Cohesin Subunit SMC1 Associates with Mitotic Microtubules at the Spindle Pole

Cohesin subunit SMC1 associates with mitotic microtubules at the spindle pole

Richard W. Wong* and Gu¨ nter Blobel†

Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10065

Contributed by Gu¨nter Blobel, August 7, 2008 (sent for review June 25, 2008) Accurate mitotic chromosome segregation depends on the forma- 24, 25). However, the molecular mechanism by which cohesin tion of a microtubule-based bipolar spindle apparatus. We report functions in spindle assembly was not addressed in these studies. that the cohesin subunit structural maintenance of chromosomes subunit 1 (SMC1) is recruited to microtubule-bound RNA export Results and Discussion factor 1 (Rae1) at the mitotic spindle pole. We locate the Rae1- SMC1 Interacts with Rae1 During Mitosis. To investigate whether binding site to a 21-residue-long region, SMC1947-967 and provide microtubule-bound Rae1 might be involved in the recruitment of several lines of evidence that phosphorylation of Ser957 and Ser966 SMC1 to mitotic spindle poles, we carried out immunoprecipi- of SMC1 stimulates binding to Rae1. Imbalances in these assembly tation of mitotic HeLa cell extracts. Coimmunoprecipitated pathways caused formation of multipolar spindles. Our data sug- proteins were separated by SDS/PAGE and proteins of interest gest that cohesin’s known bundling function for chromatids in identified by immunoblotting. We found that Rae1 antibodies mitotic and interphase cells extends to microtubules at the spindle coimmunoprecipitated both SMC1 and SMC3, along with pole. Nup98 and tubulin (Fig. 1A); conversely, SMC1 antibodies coimmunoprecipitated Rae1 and SMC3 but not Nup98 (Fig. 1B). biochemical mapping ͉ immunoﬂuorescence microscopy ͉ in vitro aster To test the strength of the associations with Rae1, we treated the ⅐ formation ͉ inhibitors of phosphorylation ͉ phosphorylation Rae1 immunoprecipitate with 2 M guanidine HCl and found that SMC3 was solubilized, whereas SMC1 remained associated with Rae1 (Fig. 1C). These data suggested that SMC1 interacts uring the cell cycle, Rae1 (also termed Gle2 or mrnp41) with Rae1. D(1–3) dynamically partitions between nuclear pore complexes (NPCs) (4), mRNPs (1), and microtubules (2, 5). As a beta SMC1 and Rae1 Colocalize at Spindle Pole. To visualize and localize propeller protein, Rae1 is ideally suited for serving as a protein these interactions, we carried out two assays, immunofluores- interaction platform. It binds to the Gle2-binding site (GLEBS) cence of permeabilized HeLa cells (Fig. 2 A and B) and in vitro of the nucleoporin Nup98 and to the Ser/Thr protein kinase assembly of asters from mitotic extracts of HeLa cells [Fig. 2 C budding uninhibited by benzimidazole (Bub1) (3, 6). Rae1– and D and supporting information (SI) Fig. S1]. In digitonin- Nup98 interaction prevents aneuploidy by inhibiting securin permeabilized HeLa cells, SMC1 localized to the spindle (Fig. degradation (7). We previously reported that microtubule- 2A), and colocalized with Rae1 at the spindle pole region (Fig. bound Rae1 recruits nuclear mitotic apparatus protein (NuMA) 2B). In the in vitro aster assembly assays, Rae1 localized along (8) to the mitotic spindle. We located NuMA’s binding site for the aster microtubules, showing a noticeable concentration in Rae1 to an N-terminal segment of NuMA and showed that in the center of the aster (Fig. 2C). Strikingly, SMC1 located almost vivo expression of this fragment and imbalances created in this exclusively to the center of the aster (Fig. 2D). In mitotic extracts assembly pathway, caused formation of multipolar spindles (2). that were immunodepleted of Rae1, SMC1, or SMC3, microtu- SMC1 has been reported to play a role in spindle pole bules failed to organize into recognizable mitotic asters (Figs.

