Rae1 interaction with NuMA is required for bipolarspindle formationRichard W. Wong, Gunter Blobel*, and Elias Coutavas*

Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10021

Contributed by Gunter Blobel, November 1, 2006 (sent for review October 8, 2006)

In eukaryotic cells, the faithful segregation of daughter chromo-somes during cell division depends on formation of a microtubule(MT)-based bipolar spindle apparatus. The Nuclear Mitotic Appa-ratus protein (NuMA) is recruited from interphase nuclei to spindleMTs during mitosis. The carboxy terminal domain of NuMA bindsMTs, allowing a NuMA dimer to function as a ‘‘divalent’’ crosslinkerthat bundles MTs. The messenger RNA export factor, Rae1, alsobinds to MTs. Lowering Rae1 or increasing NuMA levels in cellsresults in spindle abnormalities. We have identified a mitotic-specific interaction between Rae1 and NuMA and have exploredthe relationship between Rae1 and NuMA in spindle formation. Wehave mapped a specific binding site for Rae1 on NuMA that wouldconvert a NuMA dimer to a ‘‘tetravalent’’ crosslinker of MTs. Inmitosis, reducing Rae1 or increasing NuMA concentration would beexpected to alter the valency of NuMA toward MTs; the ‘‘density’’of NuMA-MT crosslinks in these conditions would be diminished,even though a threshold number of crosslinks sufficient to stabilizeaberrant multipolar spindles may form. Consistent with this inter-pretation, we found that coupling NuMA overexpression to Rae1overexpression or coupling Rae1 depletion to NuMA depletionprevented the formation of aberrant spindles. Likewise, we foundthat overexpression of the specific Rae1-binding domain of NuMAin HeLa cells led to aberrant spindle formation. These data point tothe Rae1–NuMA interaction as a critical element for normal spindleformation in mitosis.

mitotic spindle � nucleoporin

In eukaryotic cells upon entry into mitosis, interphase micro-tubules (MTs) are reorganized into the spindle apparatus, a

complex and dynamic macromolecular machine composed ofpolymerized tubulin and many interacting proteins (1). MTs arepolymers of �-�-tubulin dimers with distinct plus and minusends. The typical metaphase spindle apparatus contains twopoles at centrosomes with �-tubulin at the minus ends of MTs.Bipolar spindle assembly requires organization of MTs and theirselective local stabilization. Chromatin and kinetochores stabi-lize the plus ends of MTs and become aligned in the center ofthe spindle awaiting successful biorientation of all sister chro-matids before anaphase. In some settings, notably plant cells andoocytes, spindles form in the absence of centrosomes by MTnucleation on chromatin followed by bundling at the minusends (2–4).

The Nuclear Mitotic Apparatus protein (NuMA) is a 237-kDaprotein with an �1,500-aa discontinuous coiled-coil spacerbetween N- and C-terminal globular domains (5, 6) that can formparallel coiled-coil dimers �200 nm in length (6). The C-terminal domain of NuMA contains a site for multimerization(6), a nuclear localization sequence that interacts with karyo-pherin � (7), a MT-binding site that overlaps with a binding sitefor LGN (8) (a leucine-glycine-asparagine-repeat containingprotein) and a site for binding the polyADP-ribose polymerase,tankyrase (9). The N-terminal domain of NuMA is believed tocontain a binding site for dynactin that acts as an adaptor fordynein, a minus end-directed motor known to target NuMA tothe spindle pole (10). The WD (tryptophan-aspartic acid) repeat� propeller protein Rae1 (11), also known as gle2 (12) or mrnp41

(13), is one of �30 different proteins (14) (nucleoporins or nups)found in the nuclear pore complex. Rae1 has been shown to bindto the nucleoporin Nup98 (15) and the mitotic checkpoint kinaseBub1 (16) through their so-called GLEBS (Gle2-binding site)domains (17) and to function with Nup98 in securin degradation(18). The vesicular stomatitis virus M protein blocks host cellmRNA export by binding to Rae1 (19). Although Rae1 has beenreported to bind to MTs (20, 21), these binding sites have notbeen mapped. Interestingly, several nucleoporins uniquely lo-calize to the spindle (22), including the Nup107–160 complexrecently shown to be required for spindle assembly (23), but themechanistic aspects and functional relevance of these mitoticredistributions are largely unknown.

