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Address correspondence to Duane A. Compton, Dept. of Biochemistry, Dartmouth Medical School, 410 Remsen Bldg., Hanover, NH 03755. Tel.: (603) 650-1990. Fax: (603) 650-1128. email: duane.a.compton{at}dartmouth.edu
Abstract
Although the microtubule-depolymerizing KinI motor Kif2a is abundantly expressed in neuronal cells, we now show it localizes to centrosomes and spindle poles during mitosis in cultured cells. RNAi-induced knockdown of Kif2a expression inhibited cell cycle progression because cells assembled monopolar spindles. Bipolar spindle assembly was restored in cells lacking Kif2a by treatments that altered microtubule assembly (nocodazole), eliminated kinetochoremicrotubule attachment (loss of Nuf2), or stabilized microtubule plus ends at kinetochores (loss of MCAK). Thus, two KinI motors, MCAK and Kif2a, play distinct roles in mitosis, and MCAK activity at kinetochores must be balanced by Kif2a activity at poles for spindle bipolarity. These treatments failed to restore bipolarity to cells lacking the activity of the kinesin Eg5. Thus, two independent pathways contribute to spindle bipolarity, with the Eg5-dependent pathway using motor force to drive spindle bipolarity and the Kif2a-dependent pathway relying on microtubule polymer dynamics to generate force for spindle bipolarity.
Key Words: mitotic spindle; kinetochore; microtubule; centrosome, poleward flux
Introduction
The spindle is a microtubule-based structure responsible for accurate chromosome segregation during mitosis and meiosis (McIntosh and Koonce, 1989; Hyman and Karsenti, 1996; Compton, 2000). Organization of microtubules into a fusiform, bipolar spindle requires numerous nontubulin accessory proteins including several members of the kinesin gene family (Vale and Fletterick, 1997; Sharp et al., 2000). Kinesin-related proteins with the conserved motor domain positioned at the NH2 terminus (KinN kinesins) display plus-enddirected motility and perform diverse functions during spindle morphogenesis including kinetochoremicrotubule interactions (CENP-E; Yao et al., 1997), generation of polar ejection force (Kid; Levesque and Compton, 2001), and spindle bipolarity (Eg5; Sawin et al., 1992). Kinesin-related proteins with the conserved motor domain positioned at the COOH terminus (KinC kinesins) display minus-enddirected motility and participate in focusing microtubule minus ends at spindle poles (Endow et al., 1994; Mountain et al., 1999). Finally, kinesin-related proteins with the conserved motor domain positioned in the middle of the protein (KinI kinesins) lack motility and use their catalytic motor domains to induce conformation changes in protofilament structure at microtubule ends, which stimulates microtubule depolymerization by promoting catastrophe (Desai et al., 1999; Moores et al., 2002; Ogawa et al., 2004). MCAK/XKCM1/Kif2c (hereafter referred to as MCAK) is the best characterized member of the KinI subgroup. MCAK localizes to kinetochores and spindle microtubules (Wordeman and Mitchison, 1995; Walczak et al., 1996) where it regulates microtubule dynamics essential for proper chromosome attachment to spindle microtubules, chromosome movement, and correction of attachment errors (Maney et al., 1998, 2001; Kline-Smith and Walczak, 2002; Walczak et al., 2002; Hunter et al., 2003; Ohi et al., 2003; Kline-Smith et al., 2004). However, MCAK is not the only member of KinI kinesin-related proteins. Kif2a is a closely related KinI kinesin-related protein that plays a role in neurogenesis (Noda et al., 1995; Debernardi et al., 1997; Morfini et al., 1997; Homma et al., 2003). In this paper we demonstrate that Kif2a is essential for bipolar spindle assembly during mitosis in cultured vertebrate cells.
Results and discussion
To build Kif2a-specific antibodies we immunized rabbits with the unique 119 amino acids of the NH2-terminal end of human Kif2a. A single protein species of 110 kD was detected by immunoblot analysis with these antibodies in total cell extracts prepared from human HeLa, hamster CHO, and frog A6 cells (Fig. 1 A). The molecular mass of this protein is consistent with the predicted size of Kif2a, indicating that the antibodies are specific for Kif2a, they cross react to Kif2a from several vertebrate species, and Kif2a is expressed in cultured vertebrate cells. Kif2a localized to centrosomes in interphase and mitotic human CFPAC-1 cells (Fig. 1, BF), but also to spindle microtubules being concentrated at poles (Fig. 1, DF). In anaphase and telophase, Kif2a localized to the spindle midzone and midbody in addition to spindle poles (Fig. 1 F). We detected Kif2a at kinetochores, but the signal was just slightly above background levels and was only observed after depolymerization of microtubules with nocodazole (unpublished data). This localization pattern for Kif2a is different from that reported for the related KinI motor MCAK (Wordeman and Mitchison, 1995; Walczak et al., 1996).