formation (9). SMC1 is a large protein with a globular ATP- S2–S4). Extracts that were mock-depleted by rabbit IgG had no CELL BIOLOGY binding site at both ends and a long helical domain that is effect on aster formation (data not shown). Together with the interrupted near its center by a nonhelical region. The two long immunoprecipitation data, these results suggested that SMC1 helical regions loop back on each other and are predicted to form and SMC3 interact with microtubules and Rae1 at the spindle a 50-nm-long intramolecular coiled coil, yielding two vicinal pole region. ATP-binding sites at one end with the central nonhelical region forming a globular domain at the other end of the folded Biochemical Mapping of Rae1–SMC1 Interaction Domain. To more molecule (10–12). SMC1 pairs with a similarly built SMC3 closely examine binding of Rae1 to SMC1 that was suggested by molecule at their respective central globular regions called the the immunoprecipitation data (Fig. 1C) and to biochemically hinge domain (see Fig. 3A). The heterodimer’s ATP-binding site map the region of SMC1 that interacts with Rae1, we con- terminals are reversibly circularized by two proteins, Scc1/ structed a series of cDNA fragments covering the entire length Rad21, and Scc3/SA (12). The resulting heteromultimeric com- of SMC1 (Fig. 3A Lower) and coexpressed these constructs plex is termed cohesin and functions in tethering mitotic and together with Flag-tagged Rae1 cDNA in a cell-free reticulocyte meiotic sister chromatids (13). In addition, cohesin has been reported to function in DNA repair (14, 15), in the morphogen- Author contributions: R.W.W. and G.B. designed research; R.W.W. performed research; esis of nondividing neurons (16, 17) and in the regulation of gene R.W.W. and G.B. analyzed data; and R.W.W. and G.B. wrote the paper. transcription (18–21) in postmitotic cells. After ionizing radia- The authors declare no conﬂict of interest. tion, SMC1 is phosphorylated by the kinase Ataxia Telangiec- Freely available online through the PNAS open access option. tasia Mutated (ATM) at Ser 957 and Ser 966 (15, 22, 23). *To whom correspondence may be addressed at the present address: Frontier Science By immunofluorescence microscopy, SMC3 was colocalized Organization, Cancer Research Institute, Kanazawa University, 920-0934, Japan. E-mail: with NuMA at spindle poles, although its association with these [email protected]. structures has not been further defined (9). Moreover, immu- †To whom correspondence may be addressed. E-mail: [email protected]. nodepletion of SMC1 from mitotic extracts resulted in failure of This article contains supporting information online at www.pnas.org/cgi/content/full/ spindle aster assembly (9, 24). It was therefore suggested that 0807660105/DCSupplemental. cohesin may be involved in spindle assembly during mitosis (9, © 2008 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0807660105 PNAS ͉ October 7, 2008 ͉ vol. 105 ͉ no. 40 ͉ 15441–15445 Downloaded by guest on September 28, 2021 Fig. 1. Human cohesin subunit SMC1 interacts with Rae1 during mitosis. IP from mitotic HeLa cell extracts with anti-Rae1, anti-SMC1, or nonspeciﬁc rabbit antibodies (IgG) were analyzed by SDS/PAGE, followed by immunoblotting with Rae1, SMC1, SMC3, Nup98 or tubulin (DM1A) antibodies. In lanes marked ‘‘2% input,’’ 5 ␮lof250␮l of extract that was used per IP was analyzed directly (A and B). Anti-Rae1 IP was washed with 2.0 M guanidine⅐HCl before being analyzed by SDS/PAGE and immunoblotting with anti-SMC1 or -SMC3 antibodies (C).

