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Opposing Motor Activities Are Required for the Organization of the Mammalian Mitotic Spindle Pole Tirso Gaglio,* Alejandro Saredi,* James B. Bingham,* M. Josh Hasbani,* Steven R. Gill,* Trina A. Schroer,* and Duane A. Compton* *Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755; and*Department of Biology, 220A Mudd Hall, The Johns Hopkins University, Baltimore, Maryland 21218 Abstract. We use both in vitro and in vivo approaches in cultured cells results in the complete lack of organi- to examine the roles of Eg5 (kinesin-related protein), zation of microtubules and the failure to efficiently con- cytoplasmic dynein, and dynactin in the organization of centrate the NuMA protein despite its association with the microtubules and the localization of NuMA (Nu- the microtubules. Simultaneous immunodepletion of clear protein that associates with the Mitotic A__ppara- these proteins from the cell free system for mitotic aster tus) at the polar ends of the mammalian mitotic spindle. assembly indicates that the plus end--directed activity of Perturbation of the function of Eg5 through either im- Eg5 antagonizes the minus end--directed activity of cy- munodepletion from a cell free system for assembly of toplasmic dynein and a minus end--directed activity as- mitotic asters or antibody microinjection into cultured sociated with NuMA during the organization of the mi- cells leads to organized astral microtubule arrays with crotubules into a morphologic pole. Taken together, expanded polar regions in which the minus ends of the these results demonstrate that the unique organization microtubules emanate from a ring-like structure that of the minus ends of microtubules and the localization contains NuMA. Conversely, perturbation of the func- of NuMA at the polar ends of the mammalian mitotic tion of cytoplasmic dynein or dynactin through either spindle can be accomplished in a centrosome-indepen- specific immunodepletion from the cell free system or dent manner by the opposing activities of plus end- and expression of a dominant negative subunit of dynactin minus end-directed motors. HE mitotic spindle is a complex microtubule-based 1990; Holy and Leibler, 1994). In addition to microtubule structure that is responsible for chromosome segre- dynamics, forces exerted by both plus end- and minus T gation during cell division (Mclntosh and Koonce, end-directed microtubule-dependent motors are required 1989; Mitchison, 1989a; Rieder, 1991; Hyman and Kar- for normal assembly of the mitotic spindle. The antagonis- senti, 1996). It is assembled in a spatially and temporally tic nature of these oppositely acting motors has been ele- regulated manner during the cell cycle, and the organiza- gantly explored in Saccharomyces cerevisiae (Saunders tion of the microtubules and the movement and position- and Hoyt, 1992; Hoyt, 1994; Saunders et al., 1995). Prevail- ing of the chromosomes within the mitotic spindle are ing evidence indicates that the counteractive forces gener- achieved through a complex set of forces exerted through ated by both microtubule dynamics and microtubule mo- the microtubule lattice. These forces derive from both the tors are placing the entire mitotic spindle under tension, dynamics of microtubule polymerization (Inoue and Salmon, and the tension created by these counteracting forces ap- 1995) and the actions of microtubule-dependent motor pears to be necessary for the inactivation of a spindle- proteins (Fuller and Wilson, 1992; Barton and Goldstein, dependent cell cycle checkpoint (Rieder et al., 1994; Li 1996). Changes in microtubule polymerization occur pri- and Nicklas, 1995; Murray, 1995; Nicklas et al., 1995). marily at the plus ends of the microtubules, and these The mitotic spindle must be organized in such a way as changes have been postulated to exert force on the chro- to support dynamic changes at the microtubule ends while mosomes in a number of ways, including chromosome simultaneously integrating the forces exerted by microtu- movement mediated by the kinetochore (Koshland et al., bule-dependent motor proteins. Indeed, the observation 1988; Coue et al., 1991), the polar wind (Rieder and of poleward microtubule flux (Mitchison, 1989b; Sawin Salmon, 1994), and chromosome capture (Hayden et al., and Mitchison, 1991) demonstrates that the microtubules within the mitotic spindle are arranged in a configuration Address all correspondence to Duane Compton, Department of Biochem- that is sufficiently stable to perform chromosome move- istry, Dartmouth Medical School, Hanover, NH 03755. Tel,: (603) 650- ments while both the plus and minus ends are available to 1990. Fax: (603) 650-1128. E-mail: [email protected] exchange tubulin subunits. To accomplish this organiza- © The Rockefeller University Press, 0021-9525196/101399/16 $2.00 The Journal of Cell Biology, Volume 135, Number 2, October 1996 399~,14 399 tional task, cells have devised two distinct structural com- tained in DME containing 10% FCS, 2 mM glutamine, 100 IU/ml penicil- plexes that coordinate the dynamics at