TPX2, A Novel Xenopus MAP Involved in Spindle Pole Organization✪ Torsten Wittmann,* Matthias Wilm,‡ Eric Karsenti,* and Isabelle Vernos* *Cell Biology and Cell Biophysics Program, ‡Biochemical Instrumentation Program, E uropean Molecular Biology Laboratory, D -69117 H eidelberg, G ermany unknown function and thus defines a new family of vertebrate spindle pole components. We investigated the function of TPX2 using spindle assembly in X enopus egg extracts. Immunodepletion of TPX2 from mitotic egg extracts resulted in bipolar structures with disintegrating poles and a decreased microtubule density. A ddition of an excess of TPX2 to spindle assembly reactions gave rise to monopolar structures with abnormally enlarged poles. We conclude that, in addition to its function in targeting Xklp2 to microtubule minus ends during mitosis, TPX2 also participates in the organization of spindle poles. Key words: spindle pole • microtubules • dynein– dynactin • TPX2 • Xklp2 Introduction O nce during the cell cycle, chromosomes are segregated by a bipolar microtubule-based structure, the mitotic spindle. Spindles consist of two antiparallel arrays of microtubules. The microtubule minus ends are focused into the two spindle poles, whereas their plus ends overlap in the equatorial region. Spindle assembly and chromosome segregation depend on the function of a variety of microtubule motor proteins including kinesin-like proteins (KLPs) 1 and cytoplasmic dynein (for recent reviews see E ndow, 1999; H eald and Walczak, 1999; H eck, 1999; Vernos and Karsenti, 1996). In addition, microtubule-associated proteins (MA Ps) are required for the regulation of microtubule dynamics (reviewed by A ndersen, 2000; Cassimeris, 1999) and other molecules, like the small G TPase The online version of this article contains supplemental material. A ddress correspondence to Isabelle Vernos, E uropean Molecular Biology Laboratory, Cell Biology and Cell Biophysics Program, D -69117 H eidelberg, G ermany. Tel.: 49 6221 387 342. Fax: 49 6221 387 512. E -mail: vernos@embl-heidelberg.de 1 A bbreviations used in this paper: cdc2 kinase, cyclin-dependent kinase 2; CSF, cytostatic factor; G FP, green fluorescent protein; G ST, glutathione-S-transferase; KLP(s), kinesin-like protein(s); MA P(s), microtubule-associated protein(s); MA P kinase, mitogen-activated protein kinase; TPX2, targeting protein for Xklp2; Xklp2, X enopus kinesin-like protein 2. R an, are involved in microtubule polymerization during mitosis (Carazo-Salas et al., 1999; Kalab et al., 1999; Wilde and Z heng, 1999). Many proteins including motors, MA Ps, protein kinases, and phosphatases, as well as centrosome components become enriched at spindle poles during mitosis. In many cases both the significance of this localization and the mechanism by which these proteins reach the poles during mitosis are not well understood. Minus end–directed motor proteins like cytoplasmic dynein and XCTK2 (Walczak et al., 1997) may accumulate at microtubule minus ends simply as a consequence of their motor activity and directly transport other proteins to the poles. Indeed, dynein forms a complex with dynactin and NuMA in X enopus egg extracts, suggesting that it directly transports NuMA to the poles (Merdes et al., 2000). In addition, all of these proteins are required for the formation of a focused spindle pole (G aglio et al., 1995, 1997; H eald et al., 1996, 1997; Merdes et al., 1996). O ther microtubule-dependent mechanisms by which proteins accumulate at the spindle poles have not yet been demonstrated but they may involve interactions with proteins actively transported to the poles earlier or depend on the poleward flux of spindle microtubules (Waterman-Storer et al., 1998; Sawin and Mitchison, 1991b). In addition, there appear to be entirely different  The Rockefeller University Press, 0021-9525/2000/06/1405/14 $5.00 The Journal of Cell Biology, Volume 149, Number 7, June 26, 2000 1405–1418 http://www.jcb.org 1405 ✪ Downloaded from http://rupress.org/jcb/article-pdf/149/7/1405/1291988/0003125.pdf by guest on 10 June 2022 Abstract. TPX2, the targeting protein for X enopus kinesin-like protein 2 (Xklp2), was identified as a microtubule-associated protein that mediates the binding of the CO O H -terminal domain of Xklp2 to microtubules (Wittmann, T., H . Boleti, C. A ntony, E . Karsenti, and I. Vernos. 1998. J. Cell B iol. 143:673–685). H ere, we report the cloning and functional characterization of X enopus TPX2. TPX2 is a novel, basic 82.4-kD protein that is phosphorylated during mitosis in a microtubuledependent way. TPX2 is nuclear during interphase and becomes localized to spindle poles in mitosis. Spindle pole localization of TPX2 requires the activity of the dynein–dynactin complex. In late anaphase TPX2 becomes relocalized from the spindle poles to the midbody. TPX2 is highly homologous to a human protein of Materials and Methods Protein Sequencing TPX2 was purified as described (Wittmann et al., 1998) up to the Mono S chromatography step. The protein was in-gel digested with trypsin in a buffer containing 33% 18O water to partially label the CO O H termini of the peptides. The peptides were extracted and desalted on a Poros R 2 column assembled in a pulled glass capillary (Shevchenko et al., 1996). The total peptide mixture was eluted in 2 ml 60% methanol, 5% formic acid into a gold-coated nanoelectrospray glass capillary, and analyzed on