translation system. We found that Flag-tagged Rae1 pulled down shorter two fragments, HA-SMC1947-1025, or HA-SMC1947-967,or only the C-terminal fragment 5, containing residues 947-1233 of an empty HA-vector in HeLa cells. As shown in Fig. 3E, Rae1 SMC1 (see lanes 1–5 of Fig. 3B). This fragment, SMC1–5, was specifically pulled down by either SMC1 fragments, but not contains the entire C-terminal ATP binding domain and part of by an empty HA vector alone. We conclude that the shortest the upstream alpha-helical region (Fig. 3A Lower). To further fragment containing only 21 residues, SMC1947-967, was sufficient fine-map the Rae1 association site on SMC1–5, we shortened its to associate with Rae1, both in vitro and in vivo. Close inspection C-terminal end to obtain fragments 5A (residues 947-1100), 5B of the 21-residues-long SMC1–5C fragment revealed that it (residues 947-1025), and 5C (residues 947–967) (Fig. 3C). All contains a consensus WD-40 repeat-binding site (SXXXS) (26), three fragments of SMC1 associated with Flag-tagged Rae1 that strongly supporting our biochemical mapping data that this was cosynthesized in the reticulocyte translation system (Fig. region of SMC1 binds the WD-40 beta propeller Rae1. 3D). To test for interaction with Rae1 in vivo, we expressed the Phosphorylation of SMC1947-967. It was previously reported that the protein kinase ATM phosphorylates SMC1 at two Ser residues, S957 and S966, and that this phosphorylation is required in DNA A repair (14, 22). To investigate whether phosphorylation of SMC1 is also involved in subsequent interaction of phosphorylated SMC1 with Rae1, we used two kinase inhibitors, LY294002 and caffeine, that inhibit ATM and related kinases. We observed that the interaction between Rae1 and SMC1 in the reticulocyte translation system was abolished at the higher concentration of these inhibitors (Fig. 4A and Fig. S5). We conclude that phos- B phorylation of SMC1, whether by ATM or another kinase that is present in the reticulocyte lysate, greatly stimulates interaction with Rae1 and that this interaction is abolished in the presence of two kinase inhibitors. To investigate whether it is specifically phosphorylation of S957 or S966 alone, or of both, enhances binding to Rae1, we tested SMC1-S957A, SMC1-S966A, and the double SMC1 mutant, SMC1-S957A-S966A (14). Translation together with Flag- C tagged Rae1 in the reticulocyte system and subsequent pulldown with Flag-tagged Rae1 (Fig. 4B), showed that conversion of Ser into Ala at these sites greatly reduced, but did not completely abolish interaction with Rae1: interaction of the SMC1 mutants with Rae1 was still detected after longer exposure of the gel (compare lane 1 with lanes 2–4). To examine whether the SMC1 mutants could interact with Rae1 in vivo, we cotransfected HeLa D cells with myc-tagged wild-type or the mutant SMC1 constructs together with HA-Rae1. Myc antibody-conjugated beads were used for pulling down associated proteins that were in turn detected by immunoblotting (Fig. 4C). SMC1 mutants were clearly less efficient in pulling out Rae1, compared with wild- type SMC1. However, as observed for the in vitro experiments Fig. 2. Colocalization of SMC1 and Rae1 at the spindle pole. Confocal (Fig. 4B), the interaction of SMC1 mutants with Rae1 were not microscopy of mitotic HeLa cells that were ﬁxed and permeabilized with cold abolished. Together, these experiments suggested that in vitro methanol and 0.3% digitonin and costained with anti-SMC1 (red) and -tubulin and in vivo phosphorylation of SMC1 at Ser957 and S966 stimu- (green) (A) or anti-SMC1 (green) and -Rae1 (red) (B). DNA was visualized by using DAPI (blue). Confocal microscopy of asters that were formed in vitro lates interaction of Rae1 with SMC1. It will be interesting to from mitotic HeLa cell extracts and that were visualized by anti-tubulin determine whether the reported ATM-mediated specific phos- (green) and anti-Rae1 (red) (C) or anti-tubulin (green) and -SMC1 (red) (D). phorylation of SMC1 in a DNA repair pathway also leads to the (Scale bar, 5 ␮m.) recruitment of Rae1.