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To test if kinetochore-induced microtubule depolymerization is necessary for monopolar spindle assembly in the absence of Kif2a, we eliminated kinetochoremicrotubule attachment using siRNA to knock down expression of Nuf2, a component of a conserved, essential kinetochore complex containing Nuf2 and Hec1 (Fig. 5; DeLuca et al., 2003). We used Hec1-specific antibodies to monitor the efficiency of Nuf2 knockdown because the stability of Hec1 relies on the presence of Nuf2 (DeLuca et al., 2003), and Nuf2-specific antibodies were not available. 48 h after transfection, immunoblot analysis indicated that Kif2a and Hec1 were reduced to levels equivalent to when each protein was knocked down alone (Fig. 5 A). Most (98%; n = 77, three independent experiments) cells lacking Nuf2 alone had bipolar spindles with unattached chromosomes (Fig. 5 C), which is consistent with previous findings (DeLuca et al., 2003). In contrast to the monopolar spindles formed in cells lacking Kif2a alone (Fig. 5 D),
94% (n = 213, three independent experiments) of cells lacking both Kif2a and Nuf2 assembled bipolar spindles with unattached chromosomes (Fig. 5, E and H). Knockdown of Nuf2 failed to restore bipolarity to cells lacking Eg5 activity (Fig. 5, F, G, and I; n = 83, three independent experiments), and knockdown of both MCAK and Nuf2 showed no detectable difference from knockdown of Nuf2 alone (not depicted).
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Materials and methods
Cell culture
Human HeLa, CFPAC-1, and U2OS cells were maintained in Dulbecco's modified medium, Iscove's modified Dulbecco's medium, and McCoy's medium, respectively, containing 10% FBS, 50 IU/ml penicillin, and 50 µg/ml streptomycin.
Antibodies
The region of the cDNA encoding the NH2-terminal 119 aa of human Kif2a (GenBank/EMBL/DDBJ accession no. BG721281) was amplified using forward (5'-ATCGGATCCTGCTGCTCCAGATGAGGT-3') and reverse (5'-CTTCTCTGGGGCACGGGAAATTCTTAAG-3') primers and inserted into the BamHIEcoRI sites of pRSET-B plasmid (Invitrogen). The His-tagged recombinant protein was purified by affinity chromatography and used to immunize two rabbits (Covance Research Products).
Other antibodies used in this work included MCAK (Mack and Compton, 2001), DM1 (Sigma-Aldrich), Hec1 (Novus Biologicals), and human anticentrosome antibody (provided by J.B. Rattner, University of Calgary, Alberta, Canada).
Indirect immunofluorescence microscopy
Cells were extracted in microtubule-stabilizing buffer (4 M glycerol, 100 mM Pipes, pH 6.9, 1 mM EGTA, 5 mM MgCl2, and 0.5% Triton X-100) followed by fixation in methanol (Kif2a staining) or 1% glutaraldehyde (microtubule staining). Subsequent antibody incubations and washes were done in TBS-BSA (10 mM Tris, pH 7.5, 150 mM NaCl, and 1% BSA). Primary antibodies were detected using speciesspecific fluorescein- or Texas redconjugated secondary antibodies (Vector Laboratories). DNA was detected with 0.2 µg/ml DAPI (Sigma-Aldrich). Coverslips were mounted with ProLong Antifade mounting medium (Molecular Probes).
Fluorescent images were captured with a cooled CCD camera (model Orca II; Hamamatsu) mounted on a microscope (model Axioplan 2; Carl Zeiss MicroImaging, Inc.). A series of 0.5-µm optical sections were collected in the z plane for each channel (DAPI, fluorescein, and/or Texas red) using a 63x 1.4NA objective and deconvolved using Openlab software (Improvision Inc.) to eliminate extraneous fluorescence background.
RNA interference
Kif2a levels were reduced using the sequence 5'-GGCAAAGAGAUUGACCUGG-3'. Nuf2 and MCAK levels were reduced using published sequences (DeLuca et al., 2003; Cassimeris and Morabito, 2004). All siRNA duplexes were synthesized with 3' dTdT overhangs and were annealed (Ambion). Approximately 50,000 U2OS cells were plated on coverslips in 35-mm dishes the day before transfection and grown without antibiotics. Double stranded RNAs were transfected into cells using OligofectamineTM reagent (Invitrogen) as described previously (Elbashir et al., 2001). Samples were analyzed 48 h after transfection by either indirect immunofluorescence or immunoblot analysis.
Online supplemental material
Fig. S1 shows centrosome position during both prophase and metaphase in control cells and cells lacking Kif2a. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200404012/DC1.
Acknowledgments
We thank J. Gaetz and T. Kapoor for sharing unpublished data and J.B. Rattner for providing human anticentrosome sera.
This work was supported by National Institutes of Health grants GM51542 (D.A. Compton) and GM008704 (N. Ganem).
Submitted: 2 April 2004
Accepted: 6 July 2004
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