Aberrant expression of either Rae1 or NuMA has been linkedto formation of multipolar spindles. In the case of NuMA,multipolarity is linked to overexpression, whereas in the case ofRae1, multipolarity is linked to its depletion (21, 24). Here weidentify a mitotic interaction between Rae1 and NuMA, map thisinteraction to a specific domain of NuMA, and demonstrate thata balance of these two proteins is required for bipolar spindleformation. We propose a model wherein Rae1 modulates theMT crosslinking valency of NuMA in mitotic spindles to preventchromosome segregation defects that are commonly found incancer cells.

ResultsRae1 and NuMA Form a Transient Complex During Mitosis. To elu-cidate in greater depth the specific role of mitotic Rae1, weanalyzed the composition of purified Rae1 complexes in mitoticHeLa cells. The major Rae1-associated proteins from mitotic IPwere subjected to MALDI mass spectrometry after trypsindigestion, leading to the identification of NuMA (data notshown). By immunoblotting of anti-Rae1 immunoprecipitates,we detected coprecipitating NuMA along with Nup98 anddynein (Fig. 1 A). Conversely, using anti-NuMA antibodies, weimmunoprecipitated Rae1 and dynein but not Nup98 (Fig. 1B).These data suggested that Rae1 and NuMA interact. To furtherdefine the specificity for this mitotic Rae1–NuMA interaction,we prepared extracts from HeLa cells synchronized using adouble thymidine block followed by release into and out of theMT destabilizer nocodazole. HeLa cells were released from anS phase double thymidine block into nocodazole for 12 h andarrested in mitosis. At this time, mitotic cells were collected byshake-off and released out of nocodazole for 4 h. These exper-iments revealed a transient association of Rae1 and NuMAduring mitosis (Fig. 1C). Consistent with the IP data, we found

Author contributions: R.W.W. and E.C. designed research; R.W.W. and E.C. performedresearch; R.W.W., G.B., and E.C. analyzed data; and R.W.W., G.B., and E.C. wrote the paper.

The authors declare no conflict of interest.

Abbreviations: MT, microtubule; IP, immunoprecipitation.

*To whom correspondence may be addressed. E-mail: blobel@rockefeller.edu orcoutave@rockefeller.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0609582104/DC1.

© 2006 by The National Academy of Sciences of the USA

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that Rae1 and NuMA colocalized transiently on HeLa cellspindle poles from prophase to anaphase (Fig. 1D and SI Fig. 6).

Simultaneous Depletion of Rae1 and NuMA Rescues Bipolarity. Be-cause NuMA or Rae1 are known to individually impact spindleformation, we altered their balance in vivo by modulating theirconcentrations using RNAi and overexpression strategies andassayed the effect on spindle polarity. Consistent with previousobservations (21), reduction of Rae1 by RNAi (SI Fig. 7) led tothe formation of multipolar spindles (Fig. 2A). We quantified themitotic defects at 72 h after transfection with siRNA duplexestargeting Rae1 and found a high proportion (�30%) of cellsdisplayed strikingly altered spindle morphology compared withcontrols treated with buffer alone (transfection efficiency�90%; see Fig. 2 A and Table 1). The extra spindle polesappeared to pull chromosomes away from the main spindle,contributing to serious chromosome-alignment defects. TheRae1 siRNA-treated cells displayed NuMA localization to spin-

dle poles (data not shown) and stained positive for the centro-somal markers pericentrin and �-tubulin (Fig. 2 B and C). Givenour observation of a mitotic Rae1–NuMA interaction, we wereinterested in exploring the effect of NuMA down-regulation onthe multipolar spindle phenotype of Rae1-depleted cells. NuMAoverexpression is also linked to multipolar spindle formationthat may be rescued by reduction of NuMA levels (24). Indeed,when NuMA and Rae1 levels were reduced simultaneously bysiRNA, the incidence of multipolar spindles was greatly reduced(Fig. 2 A and Table 1).

Simultaneous Overexpression of NuMA and Rae1 Rescues Bipolarity.To further test our hypothesis that mitotic Rae1 can bind toNuMA and influence spindle formation, we explored the effectof overexpressing Rae1 in cells overexpressing NuMA and

Fig. 1. Rae1 and NuMA form a transient complex during mitosis. (A and B)IP from mitotic HeLa extracts with �-Rae1 and �-NuMA or control antibodies(IgG), followed by immunoblotting with �-NuMA, �-Nup98, �-dynein, and�-Rae1. In lanes marked ‘‘2% input,’’ 5 �l of 250 �l extract used per IP wasanalyzed directly. (C) Synchronized HeLa cells were collected at the indicatedtime points, and extracts were analyzed by immunoblotting directly (Input2%) or after IP with �-Rae1. Anti-phospho-Histone H3 and �-tubulin wereused as mitotic index and loading controls. (D) Asynchronous HeLa cellscostained with �-Rae1 (green) and �-NuMA (red); chromatin was visualizedusing DAPI (blue). The large yellow arrow points to metaphase cell, smallwhite arrowpoints to interphase, and the large white arrowpoints to latetelophase. (Scale bar, 25 �m.)