an A PI III triple quadrupole mass spectrometer (PE -Sciex; Wilm and Mann, 1996). For every peptide, two fragment spectra were acquired, the first selecting the entire 16O /18O isotopic envelope of the peptide for fragmentation and a second selecting only the 18O isotopes. Sequences were determined by identifying the CO O H -terminal y-ions throughout the fragment spectrum via their 16O /18O isotopic pattern and their different isotopic representation in both spectra. The mass differences between two adjacent y-ions correspond to a single amino acid. Several pairs of amino acids can not be distinguished due to their identical or close to identical mass (I/L and K/Q ). Cloning of TPX2 mR NA was isolated from total X enopus egg R NA using the PolyA Tract mR NA Isolation System (Promega) and first strand cD NA was synthesized with SuperScript II reverse transcriptase (G IBCO BR L) according to standard protocols. U sing the peptide sequences degenerate oligonucleotides containing up to four inosines (19–26 nucleotides, 256–576–fold degeneracy) were synthesized and used for PCR reactions with all possible primer combinations. Two different PCR products were used to screen a mature X enopus oocyte cD NA library in l-Z A P (from John Shuttleworth, U niversity of Birmingham, U K). Library screening, Northern blots and all subsequent cloning was done according to standard protocols (A usubel et al., 1995). Several independent clones were isolated and exonuclease III deletions of The Journal of Cell Biology, Volume 149, 2000 the longest clone (3.5 kb) were sequenced on both strands by the E MBL sequencing service. Recombinant Proteins U ntagged recombinant TPX2 was produced using the IMPA CT T7 onestep protein purification system (New E ngland Biolabs Inc.). The TPX2 open reading frame was amplified by PCR with PfuTurbo D NA polymerase (Stratagene) using 10 cycles of amplification introducing an NcoI and a XmaI site at its 59- and 39-end, respectively, and cloned into the pTYB4 vector (New E ngland Biolabs, Inc.) such that after cleavage only an additional proline and a glycine residue would remain at the CO O H terminus of the recombinant TPX2. The protein was expressed in E . coli BL21(D E 3)pLysS cells, purified and cleaved on a chitin column according to the manufacturer’s instructions, dialyzed against 25 mM K-H epes, pH 7.5, 250 mM KCl, 2 mM MgCl2, 10% glycerol, and 1 mM D TT, frozen in liquid nitrogen, and stored at 2808C. G ST-TPX2 fusion proteins were generated containing either the fulllength TPX2 protein or the NH 2-terminal 431 amino acids. BamH I and SalI restriction sites were introduced by PCR and the fragments were cloned into the pG E X-4T-1 vector (A mersham Pharmacia Biotech). G STfusion proteins were expressed and purified by glutathione affinity chromatography according to standard protocols. To obtain a green fluorescent protein (G FP) TPX2 fusion protein, the TPX2 cD NA was amplified by PCR introducing E coR I and BamH I restriction sites and the fragment was cloned into a modified pH A T2 vector that contained the E G FP sequence after the histidine tag (obtained from A ndrei Popov, E MBL, H eidelberg, G ermany). The histidine-tagged G FPTPX2 was expressed in E . coli BL21(D E 3)pLysS cells and purified native on TA LO N metal affinity resin (CLO NTE CH Laboratories, Inc.) according to the manufacturer’s instructions. Purified G FP-TPX2 was dialyzed, frozen, and stored the same way as untagged TPX2. p50/dynamitin was produced as described (Wittmann and H yman, 1999). Egg Extracts, Spindle Assembly and Immunodepletions Cytostatic factor arrested extracts (CSF-extracts) were prepared according to Murray (1991). H owever, energy mix was omitted in many experiments since it did not appear to improve the quality of the extracts significantly. For cycled spindle assembly, rhodamine-labeled tubulin (H yman et al., 1991) was added at 0.2 mg/ml and sperm at a concentration of z500 nuclei/ml to 15–20 ml CSF-extract and the extract was released into interphase by the addition of 0.4 mM Ca 21 (H eald et al., 1998; D esai et al., 1999). The reactions were incubated for 80–90 min at 208C and cycled back to mitosis by the addition of one volume of CSF-extract. A fter 1 h, the spindles were fixed in 1 ml BR B80 (80 mM K-Pipes, pH 6.8, 1 mM E G TA , 1 mM MgCl2) containing 30% glycerol, 0.25% glutaraldehyde, and 0.1% Triton X-100 and subsequently centrifuged (H B4 rotor, 12,000 rpm, 12 min, 168C) through a 40% glycerol cushion in BR B80 onto polyL -lysine–coated coverslips as described (Sawin and Mitchison, 1991a). For immunodepletions, 7 mg of antibody was bound to 25 ml of protein A –conjugated D ynabeads 280 (D ynal). The antibody was mixed with the beads in a total volume of 100–200 ml PBS containing 0.1% Triton X-100 and incubated for at least 1 h on a rotating wheel. The beads were then washed once with PBS, 0.1% Triton X-100, once with 0.5 M NaCl in PBS, 0.1% Triton X-100, and three times with CSF-XB (10 mM K-H epes, pH 7.7, 50 mM sucrose, 100 mM KCl, 2 mM MgCl2, 0.1 mM CaCl2, and 5 mM E G TA ). Between washing steps the beads were retrieved on a magnet and at the end the buffer was removed as much as possible. 