15442 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0807660105 Wong and Blobel Downloaded by guest on September 28, 2021 A A BC B

D E C

Fig. 4. Phosphorylation of Ser957 and Ser966 of SMC1 stimulate binding to Rae1. The kinase inhibitor LY294002 was added at the indicated concentra- Fig. 3. Mapping of the SMC1 domain interacting with Rae1. Schematic tions to the reticulocyte system that was programmed with Rae1-Flag and full drawing of the hinge-like structure known to be formed by the two cohesin length SMC1 cDNA; Rae1-Flag pulldowns were analyzed by SDS/PAGE (18% subunits, SMC1 and SMC3; purple stars indicates the location of the two Ser acryalmide gel) and autoradiography (A). (B)AsinA, except that the incuba- residues that have been reported to be phosphorylated by ATM (A Upper). tions contained the indicated SMC1 wild type or mutant constructs. HeLa cells Domain structure of SMC1 and the constructs 1–5 covering the entire length were cotransfected with Rae1-HA and Myc-SMC1-WT, Myc-SMC1-S957A, Myc- of SMC1; numbers refer to amino acid residues (A Lower). Each of the ﬁve SMC1-S966A, or Myc-SMC1- DBA (double S957A and S966A) and treated with untagged SMC1 fragments (see above) and Flag-tagged Rae1 were synthe- nocodazole (2); anti-Myc IPs of lysates were analyzed by SDS/PAGE and im- sized in 35S methionine containing reticulocyte lysate followed by pulldown munoblotted with HA antibodies (C Upper). In a control, cotransfected cell using anti-Flag coated beads. Pulldown samples were resolved in a 4–20% lysates were blotted with anti-HA and -Myc antibodies (C Lower); aster formed SDS-polyacrylamide gradient gel and visualized by autoradiography. Note in vitro and stained with tubulin antibodies (green) and ATM antibodies (red) that Rae1 pulled down SMC1 fragment 5, containing residues 947-1233 (A (D). (E)AsinD but using SMC1 antibodies (green) and ATM antibodies (red). Lower; B, lane 5). Numbers on left indicate molecular mass markers in kilo- daltons. Further mapping of SMC1–5; three constructs (5A-C) representing residues 947-1100, 947-1025, and 947–967 were made; note that all three contain Ser957 and Ser966, indicated in red (C). Fragments 5A-C were cosynthe- Abnormal Spindle Formation. Because imbalances in the concen-

sized with Flag-Rae1 as in B and pulldowns were analyzed by SDS/PAGE by tration of reaction partners in the mitotic spindle formation CELL BIOLOGY using an 18% acrylamide gel; arrows indicate pulled down fragments 5A, 5B, pathways interfere with the assembly of normal bipolar spindles, and 5C (D). In vivo expression of HA-tagged SMC1–5B and SMC1–5C and empty we carried out overexpression experiments (Fig. S8) and checked HA vector in HeLa cells, followed by anti-HA IP and immunoblotting with for the formation of supernumerary spindles (Table 1). Trans- anti-Rae1 (E). fection of HeLa cells with SMC1 or SMC3 caused an increase of multipolar spindles from a base line of 5% to Ϸ25% (Table 1). NuMA and SMC1 Bind to Different Sites of Rae1. A previously In contrast, overexpression of the corresponding S to A mutants identified fragment of NuMA, NuMA325-829, that also binds led only to a slight increase in supernumerary spindles (Table 1). Rae1 (2), did not compete, indicating that SMC1 and NuMA Strikingly, transfection with SMC1 fragments 5B or 5C yielded bind to different sites of Rae1 (Fig. S6). an increase in supernumerary spindles comparable to that of transfection with full length SMC1 or SMC3 (Table 1 and Fig. Colocalization of ATM and SMC1 at the Spindle Pole. Although ATM S8 A and B). These data suggest that overexpression of the has been shown to be localized at the spindle pole by immuno- 21-residues-long region of SMC1 might compete for phosphor- fluorescence confocal microscopy (27), we investigated whether ylation with full-length SMC1 and/or that a phosphorylated SMC1 colocalizes with ATM using the in vitro aster assembly 21-mer in turn might compete with binding of full length SMC1 assay. Strikingly, both ATM and SMC1 are colocalized at the to microtubule bound Rae1. center of the aster (Fig. 4 D and E). Moreover, after immu- When examining chromosome condensation figures as re- nodepletion of ATM from the mitotic extract, we observed only vealed by the shape of DAPI-stained metaphase chromosome a few irregular, and unbundled microtubules (Figs. S2 and S7), masses, we observed an interesting difference in the in vivo pointing to a critical function of ATM, although ATM immu- expression of the two short SMC1 fragments. Expression of the nodepletion may have codepleted other kinases. Nevertheless, longer fragment, SMC1947-1025, in addition to causing formation colocalization of ATM and SMC1 suggest that a spindle pole- of multipolar spindles, also yielded defects in the normal ar- localized ATM is a plausible candidate for phosphorylating rangement of chromosome masses; instead of the characteristic SMC1’s S957 and S966, thereby promoting binding of phosphor- metaphase plate, the chromosome masses were arranged in ylated SMC1 to microtubule-bound Rae1. circular or fragmented figures (Fig. S8A). In contrast, the shorter