Fig. 2. Simultaneous depletion of Rae1 and NuMA rescues bipolarity. (A)HeLa cells were transfected with either siRNA duplexes against Rae1 (Left) orRae1 and NuMA together (Right). After 72 h, cells were stained with �-tubulinantibody (red) and analyzed by confocal laser microscopy. Chromatin wasstained with DAPI (blue). [Scale bars, 25 �m (Upper); 5 �m (Lower).] (B and C)Representative figures of cells treated with Rae1 siRNA, fixed, and stainedwith anti-pericentrin and either �-tubulin (B) or �-tubulin (C) antibodies. DNAis counterstained with DAPI.

Table 1. RNAi and protein overexpression spindle phenotypes

Mitoticcells

Percentbipolar

Percentmonopolar

Percentmultipolar

Control 300 98.5 � 3 0 1.5 � 1Rae1 siRNA 300 67 � 7 0 33 � 3(Rae1 � NuMA) siRNA 300 91 � 3 0 9 � 6Control 200 98 � 2 0 2 � 1GFP-NuMA 200 59 � 5 12 � 3 29 � 4GFP-NuMA � HA-Rae1 200 83 � 2 4 � 2 13 � 4GFP 200 98 � 3 0 2 � 2GFP-NuMA325–829 200 65 � 2 13 � 3 22 � 2

Quantitation of spindle defects represented in Figs. 2 and 3. n � threeindependent experiments.

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displaying the multipolar spindle phenotype. First we transfectedGFP-NuMA into HeLa cells and 72 h later observed the cellsusing confocal microscopy. A variety of phenotypes was ob-served: in 29% of the cells (n � 300), additional poles wereobserved and in 12% of the cells monopolar spindles formed(Fig. 3 and SI Fig. 8). Interestingly, if we cotransfected NuMAwith Rae1, we found that additional poles were observed in only13% of the cells (n � 300); 4% of the cells remained monopolar;and most of the cells appeared normal during prometaphase/metaphase (83%; Table 1, Fig. 3 and SI Fig. 8). These datatherefore further suggest that normal spindle pole formationrequires balanced concentrations of NuMA and Rae1 duringmitosis.

Mapping the Rae1 Interaction Domain on NuMA. To investigate thebasis of the Rae1 NuMA interaction at mitosis, we generated aseries of NuMA deletion mutants and tested them for theirability to interact with Rae1. We coexpressed Rae1 and variousfragments of NuMA (see Fig. 4A) in a cell-free reticulocytetranslation system. Only the fragment NuMA325–829 interacteddirectly with Rae1 (Fig. 4B). This fragment of NuMA containsa potential GLEBS-like motif (SI Fig. 9). The Nup43 or Seh1WD-repeat � propellers did not interact with NuMA325–829 (datanot shown). We therefore conclude that a fragment of NuMAspanning amino acids 325–829, at the N-terminal end of thecoiled-coil domain, interacts specifically with Rae1.

To test whether NuMA325–829 interacts with Rae1 in vivo, weexpressed NuMA325–829-GFP in HeLa cells. We then usedanti-GFP antibodies for IPs from extracts of cells expressingeither NuMA325–829-GFP or GFP alone and tested for thepresence of Rae1. As seen in Fig. 4C, Rae1 was specificallycoimmunoprecipitated with NuMA325–829-GFP but not GFPalone (asterisks). We could also detect a small amount offull-length NuMA (arrow) in the IPs, suggesting that full-lengthNuMA associates with the NuMA325–829 fragment (arrowhead).This observation implies that at least some dimerization deter-minants reside between amino acids 325 and 829 at the beginningof the predicted coiled-coil region of NuMA.