100 ml extract was added to the beads and pipetted up and down carefully until the beads were completely resuspended. The extract was then placed on ice for 90 min and mixed by pipetting once or twice during this time. The beads were retrieved on a magnet on ice for z10 min and the recovered extract was used for spindle assembly. To analyze bound proteins, the beads were washed three times with CSF-XB, three times with PBS, 0.1% Triton X-100, boiled in SD S-PA G E sample buffer and subjected to gel electrophoresis. A lternatively, TPX2associated proteins were eluted with 0.5 M NaCl in PBS and 0.1% Triton X-100 for 15 min on ice before SD S-PA G E . Immunofluorescence, Transfections, and Microscopy XL177 cells grown on coverslips were briefly rinsed with 70% PBS and fixed in methanol at 2208C for 5 min. A ntibodies were diluted in PBS 1406 Downloaded from http://rupress.org/jcb/article-pdf/149/7/1405/1291988/0003125.pdf by guest on 10 June 2022 mechanisms for the recruitment of spindle pole proteins. g-Tubulin, for example, as well as the D rosophila centrosomal proteins CP60 and CP190 accumulate at the spindle poles at the onset of mitosis in a microtubule-independent way (O egema et al., 1995; Khodjakov and R ieder, 1999). We have previously identified a X enopus kinesin-like protein, Xklp2, that contains an NH 2-terminal motor domain and is a plus end–directed microtubule motor. Nevertheless, Xklp2 accumulates at the minus ends of spindle microtubules during mitosis (Boleti et al., 1996; Wittmann et al., 1998). Spindle pole targeting information resides in the CO O H -terminal domain of Xklp2. U sing a glutathione-S -transferase (G ST) fusion protein containing the CO O H -terminal 250 amino acids of Xklp2, G ST-Xklp2Tail, we previously examined the mechanism of Xklp2 localization to the poles. In addition to the function of the dynein–dynactin complex, the localization of G ST-Xklp2Tail requires a MA P, TPX2 (targeting protein for Xklp2), that we purified from X enopus egg extract based on its ability to mediate the binding of G ST-Xklp2-Tail to pure microtubules (Wittmann et al., 1998). To learn more about the function and localization of proteins at the mitotic spindle pole, we now describe the molecular cloning and functional characterization of TPX2. TPX2 is a novel MA P that defines a new class of vertebrate spindle pole components and is itself localized to spindle poles in a dynein-dependent way. We also show that in addition to its function in localizing Xklp2 to microtubule minus ends, TPX2 plays an important role in spindle pole organization. trometry. A full-length cD NA clone was isolated by degenerate PCR and screening of a X enopus laevis oocyte cD NA library. The PCR fragment used for library screening hybridized with a single transcript of z3 kb on a Northern blot of total X enopus egg R NA (Fig. 1 A ). The longest TPX2 cD NA clone contained an open reading frame encoding a polypeptide of 715 amino acids (Fig. 1 B). The predicted molecular mass is 82.4 kD and thus slightly lower than the apparent size of z100 kD observed during the initial purification from egg extract. The protein is highly charged (18.7% strongly basic and 14.7% strongly acidic amino acids) and very basic with a predicted isoelectric point of z9.5. D atabase searches with either the nucleotide or the predicted protein sequence did not identify any homologous protein with known function. H owever, the TPX2 protein sequence was homologous to a number of vertebrate E STs Microtubule Sedimentation Assay and In Vitro Phosphorylation Purified tubulin was polymerized as described (Wittmann et al., 1998), mixed with recombinant proteins in 100 ml 20 mM K-H epes, pH 7.5, 200 mM KCl, 2 mM MgCl2, 1 mM E G TA , 0.1% Triton X-100, 1 mM D TT, and 10 mM taxol and incubated for 20 min at 208C. The reactions were then centrifuged through a 10% sucrose cushion in BR B80 as described (Wittmann et al., 1998) and analyzed by SD S-PA G E and immunoblot. G FP-TPX2 fluorescence was measured in a Luminescence Spectrometer (A minco Bowman). In microtubule sedimentation assays with crude egg extract, the reactions were diluted 1:5 in the above buffer before centrifugation. For phosphorylation in egg extract, z50 mCi g-[32P]A TP (A mersham Pharmacia Biotech) was added to 50 ml mitotic or interphase X enopus egg extract and incubated for 20 min at 208C. The reaction was stopped by the addition of 200 ml stop buffer (100 mM NaF, 80 mM b-glycerophosphate, 20 mM Na 4P 2O 7, 20 mM E D TA , 2 mM microcystin, and 10 mg/ml aprotinin, pepstatin, leupeptin, pH 7.5) containing 5 mg anti-TPX2 antibody bound to A ffiPrep protein A beads (Bio-R ad Laboratories). The mixture was incubated for 1 h on a rotating wheel at 48C, the beads were washed twice with stop buffer and twice with PBS containing 0.1% Triton X-100 and analyzed by SD S-PA G E and autoradiography. The degree of radioactivity incorporation was quantified with a PhosphorImager (Fuji). TPX2 was purified from mitotic X enopus egg extract as described (Wittmann et al., 1998) and peptide sequences of several tryptic fragments were obtained by mass spec- Figure 1. Molecular cloning of TPX2. (A ) Northern blot of total X enopus egg R NA hybridized with a PCR fragment obtained in a reaction with degenerate oligonucleotides designed according to TPX2 peptide sequences. The amount of R NA loaded per lane is indicated on top. The migration position of the 28S rR NA (4,115 nucleotides) and the 18S rR NA (1,826 nucleotides) is shown on the left. The probe recognizes a unique transcript of 3.0–3.5 kb. (B) A mino acid sequence of TPX2. Peptide sequences obtained by mass spectrometry are underlined. D