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CENP-E) Is an Integral Component of Kinetochore Corona Fibers That Link Centromeres to Spindle Microtubules Xuebiao Yao,* Karen L

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CENP-E) Is an Integral Component of Kinetochore Corona Fibers That Link Centromeres to Spindle Microtubules Xuebiao Yao,* Karen L The Microtubule-dependent Motor Centromere–associated Protein E (CENP-E) Is an Integral Component of Kinetochore Corona Fibers That Link Centromeres to Spindle Microtubules Xuebiao Yao,* Karen L. Anderson,* and Don W. Cleveland*‡ *Laboratory of Cell Biology, Ludwig Institute for Cancer Research, ‡Division of Cellular and Molecular Medicine, School of Medicine, University of California, San Diego, La Jolla, California 92093-0660 Abstract. Centromere-associated protein E (CENP-E) oping kinetochores. After stable attachment, through- is a kinesin-related microtubule motor protein that is out chromosome congression, at metaphase, and essential for chromosome congression during mitosis. throughout anaphase A, CENP-E is a constituent of Using immunoelectron microscopy, CENP-E is shown the corona fibers, extending at least 50 nm away from to be an integral component of the kinetochore corona the kinetochore outer plate and intertwining with spin- fibers that tether centromeres to the spindle. Immedi- dle microtubules. In congressing chromosomes, CENP-E ately upon nuclear envelope fragmentation, an associ- is preferentially associated with (or accessible at) the ated plus end motor trafficks cytoplasmic CENP-E stretched, leading kinetochore known to provide the toward chromosomes along astral microtubules that en- primary power for chromosome movement. Taken to- ter the nuclear volume. Before or concurrently with gether, this evidence strongly supports a model in initial lateral attachment of spindle microtubules, which CENP-E functions in congression to tether kine- CENP-E targets to the outermost region of the devel- tochores to the disassembling microtubule plus ends. hromosome movements during mitosis are orches- ciated protein A [CENP-A]1 [attached to centromeric het- trated by the interaction of spindle microtubules erochromatin; Palmer et al., 1991; Pluta et al., 1995], C with a specialized chromosome domain located CENP-B [underneath the inner plate; Cooke et al., 1990], within the centromere. This specialized region, called the and CENP-C [a component of the inner plate; Saitoh et al., kinetochore (Luykx, 1965; Brinkley and Stubblefield, 1966), 1992]). is the site for spindle microtubule–centromere association. A generally accepted idea is that microtubule motors lo- Structurally, the kinetochore is composed of four layers: cated at or near the kinetochore power chromosome an innermost plate that apparently consists of a specialized movement during mitosis (Nicklas, 1989; Rieder and Alex- layer of chromatin, an interzone, an outer plate that has ander, 1990; Hyman and Mitchison, 1991). To date, fluo- been argued to consist of tightly packed fibers (Ris and rescence microscopy has been used to localize three mi- Witt, 1981; Rattner, 1986), and an outermost fuzzy, fibrous crotubule motor proteins to the centromere/kinetochore: corona that is most clearly seen after microtubule disas- cytoplasmic dynein (Pfarr et al., 1990; Steuer et al., 1990), sembly (e.g., Wordeman et al., 1991). Although kineto- CENP-E (Yen et al., 1992), and MCAK/XKCM1 (Worde- chore morphology has been documented in numerous ul- man and Mitchison, 1995; Walzak et al., 1996). Although trastructural studies (e.g., Brinkley and Stubblefield, 1966; cytoplasmic dynein has been implicated in transient asso- Jokelainen, 1967; Comings and Okada, 1973; Roos, 1973; ciation with kinetochores (Pfarr et al., 1990; Steuer et al., Rieder, 1982; McEwen et al., 1993), there is little informa- 1990), microinjection of specific antibodies has resulted in- tion about kinetochore composition and the respective lo- stead in spindle collapse (Vaisberg et al., 1993), rather calization of known kinetochore proteins except for three than a direct effect on chromosome attachment to spin- initially identified as human autoantigens (centromere-asso- dles, disruption of chromosome congression, or movement at anaphase. Dynein has also been shown to be involved in aster formation and spindle pole assembly in Xenopus Address all correspondence to Dr. Don W. Cleveland, 3080 CMM-East, University of California, 9500 Gilman Drive, La Jolla, CA 92093-0660. Tel.: (619) 534-7811. Fax: (619) 534-7659. 