Because the NuMA325–829 fragment seems to bind Rae1 incells, we examined the effect of expressing this domain in HeLacells. NuMA325–829-GFP localized in part to spindles (upper rowof Fig. 4D) and colocalized with tubulin (lower row of Fig. 4D).We found that additional poles were observed in 30% of these

cells (n � 200; Fig. 4D and Table 1). Although we did not observeany gross mislocalization of Rae1 in these cells (not shown), apossible interpretation of these results is that NuMA325–829 bindsto and sequesters some Rae1, making it unavailable to interactproductively with full length NuMA. This would then be anal-ogous to the multipolar spindle phenotype observed after re-duction of Rae1 by RNAi. The NuMA325–829 fragment could alsodimerize with full-length NuMA, and the resulting hybridNuMA-NuMA325–829 heterodimers would lack one C-terminaldomain and would potentially have reduced ability to link MTs.

DiscussionSpindle assembly requires the temporal and spatial coordinationof multiple overlapping pathways involving MT nucleation and

Fig. 3. Simultaneous overexpression of Rae1 and NuMA rescues bipolarity.Representative figures of HeLa cells transfected with plasmids overexpressingeither GFP-NuMA or GFP-NuMA and Rae1-HA together. After 24 h, cells werefixed, stained with �-tubulin antibody (red in overlay; GFP is green), andanalyzed by confocal laser microscopy. Chromatin was stained with DAPI(blue). (Scale bar, 5 �m.)

Fig. 4. Mapping the Rae1 interaction domain on NuMA. (A) Schematic ofNuMA and five FLAG-tagged fragments of NuMA. Numbers on the left referto amino acids (aa); all fragments are continuous (e.g., NuMA2 ends at aminoacid 829 and NuMA3 starts at amino acid 829). (B) Autoradiograph of [35S]me-thionine-labeled Rae1 and NuMA-FLAG fragments coexpressed in vitro, af-finity-purified, and separated by SDS/PAGE. Rae1 is untagged. Asterisks indi-cate the five FLAG-tag NuMA fragments expressed in varying amounts usingthis system. Numbers indicate molecular weight markers in kilodaltons. (C)Immunoblotting of �-GFP IPs from either GFP- or GFP-NuMA325–829-expressingHeLa cells. IPs are blotted with �-Rae1 or �-NuMA [using BD Biosciences clone22 monoclonal that recognizes an epitope (amino acids 658–691) withinNuMA325– 829]. (D) HeLa cells overexpressing GFP-NuMA325– 829 (green)costained with tubulin (red) and DAPI (blue).

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stabilization, pole formation and attachment and alignment ofchromosomes (3, 25). MTs are dynamically unstable structuresthat are stabilized by a variety of MT-associated proteins. Inaddition, ‘‘crosslinking’’ among MTs is required to bundle themat their minus end at the spindle pole. The C-terminal domainof NuMA has been shown to bundle MTs. If NuMA is a dimer(ref. 6 and Fig. 5), it could act as a divalent crosslinker of MTs.In addition, any NuMA-associated protein that also binds MTscould function in the MT crosslinking. Rae1 has previously beenshown to bind to MTs (21). Here we show that Rae1 alsointeracts with NuMA, and we have mapped this interaction tothe N-terminal end of the coiled-coil domain. To our knowledge,this is a previously undescribed biochemical mapping of aspecific interaction between a nucleoporin and any componentof the mitotic spindle. We suggest that by interacting with NuMAand MTs, Rae1 could increase the MT crosslinking valency ofNuMA (Fig. 5) and further stabilize MTs at their minus ends. Wepropose this interaction is critically required for normal bipolarspindle formation. We observed the presence of centrosomalmarkers in the aberrant spindle poles of Rae1-depleted cells, andthe same has been reported in the case of NuMA overexpressionin cancer cell lines (24); therefore, we cannot preclude thepossibility that Rae1 and NuMA are involved in centrosomeduplication or stabilization.

Our data suggest that a balance between NuMA and Rae1 iscritical for bipolar spindle formation. Specifically, in the case ofoverexpression of NuMA coupled with concomitant overexpres-sion of Rae1 and, conversely, in the case of depletion of Rae1coupled with concomitant depletion of NuMA, formation ofsupernumerary spindle poles is suppressed (Figs. 3 and 4D). Wespeculate that the additional crosslinking valency of NuMA, byvirtue of its interaction with MT-bound Rae1, increases the‘‘density’’ of crosslinks and therefore enhances the bundling ofMTs at their minus ends. In the case of NuMA overexpressionor Rae1 depletion, many of the crosslinks at the minus end ofMTs would be divalent rather than tetravalent (Fig. 5). Never-theless, if a critical number of these divalent crosslinks has been