ifferences between the predicted protein sequence and the peptide sequences are indicated by dotted underlines. Putative cdc2 kinase sites, (S/T)PX(K/R ), are shaded in dark gray and MA P kinase phosphorylation sites, PX(S/T)P, are shaded in light gray, nuclear localization signals are shown in bold and the predicted coiled-coil regions are in italics. The complete cD NA sequence of X enopus laevis TPX2 is available from G enBank/E MBL/D D BJ under accession number A F244546. (C) Sequence comparison of a region from TPX2 (corresponding to amino acids 297–356) with the human protein fls353 (amino acids 328–387) and predicted protein sequences from E STs from mouse, chicken and zebrafish (these data are available from G enBank/E MBL/D D BJ under accession number A I415194, A J394859, and A I883915). Conserved residues are shaded in gray and boxes indicate the highly conserved cdc2 and MA P kinase phosphorylation sites within this region. Wittmann et al. TPX2, A Novel Spindle Pole Component 1407 Online Supplemental Material Time-lapse imaging of a mitotic XL177 cell expressing low levels of G FPTPX2 as shown in Fig. 4 B. The sequence contains images taken every minute over a total time of 60 min. Supplemental materials are viewable at http://www.jcb.org/cgi/content/full/149/7/1405/D C1. Results Molecular Characterization of TPX2 Downloaded from http://rupress.org/jcb/article-pdf/149/7/1405/1291988/0003125.pdf by guest on 10 June 2022 containing 0.1% Triton X-100 and 2% BSA . Primary and secondary antibodies were applied to the coverslips for 20 min at room temperature, coverslips were washed in between incubations with PBS for at least three times and finally embedded in mowiol (H oechst). Pictures were taken with an LSM 510 confocal microscope (Z E ISS). The TPX2 cD NA sequence was amplified by PCR introducing an E coR I and a BamH I restriction site and cloned into the pE G FP-N1 eukaryotic expression vector (CLO NTE CH Laboratories, Inc.). XL177 cells were transfected by electroporation essentially as described (Tuma et al., 1998). In brief, XL177 cells were electroporated in 0.2-cm cuvettes at 220 V, 200 V, and 250 mF using a G enePulser II (Bio-R ad Laboratories). A fter electroporation the cells were directly plated onto poly-L -lysine– coated coverslips and mitotic transfected cells were observed 30–60 h after transfection. Spindles assembled in egg extract were processed for immunofluorescence as described (Wittmann et al., 1998). A ll pictures of in vitro assembled structures were taken with a 12-bit grayscale cooled CCD camera (F-View, Soft Imaging System) mounted on an A xioskop-2 (Z E ISS) and digital images were processed with the analysis software (Soft Imaging System) and A dobe Photoshop. For quantification, images were taken at 163 magnification, printed, and the structures were scored from the printouts. For fluorescence intensity measurements, images were taken at 633 magnification with identical camera and illumination settings below saturation of the camera. The gray values of all pixels in a 50 3 50–pixel area were integrated and a background value was subtracted determined from an equal-sized square on the same image. R abbit polyclonal antibodies were raised against the G ST-TPX2 fusion proteins. The antibodies were affinity-purified on a column of untagged TPX2 covalently linked to Sepharose. A nti-G ST and anti-Xklp2 antibodies were as described (Wittmann et al., 1998). Monoclonal anti–a-tubulin antibodies were from A mersham. Fluorescent- and horseradish peroxidase–conjugated antibodies were from Jackson ImmunoR esearch Laboratories, A mersham Pharmacia Biotech (CyD ye), and Molecular Probes (A lexa). TPX2 Binds to Microtubules with High Affinity To estimate the affinity of TPX2 for microtubules, bacterially expressed G FP-TPX2 was mixed with different concentrations of pure, prepolymerized microtubules. The microtubule-bound fraction was removed by centrifugation, and the amount of G FP-TPX2 that remained soluble was determined by fluorescence spectrometry. Plotting of the fraction of G FP-TPX2 bound to microtubules versus the microtubule concentration and direct hyperbolic curve fitting yielded a dissociation constant of below 0.5 mM (Fig. 2 C). H owever, even with saturating amounts of microtubules only about half of the G FP fluorescence was recovered in the microtubule pellet which is probably due to the relatively high amount of truncated G FP-TPX2 present in the protein preparation (see Fig. 3 B, last lane). Since TPX2 was phosphorylated in a cell cycle–dependent manner, we analyzed whether the cell cycle state of the extract affected the association of TPX2 with microtu- The Journal of Cell Biology, Volume 149, 2000 Figure 2. TPX2 is phosphorylated in mitotic egg extract and binds to microtubules with high affinity. (A ) Immunoblot of lowspeed X enopus egg extracts, mitotic microtubule-associated proteins purified as described (Wittmann et al., 1998) and XL177 SD S lysate probed with affinity-purified anti-TPX2N. The molecular mass of marker proteins is indicated on the left. (B) Phosphorylation of TPX2 in mitotic, M, and interphase, I, egg extract incubated in the presence of g-[32P]A TP. Taxol was added to 2 mM and the extracts were incubated for 30 min at 208C. The immunoprecipitates were analyzed on a 6% SD S-PA G E and subjected to autoradiography. TPX2 is phosphorylated in mitotic extract and hyperphosphorylated