The Kinesin-related Protein, HSET, Opposes the Activity of Eg5 and Cross-links Microtubules in the Mammalian Mitotic Spindle

doi:10.1083/jcb.147.2.351

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© The Rockefeller University Press, 0021-9525/1999/10/351/ $5.00
The Journal of Cell Biology, Volume 147, Number 2, October 18, 1999 351-366

Original Article

The Kinesin-related Protein, HSET, Opposes the Activity of Eg5 and Cross-links Microtubules in the Mammalian Mitotic Spindle

Vicki Mountain^a, Calvin Simerly^b, Louisa Howard^c, Asako Ando^d, Gerald Schatten^b, and Duane A. Compton^a
^a Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755
^b Departments of Cell-Developmental Biology, Obstetrics-Gynecology, and Oregon Regional Primate Research Center, Oregon Health Sciences University, Beaverton, Oregon 97006
^c Rippel Electron Microscope Facility, Dartmouth College, Hanover, New Hampshire 03755
^d Department of Genetic Information, Division of Molecular Life Science, University School of Medicine, Bohseidai, Isehara, Kanagawa 259-1193, Japan

Correspondence to: Duane A. Compton, Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755. Tel:(603) 650-1990 Fax:(603) 650-1128 E-mail:duane.a.compton{at}dartmouth.edu.

We have prepared antibodies specific for HSET, the human homologueof the KAR3 family of minus end-directed motors. Immuno-EM withthese antibodies indicates that HSET frequently localizes betweenmicrotubules within the mammalian metaphase spindle consistentwith a microtubule cross-linking function. Microinjection experimentsshow that HSET activity is essential for meiotic spindle organizationin murine oocytes and taxol-induced aster assembly in culturedcells. However, inhibition of HSET did not affect mitotic spindlearchitecture or function in cultured cells, indicating thatcentrosomes mask the role of HSET during mitosis. We also showthat (acentrosomal) microtubule asters fail to assemble in vitrowithout HSET activity, but simultaneous inhibition of HSET andEg5, a plus end-directed motor, redresses the balance of forcesacting on microtubules and restores aster organization. In vivo,centrosomes fail to separate and monopolar spindles assemblewithout Eg5 activity. Simultaneous inhibition of HSET and Eg5restores centrosome separation and, in some cases, bipolar spindleformation. Thus, through microtubule cross-linking and oppositelyoriented motor activity, HSET and Eg5 participate in spindleassembly and promote spindle bipolarity, although the activityof HSET is not essential for spindle assembly and function incultured cells because of centrosomes.

Key Words: mitotic spindle, HSET, Eg5, kinesin, microtubule

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