established, the resulting supernumerary poles may be suffi-ciently stable to persist, because none of the spindle poles haveaccumulated enough of the tetravalent crosslinks to competewith each other for stability. In this scenario, the minus ends ofMTs are capped with �-tubulin and surrounded by pericentrin.In contrast, if NuMA overexpression is accompanied by Rae1overexpression, the formation of a high density of tetravalentcrosslinks may kinetically favor the formation of a bipolarspindle destabilizing any supernumerary poles that lack a criticaldensity of tetravalent crosslinks. A spindle with a higher densityof tetravalent crosslinks could successfully compete for NuMAand Rae1 against spindles with low-density crosslinks that areless stable. Our results with the overexpression of a Rae1-bindingfragment of NuMA (NuMA325–829) would be entirely consistentwith our speculations regarding the valency and density ofNuMA-mediated minus-end MT bundling.

The kinetics of spindle formation are influenced by manyother components, including MT-based motors (26), MT dy-namics (27), gradients of Ran and kinases (28), and polyADP-ribosylation (29), that will likely modify the effects we reportedhere for Rae1 and NuMA. In any case, our results should providea useful framework for further testing the dynamics of MTbundling in mitosis and elucidating the role of a Rae1–NuMAimbalance in chromosome segregation defects leading toaneuploidy.

Materials and MethodsPlasmids. The plasmid-encoding full length human Rae1 (ImageID LIFESEQ95168410; Open Biosystems, Huntsville, AL) wassubcloned into pcDNA3 with HA tag and pET28a. The NuMAdomains were subcloned by PCR from pCDNA3-GFP-NuMAinto pET28a with a C-terminal simian virus 40 T antigen nuclearlocalization sequence and a FLAG tag. All constructs wereconfirmed by DNA sequencing.

Cell Culture, Transfections, and Synchronization. HeLa cells weretransfected with Rae1 and NuMA siRNAs using Oligofetamine andwith GFP-NuMA and HA-Rae1 plasmids using Lipofectamine2000 following the manufacturer’s protocol (Invitrogen, Carlsbad,CA). Cells were synchronized in S phase by double thymidine block(30) using 2 mM thymidine with the following modifications. Inexperiments involving siRNA oligos [Fig. 2 and supporting infor-mation (SI) Fig. 7], the cells were transfected 24 h before theinitiation of the first thymidine block and collected or imaged after72 h. In experiments involving plasmid-mediated protein overex-pression (Figs. 2 and 3 and SI Fig. 8), the transfection was initiatedbefore the second thymidine block for 4 h. For experimentsrepresented in Fig. 1C, cells were released into 30 ng/ml Nocoda-zole after the second thymidine block, harvested for analysis athourly intervals for 12 h during Nocodazole incubation, and thencollected by mitotic shakeoff, replated in fresh medium, andharvested for analysis at hourly intervals for 4 h. For the RNAiexperiments, siRNA duplexes targeting Rae1 [5�-GCAGUAAC-CAAGCGAUACA-3�] (21) or NuMA [5�-GGCGUGGCAG-GAGAAGUUC-3�] (31) were purchased from Integrated DNATechnologies (Coralville, IA). Mock transfection was with bufferalone (control). Transfection efficiency was monitored withBlock-iT (Invitrogen).

Antibodies and Immunofluorescence. In initial experiments, anti-Rae1 antibodies from K. Weis (University of California, Berke-ley, CA) (21) and J. van Deursen (Mayo Clinic, Rochester, MN)(32) were used (Fig. 1 A and B). For all subsequent experiments,peptides based on human Rae1 residues 313–327 (FYNPQKK-NYIFLRNAAEE) (21), with N-terminal acetylation and C-terminal amidation, were injected in rabbits (Cocalico Biologi-cals, Reamstown, PA). Antibodies were affinity-purified beforeuse. Anti-NuMA polyclonal antibody used in Fig. 1 A and B was

Fig. 5. A ‘‘valency’’ model of MTs interacting with NuMA and Rae1. NuMAis assumed to be a dimer (6) with the C-terminal (C) indicated to directlyinteract with MTs (7). A region (residues 325–829) at the N-terminal end of thecoiled coil of NuMA interacts with Rae1 (data in this paper) and thereforeconverts NuMA from a divalent to a tetravalent MT ‘‘crosslinker.’’