upon assembly of microtubules. (C) Microtubule sedimentation assay of G FP-TPX2 with pure microtubules. The fraction of G FP-TPX2 bound to microtubules is plotted versus the microtubule concentration. The solid line represents the hyperbolic curve fit. (D ) Microtubule sedimentation assay from crude extract. Interphase, I, and mitotic, M, extract were supplemented with either 20 mM nocodazole or 2 mM taxol, incubated for 20 min at 208C and centrifuged through a 10% sucrose cushion. The immunoblots show the amount of TPX2 present in equivalent amounts of the soluble and pelleted fraction. bules. Interphase and mitotic egg extract were incubated in the presence of either nocodazole or taxol, and the insoluble proteins were recovered by centrifugation through a sucrose cushion (Fig. 2 D ). R egardless of the cell cycle state of the extract, the major amount of TPX2 was recovered in the microtubule pellet. This suggests that global cell cycle–dependent phosphorylation does not regulate the affinity of TPX2 for microtubules. TPX2 Is Required for the Targeting of Xklp2 to Microtubule Minus Ends To verify that the protein we have cloned was sufficient for the binding of the CO O H -terminal domain of Xklp2 to microtubules we used a sedimentation assay with pure, prepolymerized microtubules. Microtubules were mixed with bacterially expressed TPX2 and G ST-Xklp2-Tail and centrifuged through a sucrose cushion (Fig. 3 A ). In the absence of microtubules both TPX2 and G ST-Xklp2-Tail 1408 Downloaded from http://rupress.org/jcb/article-pdf/149/7/1405/1291988/0003125.pdf by guest on 10 June 2022 and highly similar to a hypothetical human 747–amino acid protein (fls353 or D IL-2, respectively; these data are available from G enBank/E MBL/D D BJ under accession number A B024704 and A B027467; see also Fig. 1 C). TPX2 and fls353 are 52% identical over the whole protein sequence. The degree of identity increases to 63% when the NH 2-terminal 180 amino acids are not considered. A nalysis of the TPX2 amino acid sequence revealed that it contained three putative cdc2 phosphorylation sites as well as five putative sites for phosphorylation by MA P kinase. Most of these phosphorylation sites were conserved between the frog and human protein. The TPX2 protein also contained seven potential nuclear localization signals and two short regions (amino acids 171–208 and 650–684) that showed a significant probability for coiled-coil formation. To investigate the function of TPX2 we raised polyclonal antibodies against the full-length protein, anti-TPX2, and against the NH 2-terminal 431 amino acids, antiTPX2N. Both affinity-purified antibodies recognized a single protein band of z100 kD in mitotic as well as in interphase X enopus egg extracts, in MA Ps purified from mitotic egg extract and in a lysate prepared from unsynchronized X enopus tissue culture cells (Fig. 2 A ). In mitotic egg extract and in the mitotic MA Ps fraction the TPX2 band was slightly upshifted indicating a posttranslational modification during mitosis. To test whether this upshift was due to phosphorylation, TPX2 was immunoprecipitated from interphase and mitotic extract after incubation with radiolabeled A TP (Fig. 2 B). The protein immunoprecipitated from mitotic extract showed a retardation in gel electrophoresis and a three- to fivefold increased incorporation of radioactivity indicating that it was more phosphorylated in mitotic compared with interphase extract. Interestingly, additional phosphorylation events were observed only in mitotic but not in interphase extract upon microtubule polymerization. A ddition of taxol to mitotic extract caused a further upshift of the TPX2 band and another two- to threefold increase in incorporation of radioactivity. These results show that TPX2 can be phosphorylated in mitotic extract on several phosphorylation sites in distinct steps. TPX2 Is Localized to Mitotic Spindle Poles We next determined the localization of TPX2 both in X enopus tissue culture cells and in spindles assembled in vitro. D uring interphase a small subset of cells displayed very faint nuclear staining. No localization was observed along interphase microtubules (Fig. 4). D uring prophase, TPX2 staining became apparent at the center of the mitotic asters around the time of nuclear envelope break- Figure 3. TPX2 is required for the binding of Xklp2 to microtubules. (A ) Microtubule sedimentation assay. Pure prepolymerized microtubules (1 mM) were mixed with bacterially expressed TPX2 and G ST-Xklp2-Tail (both 0.1–0.2 mM), T, or G ST-Xklp2CD el2, C (Wittmann et al., 1998), as indicated on top, incubated for 20 min at 208C and centrifuged through a 10% sucrose cushion. E quivalent amounts of the soluble and pelleted fraction were analyzed by immunoblot probed with anti-TPX2N or anti-Xklp2 antibodies as indicated. G ST-Xklp2-Tail is only recovered in the pellet in the presence of both microtubules and TPX2. (B) Immunodepletion of TPX2 and G FP-TPX2 addback. 