Wong and Blobel PNAS ͉ October 7, 2008 ͉ vol. 105 ͉ no. 40 ͉ 15443 Downloaded by guest on September 28, 2021 independent 3 ؍ Table 1. Spindle phenotypes in HeLa cells overexpressing SMC3 or various SMC1 constructs (n experiments) Vectors Mitotic cells Percent bipolar Percent monopolar Percent multipolar

Control 100 95 Ϯ 205Ϯ 1 Myc-SMC1 100 67 Ϯ 55Ϯ 328Ϯ 2 Myc-SMC1-DBA 100 90 Ϯ 1010Ϯ 1 Myc-SMC1-S957A 100 91 Ϯ 309Ϯ 2 Myc-SMC1-S966A 100 89 Ϯ 2011Ϯ 3 HA-SMC3 100 71 Ϯ 52Ϯ 227Ϯ 4 HA-SMC1–5B 100 73 Ϯ 52Ϯ 225Ϯ 4 (22 Ϯ 2) HA-SMC1–5C 100 76 Ϯ 21Ϯ 223Ϯ 3(4Ϯ 1)

Numbers in brackets in the last two lines indicate percentage of cells with irregular chromosome masses.

fragment SMC1947-967 yielded chromosome masses arranged in a Ͼ90% and we typically obtained 107 cells from eight 15-cm2 tissue culture more normal metaphase plate (Fig. S8B). One possible inter- dishes. The cells were then collected by centrifugation at 2,000 ϫ g and pretation of these observations is that the shorter fragment washed twice with cold PBS containing 20 ␮g/ml cytochalasin B. Cells were interferes only with phosphorylation and the resulting bundling washed one last time in cold KHM buffer (78 mM KC1/50 mM Hepes, pH 7.0/4 mM MgC12/2 mM EGTA/1 mM DTT) containing 20 ␮g/ml cytochalasin B and of microtubules at the spindle pole, whereas the longer fragment, finally sonicated at a concentration of Ϸ3 ϫ 106 cells per ml in KHM buffer perhaps by invading the coiled-coil domain of SMC1, also containing 20 ␮g/ml cytochalasin B, 20 ␮g/ml phenylmethylsulfonyl fluoride interferes with cohesin’s function in chromosome condensation and 1/4 pill of Protease inhibitor (Roche). The crude cell extract was then pathways and in the formation of a normal metaphase plate subjected to sedimentation at 100,000 ϫ g for 30 min at 4°C (Beckman airfuge, arrangement of the chromosome masses. 25 psi). The supernatant was recovered and supplemented with 2.5 mM In conclusion, we have detected a distinct pathway that ATP-Mg and 10 mM taxol. The total protein concentration of the extract participates in spindle pole formation and that involves recruit- prepared in this fashion was routinely Ϸ0.8–1.0 mg/ml. The mitotic extract ment of SMC1 by microtubule-bound Rae1 at the spindle was incubated at 33°C for 30–60 min to form asters. After incubation, 5 ␮lof ␮ pole (Figs. 1 and 2). The region of SMC1 that interacts with Rae1 the extract was diluted into 25 l of KHM buffer, spotted onto a polyL-lysine- coated glass coverslip, fixed by immersion in Ϫ20°C methanol, and then has been mapped to a 21-residue-long segment of SMC1, processed for confocal microscopy as described (2). SMC1947-967 (Fig. 3). The spindle-pole-anchored ATM or a related kinase presumably phosphorylates two Ser residues, Immunodepletion of Mitotic Extracts. Antibodies to Rae1, SMC1, SMC3, ATM, 957 967 947–967 Ser and Ser of SMC1 (Fig. 4), which in turn stimulates or for mock depletion, nonspecific rabbit IgG, were each coupled to protein-A binding of SMC1 to microtubule bound Rae1 (Fig. 4 and Figs. Dynabeads (Dynal, Invitrogen). Using a 1:10 ratio of beads to extract, 500-␮l S2, S5, and S7). We propose that only those SMC1/SMC3 aliquots were incubated with agitation for 1 h and the beads removed by heterodimers that have access to a spindle pole-anchored ATM sedimentation at 15,000 ϫ g for 10 s. After three repeat cycles, leached will be phosphorylated in situ. We envision that such assemblies antibodies were removed by one cycle of incubation with 10 ␮l of washed occlude further access by additional SMC1/SMC3 heterodimers protein-A Dynabeads. The immunodepleted extracts were then incubated for to spindle pole-anchored ATM thereby preventing the spread of aster formation as described above. Depletion efficiency was assayed by SDS/PAGE and immunoblot. SMC1/SMC3 interactions along microtubule-bound