Rae1 interaction with NuMA is required for bipolar spindle formation Richard W. Wong, Gu¨ nter Blobel*, and Elias Coutavas* Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10021 Contributed by Gu¨nter Blobel, November 1, 2006 (sent for review October 8, 2006) In eukaryotic cells, the faithful segregation of daughter chromo- (13), is one of Ϸ30 different proteins (14) (nucleoporins or nups) somes during cell division depends on formation of a microtubule found in the nuclear pore complex. Rae1 has been shown to bind (MT)-based bipolar spindle apparatus. The Nuclear Mitotic Appa- to the nucleoporin Nup98 (15) and the mitotic checkpoint kinase ratus protein (NuMA) is recruited from interphase nuclei to spindle Bub1 (16) through their so-called GLEBS (Gle2-binding site) MTs during mitosis. The carboxy terminal domain of NuMA binds domains (17) and to function with Nup98 in securin degradation MTs, allowing a NuMA dimer to function as a ‘‘divalent’’ crosslinker (18). The vesicular stomatitis virus M protein blocks host cell that bundles MTs. The messenger RNA export factor, Rae1, also mRNA export by binding to Rae1 (19). Although Rae1 has been binds to MTs. Lowering Rae1 or increasing NuMA levels in cells reported to bind to MTs (20, 21), these binding sites have not results in spindle abnormalities. We have identified a mitotic- been mapped. Interestingly, several nucleoporins uniquely lo- specific interaction between Rae1 and NuMA and have explored calize to the spindle (22), including the Nup107–160 complex the relationship between Rae1 and NuMA in spindle formation. We recently shown to be required for spindle assembly (23), but the have mapped a specific binding site for Rae1 on NuMA that would mechanistic aspects and functional relevance of these mitotic convert a NuMA dimer to a ‘‘tetravalent’’ crosslinker of MTs. In redistributions are largely unknown. mitosis, reducing Rae1 or increasing NuMA concentration would be Aberrant expression of either Rae1 or NuMA has been linked expected to alter the valency of NuMA toward MTs; the ‘‘density’’ to formation of multipolar spindles. In the case of NuMA, of NuMA-MT crosslinks in these conditions would be diminished, multipolarity is linked to overexpression, whereas in the case of even though a threshold number of crosslinks sufficient to stabilize Rae1, multipolarity is linked to its depletion (21, 24). Here we CELL BIOLOGY aberrant multipolar spindles may form. Consistent with this inter- identify a mitotic interaction between Rae1 and NuMA, map this pretation, we found that coupling NuMA overexpression to Rae1 interaction to a specific domain of NuMA, and demonstrate that overexpression or coupling Rae1 depletion to NuMA depletion a balance of these two proteins is required for bipolar spindle prevented the formation of aberrant spindles. Likewise, we found formation. We propose a model wherein Rae1 modulates the that overexpression of the specific Rae1-binding domain of NuMA MT crosslinking valency of NuMA in mitotic spindles to prevent in HeLa cells led to aberrant spindle formation. These data point to chromosome segregation defects that are commonly found in the Rae1–NuMA interaction as a critical element for normal spindle cancer cells. formation in mitosis. Results ͉ mitotic spindle nucleoporin Rae1 and NuMA Form a Transient Complex During Mitosis. To elu- cidate in greater depth the specific role of mitotic Rae1, we n eukaryotic cells upon entry into mitosis, interphase micro- analyzed the composition of purified Rae1 complexes in mitotic Itubules (MTs) are reorganized into the spindle apparatus, a HeLa cells. The major Rae1-associated proteins from mitotic IP complex and dynamic macromolecular machine composed of were subjected to MALDI mass spectrometry after trypsin polymerized tubulin and many interacting proteins (1). MTs are digestion, leading to the identification of NuMA (data not polymers of ␣-␤-tubulin dimers with distinct plus and minus shown). By immunoblotting of anti-Rae1 immunoprecipitates, ends. The typical metaphase spindle apparatus contains two we detected coprecipitating NuMA along with Nup98 and poles at centrosomes with ␥-tubulin at the minus ends of MTs. dynein (Fig. 1A). Conversely, using anti-NuMA antibodies, we Bipolar spindle assembly requires organization of MTs and their immunoprecipitated Rae1 and dynein but not Nup98 (Fig. 1B). selective local stabilization. Chromatin and kinetochores stabi- These data suggested that Rae1 and NuMA interact. To further lize the plus ends of MTs and become aligned in the center of define the specificity for this mitotic Rae1–NuMA interaction, the spindle awaiting successful biorientation of all sister