesin-related protein HSET, which has been shown to cooperate with NuMA and cytoplasmic dynein in spindle organization (Walczak et al, 1998;Mountain et al, 1999;Gordon et al, 2001). To test this, we simultaneously injected cells with antibodies to NuMA, Kid, and HSET, and found that 90% of cells injected with all three antibodies displayed disorganized poles and unaligned chromosomes ( Figure 3E and Table 1).…”

“…We previously demonstrated that injection of HSET-specific an- tibodies perturbed HSET function, which disrupted spindle pole organization in the absence of either centrosomes or NuMA activity (Mountain et al, 1999;Gordon et al, 2001). However, perturbation of HSET alone did not significantly alter spindle organization, chromosome movement and alignment, or anaphase onset (Mountain et al, 1999;Gordon et al, 2001).…”

“…We previously demonstrated that injection of HSET-specific an- tibodies perturbed HSET function, which disrupted spindle pole organization in the absence of either centrosomes or NuMA activity (Mountain et al, 1999;Gordon et al, 2001). However, perturbation of HSET alone did not significantly alter spindle organization, chromosome movement and alignment, or anaphase onset (Mountain et al, 1999;Gordon et al, 2001). Similarly, simultaneous injection of antibodies to Kid and HSET had no detectable effect on chromosome movement and alignment or spindle organization although the metaphase plate was ϳ10% broader than control cells similar to when cells were injected with Kid antibodies alone ( Figure 3F and Table 1).…”

“…To test whether the lack of efficient chromosome alignment was specific to the perturbation of both Kid and NuMA, we simultaneously injected antibodies to Kid and either HSET or CENP-E. HSET is a kinesin-related protein that cooperates with NuMA and cytoplasmic dynein in organizing microtubule minus ends at spindle poles (Walczak et al, 1998;Mountain et al, 1999;Gordon et al, 2001). We previously demonstrated that injection of HSET-specific an- tibodies perturbed HSET function, which disrupted spindle pole organization in the absence of either centrosomes or NuMA activity (Mountain et al, 1999;Gordon et al, 2001).…”

“…CENP-E was detected with a rabbit polyclonal antibody provided by Tim Yen (Fox Chase Cancer Center, Philadelphia, PA). NuMA-, HSET-, and Kid-specific antibodies used for injections were as described previously (Gaglio et al, 1995;Mountain et al, 1999;Levesque and Compton, 2001). For immunofluorescence staining, NuMA was detected using a human NuMA-specific antibody provided by D. Pettijohn (University of Colorado, Boulder, CO).…”