Rae1 away from the spindle pole. It is likely that the spindle pole bound Preparation of Flag-Rae1. Flag-Rae1 was expressed in 600 ␮l of rabbit reticu- SMC1/SMC3 heterodimer recruits the other members of the locyte lysate by using the Promega TNT coupled transcription/translation cohesin complex, Scc1 and Scc3, thereby completing closure into system according to the manufacturer’s protocol; after posttranslational in- a circular structure. Hence, cohesin would function to embrace cubation with 20 ␮l of anti-Flag beads (Sigma-Aldrich), the beads were microtubules at the spindle pole, extending its known function in washed three times with Binding buffer (20 mM Hepes, pH 7.5/100 mM KCl/5 ϫ encircling chromatids. mM MgCl2/0.1% Tween 20/20% glycerol/0.01% BSA/1 mM DTT/1 mM PMSF/1 A detailed biochemical characterization of the interactions complete protease inhibitor mixture) and Flag-Rae1 was eluted by 100 ␮g/ml ⅐ between the numerous proteins that have so far been reported Flag peptide (Sigma-Aldrich) or with 0.1 M glycine HCl, pH 3.5, according to to be associated with normal bipolar spindles by microscopical the manufacturer’s protocol. Ten micrograms per milliliter of Flag-Rae1 was routinely collected by the above methods. Flag-Rae1 was further concen- method, will considerably advance our understanding of the trated to 100 ␮g/ml by using Microcon-30 concentrators (Amicon), and where mechanisms underlying the formation and the dynamic structure indicated, was added to Rae1 immunodepleted extracts at 1:10 in volume. of bipolar spindles. Immunoprecipitations (IPs). HeLa cells (107) were seeded and synchronized as Methods described in ref. 2. Mitotic cells were collected, washed with PBS, spun at 400 ϫ Plasmids. The mammalian expression constructs encoding full-length human g for 10 min, and lysed in 1 ml of cold lysis buffer [50 mM Tris⅐HCl, pH 7.2/300 SMC1 and its variants at the serine phosphorylation sites [SMC1 S957A, SMC1 mM NaCl/0.1% Nonidet P-40/5 mM MgCl2/2 mM EDTA/10% glycerol) containing S966A, and double-mutant SMC1 S957A:S966A (SMC1-DBA) were a generous 1x protease inhibitor mixture (Roche)] and 1 mM PMSF. Lysates were centrifuged gift of Michael B. Kastan (St. Jude Children’s Research Hospital, Memphis, TN]. for 30 min at 4°C at 14,000 ϫ g. The supernatant was cleared with 50 ␮l of Protein Rae1 with Flag tag was subcloned into pET 28a vector. The SMC1 domains A/G bead slurry (Santa Cruz Biotechnology), then mixed with 10 ␮l of various were subcloned by PCR from pJET vector. All constructs were confirmed by antibodies as specified, and incubated for 1 h at 4°C with rocking. The beads were DNA sequencing. then washed five times with 500 ␮l of lysis buffer. After the last wash, proteins were extracted by 50 ␮lof1ϫ SDS/PAGE Blue Loading buffer (New England Preparation of Mitotic Extracts for Mitotic Aster Formation. Mitotic extracts Biolabs) and analyzed by SDS/PAGE. from HeLa cells were prepared according to refs. 28 and 29 (for overview, see Fig. S1). Briefly, HeLa cells were synchronized by double block with 2 mM Immunofluorescence Confocal Microscopy. Synchronized HeLa cells were thymidine. After a release from thymidine block, the cells were allowed to washed in PBS and fixed for 10 min in methanol at Ϫ20°C. Cells were then grow for 7 h, and then nocodazole was added to a final concentration of 40 permeabilized with 0.1% Triton X-100 or 0.3% Digitonin in PBS for 10 min at ng/ml. The mitotic cells that accumulated over the next 4 h were collected by room temperature. Samples were examined on a Zeiss LSM510 MEGA confocal mitotic shake-off and incubated for 30 min at 37°C with 20 ␮g/ml cytochalasin microscope, and all images were acquired by using a plan-Apochromat 100 ϫ B. The mitotic index of the population of cells isolated in this fashion was 1.4-n.a. objective.