chro- we prepared extracts from HeLa cells synchronized using a matids before anaphase. In some settings, notably plant cells and double thymidine block followed by release into and out of the oocytes, spindles form in the absence of centrosomes by MT MT destabilizer nocodazole. HeLa cells were released from an nucleation on chromatin followed by bundling at the minus S phase double thymidine block into nocodazole for 12 h and ends (2–4). arrested in mitosis. At this time, mitotic cells were collected by The Nuclear Mitotic Apparatus protein (NuMA) is a 237-kDa Ϸ shake-off and released out of nocodazole for 4 h. These exper- protein with an 1,500-aa discontinuous coiled-coil spacer iments revealed a transient association of Rae1 and NuMA between N- and C-terminal globular domains (5, 6) that can form during mitosis (Fig. 1C). Consistent with the IP data, we found parallel coiled-coil dimers Ϸ200 nm in length (6). The C- terminal domain of NuMA contains a site for multimerization (6), a nuclear localization sequence that interacts with karyo- Author contributions: R.W.W. and E.C. designed research; R.W.W. and E.C. performed pherin ␣ (7), a MT-binding site that overlaps with a binding site research; R.W.W., G.B., and E.C. analyzed data; and R.W.W., G.B., and E.C. wrote the paper. for LGN (8) (a leucine-glycine-asparagine-repeat containing The authors declare no conflict of interest. protein) and a site for binding the polyADP-ribose polymerase, Abbreviations: MT, microtubule; IP, immunoprecipitation. tankyrase (9). The N-terminal domain of NuMA is believed to *To whom correspondence may be addressed. E-mail: [email protected] or contain a binding site for dynactin that acts as an adaptor for [email protected]. dynein, a minus end-directed motor known to target NuMA to This article contains supporting information online at www.pnas.org/cgi/content/full/ the spindle pole (10). The WD (tryptophan-aspartic acid) repeat 0609582104/DC1. ␤ propeller protein Rae1 (11), also known as gle2 (12) or mrnp41 © 2006 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0609582104 PNAS ͉ December 26, 2006 ͉ vol. 103 ͉ no. 52 ͉ 19783–19787 Downloaded by guest on September 26, 2021 Fig. 2. Simultaneous depletion of Rae1 and NuMA rescues bipolarity. (A) HeLa cells were transfected with either siRNA duplexes against Rae1 (Left)or Rae1 and NuMA together (Right). After 72 h, cells were stained with ␣-tubulin antibody (red) and analyzed by confocal laser microscopy. Chromatin was stained with DAPI (blue). [Scale bars, 25 ␮m(Upper); 5 ␮m(Lower).] (B and C) Representative figures of cells treated with Rae1 siRNA, fixed, and stained with anti-pericentrin and either ␥-tubulin (B)or␣-tubulin (C) antibodies. DNA is counterstained with DAPI. dle poles (data not shown) and stained positive for the centro- 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 somal markers pericentrin and -tubulin (Fig. 2 B and C). Given (IgG), followed by immunoblotting with ␣-NuMA, ␣-Nup98, ␣-dynein, and our observation of a mitotic Rae1–NuMA interaction, we were ␣-Rae1. In lanes marked ‘‘2% input,’’ 5 ␮lof250␮l extract used per IP was interested in exploring the effect of NuMA down-regulation on analyzed directly. (C) Synchronized HeLa cells were collected at the indicated the multipolar spindle phenotype of Rae1-depleted cells. NuMA time points, and extracts were analyzed by immunoblotting directly (Input overexpression is also linked to multipolar spindle formation ␣ ␣ 2%) or after IP with -Rae1. Anti-phospho-Histone H3 and -tubulin were that may be rescued by reduction of NuMA levels (24). Indeed, used as mitotic index and loading controls. (D) Asynchronous HeLa cells costained with ␣-Rae1 (green) and ␣-NuMA (red); chromatin was visualized when NuMA and Rae1 levels were reduced simultaneously by using DAPI (blue). The large yellow arrow points to metaphase cell, small siRNA, the incidence of multipolar spindles was greatly reduced white arrowpoints to interphase, and the large white arrowpoints to late (Fig. 2A and Table 1). telophase. (Scale bar, 25 ␮m.) Simultaneous Overexpression of NuMA and Rae1 Rescues Bipolarity. To further test our hypothesis that mitotic Rae1 can bind to that Rae1 and NuMA colocalized transiently on HeLa cell NuMA and influence spindle formation, we explored the effect spindle poles from prophase to anaphase (Fig. 1D and SI Fig. 6). of overexpressing Rae1 in cells overexpressing NuMA and Simultaneous Depletion of Rae1 and NuMA Rescues Bipolarity. Be- cause NuMA or Rae1 are known to individually impact spindle Table 1. RNAi and protein overexpression spindle phenotypes formation, we altered their balance in vivo by modulating their Mitotic Percent