1. Abbreviation used in this paper: CENP, centromere-associated protein. The Rockefeller University Press, 0021-9525/97/10/435/13 $2.00 The Journal of Cell Biology, Volume 139, Number 2, October 20, 1997 435–447 http://www.jcb.org 435 (Verde et al., 1991; Heald et al., 1996; Merdes et al., 1996) cholate). The extracts were then sonicated and centrifuged to remove the and HeLa cell (Gaglio et al., 1996) extracts, while evidence residual insoluble materials. The chromosome scaffolds were prepared as described below. Before electrophoresis, an appropriate amount of ex- from budding yeast has proven its role in spindle position- tract was diluted with 43 sample buffer and boiled for 2 min. After sepa- ing (Eshel et al., 1993; Li et al., 1993) with a possible in- ration in SDS-PAGE, the proteins were transferred onto a nitrocellulose volvement in anaphase chromosome segregation (Saun- membrane (Micron Separation Inc., Westborough, MA) and incubated ders et al., 1995). Echeverri et al. (1996) have localized a with anti–CENP-E antibody followed by 125I–protein A. Immunoreactive proportion of p50, a component of the dynactin complex signals were visualized by autoradiography on Kodak BioMAX MS film (Rochester, NY) for 6–8 h at 2808C with an intensifying screen. that can activate cytoplasmic dynein (Steuer et al., 1990), to prometaphase kinetochores followed by release at or after bipolar attachment to spindles. Overexpression of Cell Culture p50 using DNA transfection disrupts spindle assembly and HeLa cells, from American Type Culture Collection (Rockville, MD), eliminates kinetochore-associated cytoplasmic dynein but were maintained as subconfluent monolayers in RPMI 1640 media does not block microtubule attachment to centromeres. (GIBCO BRL; Life Technologies, Gaithersburg, MD) with 10% FCS (Gemini Bio-Products, Inc. Calabasas, CA) and 100 U/ml penicillin plus Rather, the aberrant spindles generally display monopolar 100 mg/ml streptomycin (GIBCO BRL; Life Technologies). attachment of chromosomes near microtubule plus ends, findings demonstrating that initial kinetochore attachment Chromosome Scaffold Preparation to microtubules is mediated, at least in part, by compo- nents other than dynein. Logarithmically growing HeLa cells were treated with 10 ng/ml nocoda- For CENP-E, whose cell cycle–dependent accumulation zole (Sigma Chemical Co.) for 18 h. After arrest, mitotic HeLa cells were harvested by mitotic shake-off and washed with ice-cold PBS. Chromo- yields a maximum of z5,000 molecules per HeLa cell in somes were isolated by the protocol described by Mitchison and Kirschner G2/M, (i.e., about 50 molecules per kinetochore; Brown et (1985). Briefly, mitotic HeLa cells were hypotonically swollen for 5 min at al., 1994), there is evidence that altering its action can af- room temperature in 10 vol of PEM buffer containing 5 mM Pipes, pH 7.2, fect chromosome movements: (a) Antibodies to CENP-E 0.5 mM EDTA, 5 mM MgCl2, 5 mM NaCl, and a protease inhibitor cock- tail (1 mM PMSF, 2 mg/ml aprotinin, 2 mg/ml pepstatin A, and 2 mg/ml leu- do inhibit poleward chromosome movements powered by peptin). The hypotonically swollen cells were harvested by centrifugation microtubule disassembly in vitro (Lombillo et al., 1995a); and homogenized in PEM buffer containing 0.1% digitonin (Sigma Chem- (b) antibody injection into cells slows the metaphase- ical Co.). The homogenates were clarified to remove nuclei and the super- to-anaphase transition (Yen et al., 1992); (c) antibody in- natant was loaded onto a stepwise gradient containing 30, 40, 50, and 60% jection into mouse eggs completely blocks meiosis I at sucrose in PEM buffer and centrifuged (2,500 g for 15 min) at 48C. After centrifugation, a visible, flocculent band migrating at the 50–60% prometaphase/metaphase (Duesbery et al., 1997); and (d) sucrose interphase was harvested and suspended in 3 vol of PEM buffer. immunodepletion of CENP-E from Xenopus egg extracts A subsequent chromosome scaffold preparation was performed according blocks chromosome congression but not attachment to to the protocol described by Lewis and Laemmli (1982). spindles assembled in vitro (Wood et al., 1997). The sum of this evidence suggests that CENP-E functions as a kinet- Immunofluorescence Microscopy ochore-associated microtubule motor, but to better under- For immunolabeling, cells were trypsinized and seeded onto acid-treated stand the exact molecular function of the motor, it is im- sterile 18-mm coverslips in six-well dishes (Corning Glass Works, Corn- portant to know in which of the four layers of the kinetochore ing, NY). After reaching 75% confluence in z36 h, cells were rinsed for 1 CENP-E is located, and whether or how CENP-E distribu- min with PHEM buffer (100 mM Pipes, 20 mM Hepes, pH 6.9, 5 mM tion changes during the various phases of chromosome EGTA, 2 mM MgCl2, and 4 M glycerol) and permeabilized for 1 min with movement in mitosis. Using immunoelectron microscopy, PHEM plus 0.1% Triton X-100 as described (Compton et al., 1992). In some instances, 5 mM Taxol (Sigma Chemical Co.) was included to mini- we now show that CENP-E binds to the outer surface of mize the depolymerization of microtubules during extraction. Extracted the immature kinetochores early in prometaphase, consis- cells were then fixed in 4% freshly made paraformaldehyde (Polysciences, tent with
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