1- ml aliquots of extract were analyzed by immunoblot and probed with antiTPX2N. The molecular mass of marker proteins is indicated on the left. A dditional bands in the addback lane are due to degradation products of G FP-TPX2. (C) Mitotic asters assembled for 30 min at 208C in the presence of 5% D MSO , 1 mM G ST-Xklp2Tail, and rhodamine-labeled tubulin in control- and TPX2depleted extract supplemented with bacterially expressed G FPTPX2. The asters were sedimented onto coverslips and stained with an anti-G ST and an A lexa350-conjugated anti–rabbit antibody. (D ) Mitotic asters assembled in the presence of Cy5labeled tubulin as in C but without G ST-Xklp2-Tail added. The asters were stained with an anti-Xklp2 and a rhodamine-conjugated anti–rabbit antibody. Bars, 10 mm. Wittmann et al. TPX2, A Novel Spindle Pole Component 1409 Downloaded from http://rupress.org/jcb/article-pdf/149/7/1405/1291988/0003125.pdf by guest on 10 June 2022 remained soluble. H owever, in the presence of microtubules, TPX2 was quantitatively recovered in the microtubule pellet. G ST-Xklp2-Tail cosedimented with microtubules only when TPX2 was included in the reaction (Fig. 3 A , lane 5). The specificity of this interaction was further demonstrated by the observation that a truncated version of G ST-Xklp2-Tail that does not localize to mitotic structures (Wittmann et al., 1998) did not cosediment with microtubules in the presence of TPX2 (Fig. 3 A , last lane). The minor amount of TPX2 that pelleted in the presence of unpolymerized tubulin was probably due to some tubulin aggregation (Fig. 3 A , second lane). The above results confirmed that TPX2 was required to bind the CO O H -terminal domain of Xklp2 to microtubules. H owever, in our previous work we could not determine whether TPX2 was required for targeting Xklp2 to microtubule minus ends in mitotic egg extracts. To address this question we used the anti-TPX2N antibody to deplete the protein from extract and analyzed the localization of Xklp2 in mitotic asters assembled by the addition of D MSO (Stearns and Kirschner, 1994). D MSO -induced asters formed both in control and TPX2 extracts (Fig. 3). H owever, in TPX2-depleted extract G ST-Xklp2-Tail failed to localize to the center of these asters, whereas it did localize perfectly well in control-depleted extracts. G FP-TPX2 added to the TPX2-depleted extract became localized to the center of the asters and rescued the localization of G ST-Xklp2-Tail (Fig. 3 C). The endogenous Xklp2 showed essentially the same behavior as exogenously added G ST-Xklp2-Tail (Fig. 3 D ). The Journal of Cell Biology, Volume 149, 2000 1410 Downloaded from http://rupress.org/jcb/article-pdf/149/7/1405/1291988/0003125.pdf by guest on 10 June 2022 Figure 4. (A ) Immunolocalization of TPX2 throughout the cell cycle. XL177 X enopus tissue culture cells were fixed in cold methanol and stained with 0.6 mg/ml affinity-purified antiTPX2N, monoclonal anti-tubulin, and H oechst 33258 followed by FITC-labeled anti–mouse and TR ITC-labeled anti–rabbit antibodies. Images represent single confocal slices. The same staining pattern was observed with anti-TPX2 or when the cells were fixed with glutaraldehyde. TPX2 staining appears at the end of prophase, persists at the spindle poles throughout mitosis until late anaphase. It then relocalizes to the midbody where it disappears in late telophase. (B) Live images of a mitotic XL177 cell expressing low amounts of G FP-TPX2. Note the nuclear fluorescence in telophase. The elapsed time is indicated in the upper right corner. Supplemental video is viewable at http:// www.jcb.org/cgi/content/full/149/ 7/1405/D C1. Bars, 10 mm. bled in cycled mitotic extract and recombinant p50/dynamitin was added to the reactions when the extract was cycled back from interphase to mitosis. The addition of p50/dynamitin to egg extract leads to the dissociation of the dynactin complex (Wittmann and H yman, 1999). A lmost all of the bipolar structures formed had unfocused poles, a result expected when dynein is inhibited during spindle assembly (H eald et al., 1997). Moreover, spindles assembled in the presence of p50/dynamitin were z30% longer than in the control reaction (in average 33 6 3.0 mm versus 26 6 3.6 mm, n 5 20). In control spindles, TPX2 was concentrated close to the spindle poles. H owever, in the presence of p50/dynamitin, TPX2 staining was less bright and spread over the whole length of the spindle (Fig. 6 A ). This observation was confirmed by measuring the TPX2 fluorescence intensity profile along the spindle axis. The two prominent peaks close to the spindle poles corresponding to the highest TPX2 concentration disappeared in the presence of p50/dynamitin and the intensity profile assumed a shape more similar to that of tubulin (Fig. 6 B). The total TPX2 fluorescence was reduced to z70% compared with the control. In contrast, the total tubulin fluorescence remained roughly the same in p50/ dynamitin-treated spindles and the intensity profile was only slightly elongated. This indicates that dynein–dynactin function is necessary for TPX2-localization to the spindle poles. TPX2 Is Involved in Spindle Pole Organization Since we had observed that the localization of the G STXklp2-Tail protein was dependent on a functional dynein–dynactin complex (Wittmann et al., 1998) it was likely that TPX2-localization would also require this motor protein. To test this hypothesis, spindles were assem- To further analyze the function of TPX2, we depleted the protein from egg extract and tested the effect on spindle assembly. In our hands, spindles assembled in extracts cycled from interphase to mitosis yielded more reproducible results and a higher percentage of bipolar spindles than half-spindle reactions in CSF extracts (Sawin and Mitchison, 1991a). H owever, published immunodepletion protocols most often resulted in extracts