15444 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0807660105 Wong and Blobel Downloaded by guest on September 28, 2021 In Vitro Binding Assays. Proteins were expressed by using the Promega TNT- mouth Medical School, Hanover, NH); anti-NuMA monoclonal antibodies coupled transcription/translation system according to the manufacturer’s (clone 22) from BD Biosciences; anti-Myc monoclonal antibodies, DM1A protocol. Five microliters of Flag beads (ANTI-FLAG M1 Agarose Afﬁnity Gel monoclonal ␣-tubulin antibodies were from Sigma–Aldrich; anti-hSMC1, anti- from Sigma–Aldrich) was washed three times with binding buffer (see above), hSMC3 polyclonal antibodies were from Chemicon; anti-SMC1(C2M) mono- preblocked for 10 min with 10 ␮l of nonspeciﬁc rabbit IgG, washed with clonal antibody was from Santa Cruz Biotechnology; anti-Histone H3, anti- binding buffer, and resuspended in 60 ␮l of binding buffer. Then, 10 ␮lof Phospho-Histone H3 antibodies were from Upstate Biotechnology; anti-ATM, various indicated combinations of in vitro transcribed and translated [35S] anti-HA, anti-GFP antibodies were from Abcam. Secondary antibodies were methionine-labeled SMC1, SMC3, SMC1S957A, SMC1S966A, or double S/A from Molecular Probes. mutants and Flag-Rae1 were incubated with beads at 4°C for 1 h. Beads were washed six times with binding buffer and boiled in 15 ␮l of SDS/PAGE sample ACKNOWLEDGMENTS. We thank D. Compton (Dartmouth Medical School, buffer. Samples were analyzed by SDS/PAGE (4–20% Tris-glycine gels; Invitro- Hanover, NH) for NuMA antibodies and M. Kastan (St. Jude Children’s Re- gen), followed by autoradiography. All other chemicals were from Sigma- search Hospital, Memphis, TN) for the SMC1 plasmids. We also thank the staff Aldrich, including kinase inhibitors (LY294002 and Caffeine). at The Rockefeller University Bio-imaging facility for help with confocal microscopy. We thank Elias Coutavas for help in the preparation of this manuscript. This work was supported by Howard Hughes Medical Institute Mammalian Cell Culture and Transfection. Mammalian cell culture, preparation and a grant from the Leukemia and Lymphoma Society (to G.B.). R.W. was also of mitotic HeLa cells, and plasmid transfection of HeLa cells, were as described (2). supported in part by the Program for Improvement of Research Environment for Young Researchers by Special Coordination Funds for Promoting Science Antibodies. Anti-Rae1 and -Nup98 polyclonal antibodies were prepared as and Technology (SCF), Grants-in-Aid (Start-Up), and the 21st Century COE described (2). Anti-NuMA polyclonal antibodies were from D. Compton (Dart- program from MEXT, Japan.

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Wong and Blobel PNAS ͉ October 7, 2008 ͉ vol. 105 ͉ no. 40 ͉ 15445 Downloaded by guest on September 28, 2021