microtubule ends lin, and 0.1 ~g/ml streptomycin. Cells were grown at 37°C in a humidified with motor-dependent force production (Desai and Mitch- incubator with a 5% CO2 atmosphere. ison, 1995). The best characterized of these is the kineto- chore (Earnshaw and Tomkiel, 1992; Pluta et al., 1995). Antibodies Kinetochores are located at the chromosome centromeres NuMA was detected with either a human-specific mouse mAb (mAblF1; and are responsible for both the attachment of microtu- Compton et al., 1991), a human autoimmune serum (courtesy of Dr. D. bule plus ends to chromosomes and force production dur- Pettijohn, University of Colorado, Boulder, CO), or the rabbit polyclonal ing chromosome movements (Koshland et al., 1988; Nick- antibody (Gaglio et al., 1995). Tubulin was detected using the mAb DMlct (Blose et al., 1984). Eg5 was detected using an affinity purified rabbit las, 1989; Rieder et al., 1990; Coue et al., 1991; Hyman and polyclonal antibody (Sawin et al., 1992a). Dynactin was detected using ei- Mitchison, 1991). Thus, kinetochores must not only serve ther an mAb directed against the Arpl subunit (mAb45A; Schafer et al., as the sites for the localization (and function) of the mo- 1994) or an mAb specific for the pl50B subunit (mAbl50B; Gill, S.R., and tors that power chromosome movement, but they must T.A. Schroer, unpublished results). Cytoplasmic dynein was detected us- ing an mAb specific for IC74 intermediate chain (mAb70.1; Steuer et al., also serve to maintain the attachment of microtubule plus 1991). Finally, pericentrin was detected using a rabbit polyclonal antibody ends with the chromosomes as the microtubules convert generated against the purified recombinant pericentrin protein (Doxsey between growing and shrinking states. et al., 1994). The minus ends of microtubules focused at the spindle pole must also be organized by a unique structural com- Immunological Techniques plex. This putative complex would maintain the organiza- tion of microtubule minus ends in a morphologic pole Indirect immunofluorcscence microscopy was performed on cultured cells while permitting them to release from the centrosome and by immersion in microtubule stabilization buffer (MTSB; 4 M glycerol, 100 mM Pipes, pH 6.9, 1 mM EGTA, and 5 mM MgCl2) for I rain at room allow for tubulin subunit exchange during poleward mi- temperature, extraction in MTSB + 0.5% Triton X-100 for 2 rain, fol- crotubule flux. The identity of the proteins contained lowed by MTSB for 2 rain. Cells were then fixed in -20°C methanol for 10 within this hypothetical mitotic spindle pole organizing rain. Indirect immunofluorescence microscopy on mitotic asters assem- complex are currently unknown, although there is substan- bled in the cell free mitotic extract was performed by dilution of 5 }xl of the extract into 25 ~l of KHM buffer (78 mM KC1, 50 mM Hepes, pH 7.0, tial experimental evidence indicating that a key compo- 4 mM MgCI2, 2 mM EGTA, 1 mM DTT; Burke and Gerace, 1986). The nent is the NuMA protein N(_~_clear protein that associates diluted sample was then spotted onto a poly-L-lysine--coated glass cover- with the Mitotic A_pparatus) 1 (for reviews see Compton slip and fixed by immersion in -20°C methanol. Both the fixed ceils and and Cleveland, 1994; Cleveland, 1995). NuMA is localized mitotic asters were rehydrated in TBS (10 mM Tris-HC1, pH 7.5, 150 mM in the nucleus during interphase but concentrates at the NaCI) containing 1% albumin and all antibody incubations and washes were performed in TBS + 1% albumin. Each primary antibody was incu- polar ends of the mitotic spindle during mitosis (Lyderson bated on the coverslip for 30 min followed by 5-min washes in TBS + 1% and Pettijohn, 1980; Kallajoki et al., 1991; Maekawa et al., albumin, and the bound antibodies were detected using either fluorescein- 1991; Tousson et al., 1991; Compton et al., 1992; Yang et al., or Texas red-conjugated species-specific secondary antibodies at dilutions 1992). Both in vitro and in vivo experiments indicate that of 1:500 (Vector Labs, Inc., Burlingame, CA). The DNA was detected us- ing 4',6-diamidino-2-phenylindole (DAPI) at 0.4 p,g/ml (Sigma Chemical NuMA is required for the organization of the mitotic spin- Co., St. Louis, MOO. After a final wash, the coverslips were mounted in dle (Kallajoki et al., 1991, 1992, 1993; Yang and Snyder, FITC-guard mounting medium (Testog, Inc., Chicago, IL) and observed 1992; Compton and Cleveland, 1993; Compton and Luo, on a microscope equipped for epifluorescence (model Optiphot; Nikon 1995; Gaglio et al., 1995); however, it is unclear if NuMA Inc., Melville, NY). Proteins from the mitotic extracts were solubilized directly with SDS- interacts with any of the microtubule-dependent motors PAGE sample buffer. The proteins were then separated by size using involved in the organization of the microtubules at the mi- SDS-PAGE (Laemmli, 1970) and transferred to polyvinylidene fluoride totic spindle poles or how it becomes concentrated at the (PVDF) membrane (Millipore Corp., Bedford, MA).

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