incapable of cycled spindle assembly (D esai et al., 1999). We therefore used protein A –conjugated magnetic beads and minimized the agitation of the extract during incubation to obtain depleted extracts that reliably formed bipolar spindles in cycled extracts. With this method, z95% of TPX2 could be reproducibly removed from egg extract (see Fig. 8 A ). In addition to TPX2, a polypeptide of z90 kD was recovered almost in stoichiometric amounts in TPX2 immunoprecipitations (Fig. 7). This protein could be eluted from the beads with a high salt buffer (Fig. 7 A ) and was not recognized by anti-TPX2 antibodies demonstrating that it was not a TPX2 degradation product. Furthermore, when an excess of G ST-TPX2 fusion protein was added to mitotic extract and immunoprecipitated with an anti-G ST antibody, the 90-kD band was again recovered in stoichiometric amounts (Fig. 7 B). This indicates that the 90-kD protein may form a 1:1 complex with TPX2. Since the 90-kD protein appears to be more abundant in egg extract than TPX2 it seems unlikely that its amount is significantly reduced in TPX2-depleted extract. The identification and analysis of this protein will be the subject of future studies. By contrast, TPX2 did not coimmunoprecipitate with G ST-TPX2. NuMA , subunits of the dynein–dynactin complex or Xklp2 could not be detected in TPX2 immunopre- Wittmann et al. TPX2, A Novel Spindle Pole Component 1411 TPX2 Localization Requires the Dynein–Dynactin Complex Downloaded from http://rupress.org/jcb/article-pdf/149/7/1405/1291988/0003125.pdf by guest on 10 June 2022 down and in prometaphase the center of both asters was labeled. The labeling intensity increased until metaphase and spindle microtubules were brightly stained. The signal appeared to be more intense towards the spindle poles and astral microtubules were almost unlabeled. A t the onset of anaphase, TPX2 remained concentrated at the spindle poles. In late anaphase, a relocalization to the midzone occurred that resulted in a fairly strong staining of the midbody so that in late telophase no staining was left in the centrosome area (Fig. 4). Since TPX2 was present in comparable amounts both in mitotic and in interphase egg extract we performed immunofluorescence on in vitro interphase nuclei and on spindles assembled in interphase-to-mitotic extracts (Sawin and Mitchison, 1991a). In interphase extract, TPX2 showed a diffuse nuclear localization as well as an association with the nuclear envelope and a branched tubular structure within the nucleus. This staining became more prominent after longer incubation times when the chromatin started to condense (Fig. 5 A ). Patches of condensing chromatin were in close proximity to the TPX2-labeled structures. This nuclear staining was not observed in TPX2-depleted extracts or with secondary antibodies alone. A t very early time points (5–10 min) after cycling the extract back into mitosis we could observe half-spindle–like structures where microtubules had just begun to interact with the condensing chromatin. Interestingly, in these structures TPX2 staining was continuous between the remains of the nucleus and microtubules in the proximity of the chromatin (Fig. 5 B). A fter z60 min, a high percentage of bipolar spindles had formed. In most spindles TPX2 was strongly concentrated in a crescent-shaped area at both spindle poles (Fig. 5 B). Similar spindle pole staining was observed in spindles assembled in the absence of centrosomes around chromatin beads (data not shown). To compare TPX2 localization to another well-characterized pole protein, spindles were assembled and fixed under identical conditions and then stained either for TPX2 or NuMA (Fig. 5 C). In contrast to NuMA , which was concentrated in a very small area at spindle poles, TPX2 was also present on microtubules throughout the spindle. A s an alternative way to examine the localization of TPX2, G FP-tagged TPX2 was either added to egg extracts or transfected into X enopus tissue culture cells. In interphase and mitotic extracts, G FP-TPX2 showed the same behavior as the endogenous protein detected by immunofluorescence (data not shown). In transfected tissue culture cells, we could observe that G FP-TPX2 remained at the spindle poles until late anaphase. It then became redistributed to the midbody along spindle microtubules and was imported into the reforming nuclei (Fig. 4 B and video). Taken together, these results show that TPX2 exhibits a complex pattern of localization during the cell cycle. cipitations either, suggesting that microtubules are required for the binding of Xklp2 to TPX2. We then analyzed mitotic structures formed in TPX2depleted extracts. TPX2 depletion did not significantly af- fect the formation of bipolar microtubule arrays. 