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from D. Compton (Dartmouth Medical School, Hanover, NH)(33); anti-NuMA monoclonal antibody (clone 22) from BDBiosciences (Franklin Lakes, NJ) was used in all other experi-ments. DM1A monoclonal �-tubulin antibody and �-tubulinantibody were from Sigma–Aldrich (St. Louis, MO). The dyneinmonoclonal antibody (clone 74.1) was from Chemicon (Te-mecula, CA), Phospho-Histone H3 antibody was from UpstateBiotechnology (Lake Placid, NY). �-HA, �-GFP, and �-peri-centrin antibodies were from Abcam (Cambridge, U.K.). Sec-ondary antibodies were from Molecular Probes (Eugene, OR).

For immunofluorescence, synchronized HeLa cells werewashed in PBS and fixed for 10 min in methanol at �20°C. Cellswere then permeabilized with 0.2% Triton X-100 in PBS for 10min at room temperature. Samples were examined on a Zeiss(Oberkochen, Germany) LSM510 MEGA confocal microscope,and all images were acquired by using a plan-Apochromat 100 �1.4-N.A. objective.

Immunoprecipitations (IPs). For IPs, �107 cells were seeded andsynchronized as described above. Mitotic HeLa cells were col-lected, washed with PBS, spun at 400 � g for 10 min, and lysedin 1 ml of cold Lysis buffer (50 mM Tris�HCl, pH 7.2/250 mMNaCl/0.1% Nonidet P-40/2 mM EDTA/10% glycerol) containing1� protease inhibitor mixture (Roche, Indianapolis, IN) and 1mM PMSF. Lysates were centrifuged for 30 min at 4°C at14,000 � g. The resulting lysate supernatants were preclearedwith 50 �l of Protein A/G bead slurry (Santa Cruz Biotechnol-ogy, Santa Cruz, CA), mixed with 5–10 �l of various antibodiesas specified, and incubated for 1 h at 4°C with rocking. The beadswere then washed five times with 500 �l of Lysis buffer. After thelast wash, 50 �l of 1� SDS/PAGE blue loading buffer (NewEngland Biolabs, Ipswich, MA) was added to the bead pelletbefore loading.

In Vitro Binding Assays. Proteins were expressed by using thePromega (Madison, WI) TNT coupled transcription/translationsystem according to the manufacturer’s protocol. Five microli-ters of Flag beads (ANTI-FLAG M1 Agarose Affinity Gel fromSigma–Aldrich) were washed three times with binding buffer (20mM Hepes, pH 7.5/100 mM KCl/5 mM MgCl2/0.1% Tween20/20% glycerol/0.01% BSA/1 mM DTT/1 mM PMSF/1� com-plete protease inhibitor mixture), preblocked for 10 min with 10�l of nonspecific rabbit serum, washed with binding buffer, andresuspended in 60 �l of binding buffer. Then, 10 �l of in vitrotranscribed and translated [35S]methionine labeled Rae1 andNuMA-Flag mutants were added to the beads, and the mixturewas incubated at 4°C for 1 h. Beads were washed six times withbinding buffer and boiled in 15 �l of SDS/PAGE sample buffer.Samples were analyzed by SDS/PAGE (4–20% Tris-glycine gels;Invitrogen), followed by autoradiography.

We thank M. Blower and K. Weis (University of California, Berkeley,CA) and J. van Deursen (Mayo Clinic, Rochester, MN) for the initialsupply of human Rae1 antibodies, D. Compton (Dartmouth MedicalSchool, Hanover, NH) for NuMA antibodies, A. Merdes (Centre Na-tional de la Recherche Scientifique) for the GFP–NuMA construct, andM. Kastan (St. Jude Children’s Research Hospital, Memphis, TN) for thepcDNA3-HA tag vector. The FLAG tag pET28a plasmid was a kind giftof K. Yoshida (the Blobel laboratory). We thank K. Hsia (the Blobellaboratory) for various � propeller constructs. We also thank HaitengDeng and Joseph Fernandez for the mass spectrometry analysis, HenryZebroski for the Rae1 peptide synthesis, and A. North for support withconfocal microscopy at The Rockefeller University Bioimaging facility.We thank members of the Blobel laboratory for helpful discussions andMegan King, Patrick Lusk, Joe Glavy, and Hang Shi for critical readingof the manuscript. This work was supported in part by the NationalInstitutes of Health (to E.C.) and by a grant from the Leukemia andLymphoma Society (to G.B).

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