1 h after cycling back into mitosis the total number of bipolar structures formed in control- and TPX2-depleted extract was about the same but TPX2-depleted bipolar arrays had The Journal of Cell Biology, Volume 149, 2000 1412 Downloaded from http://rupress.org/jcb/article-pdf/149/7/1405/1291988/0003125.pdf by guest on 10 June 2022 Figure 5. Immunolocalization of TPX2 in X enopus egg extract. (A ) Sperm nuclei were incubated in extract released into interphase by the addition of calcium for the indicated time, fixed and sedimented onto coverslips. (B) Mitotic spindles assembled in the presence of rhodamine-labeled tubulin in cycled CSF-extract. R eactions were fixed at the indicated times after reentry into mitosis and sedimented onto coverslips. Spindles and nuclei were stained with 3 mg/ml anti-TPX2N, a Cy2labeled anti–rabbit antibody and H oechst 33258. TPX2 localizes to a branched structure in interphase nuclei and migrates to the spindle poles in mitosis. (C) Mitotic spindles centrifuged onto coverslips, postfixed in cold methanol and stained for either TPX2 or NuMA . Images were taken under identical conditions. Bars, 10 mm. abnormal morphologies (Fig. 8, B and C). In contrast to control spindles, TPX2 staining was absent from these structures. H owever, more normal looking spindles often showed some remaining TPX2 staining at the poles. In general, TPX2-depleted structures appeared less compact than control spindles and displayed a variety of pole defects. Some bipolar structures had completely unfocused microtubules reminiscent of spindles formed in dynein– dynactin-inhibited extracts. Many others displayed multi- Figure 7. Immunoprecipitation of TPX2 from egg extract. (A ) Immunoprecipitated proteins using a control rabbit IgG or antiTPX2N were eluted with a high-salt buffer and proteins remaining on the beads or recovered in the eluate were analyzed on a Coomassie-stained SD S-PA G E . (B) G ST-TPX2 was added to mitotic egg extract, incubated for 30 min at 208C and immunoprecipitated with an anti-G ST antibody. For both gels, the molecular mass of marker proteins is indicated on the left. A polypeptide of z90 kD (arrowhead) is specifically immunoprecipitated together with the endogenous TPX2 or with G ST-TPX2. ple asters loosely attached to the disintegrating spindle poles (Fig. 8 C). These asters often appeared as the brightest areas indicating a reduced microtubule density. This was confirmed by quantification of the rhodamine-tubulin fluorescence in a small area between poles and metaphase plate showing that the microtubule density was reduced by z50% in TPX2-depleted structures (Fig. 8 D ). The overall size of bipolar structures assembled in TPX2-depleted extract (length 33 6 3.4 mm, width 16 6 3.3 mm, n 5 100) was only marginally increased compared with control spindles (length 29 6 3.0 mm, width 15 6 2.1 mm, n 5 100). The TPX2 depletion phenotype could be rescued by the addition of bacterially expressed TPX2: the microtubule density in bipolar spindles formed was indistinguishable from the control and the added protein localized to the spindle poles. Most of the bipolar spindles had a normal pole morphology, but some showed open ring-like poles (Fig. 8, C and D ). H owever, the number of bipolar structures was greatly reduced compared with control- and TPX2-depleted reactions and many monopolar and rosette-like structures formed that were not quantified due to a great variability in different experiments. A previous analysis of Xklp2 function using the G STXklp2-Tail as a dominant negative inhibitor resulted predominantly in the formation of monopolar structures (Boleti et al., 1996). Since TPX2 is required for the localization of Xklp2 to the poles, we were surprised to find mostly bipolar structures in TPX2-depleted extracts. Therefore, we decided to reexamine Xklp2 function by analyzing cycled spindle assembly in Xklp2-depleted extracts. A fter depletion of .98% of Xklp2 from mitotic egg extract (Fig. 9 A ), the percentage of normal bipolar spindles formed was indistinguishable from controls (Fig. 9 B). TPX2 remained localized to the spindle poles in Xklp2- Wittmann et al. TPX2, A Novel Spindle Pole Component 1413 Downloaded from http://rupress.org/jcb/article-pdf/149/7/1405/1291988/0003125.pdf by guest on 10 June 2022 Figure 6. The dynein–dynactin complex is required for the localization of TPX2 to spindle poles. (A ) R ecombinant p50/dynamitin was added to cycled spindle reactions at z0.7 mg/ml. The spindles were sedimented onto coverslips and stained with anti-TPX2N and a Cy2conjugated anti–rabbit antibody. In the overlay, also the D NA visualized by H oechst 33258 staining is shown in blue. Fluorescence intensity of the TPX2 staining and the rhodamine-labeled tubulin was measured in a 100-pixel wide stripe (width is indicated by arrowheads on the right) along the spindle axis. (B) The average fluorescence intensity of eleven spindles with (red) and without (black) p50/dynamitin added was plotted against the distance from the metaphase plate. depleted extract (Fig. 9 C). In extracts depleted of both Xklp2 and TPX2, we observed the same phenotype as when TPX2 was depleted alone (Fig. 9 C). From the immunoprecipitated material it was also evident that Xklp2 is more abundant in egg extract than TPX2 (data not shown). These experiments raised the question of the actual function of Xklp2 in spindle pole separation, but indicated that in addition to localizing Xklp2 to the poles, TPX2 plays a role in spindle pole organization. To determine the fate of another spindle pole component when TPX2 is inhibited, TPX2-depleted structures were stained with anti-NuMA antibodies (Fig. 9 D ). NuMA was clearly detectable in these structures and concentrated in the multiple microtubule foci observed in TPX2-depleted structures indicating that they may represent fragments of spindle pole material. H owever, the total intensity of NuMA staining was somewhat reduced compared with control spindles. The Journal of Cell Biology, Volume 149, 2000 1414 Downloaded from http://rupress.org/jcb/article-pdf/149/7/1405/1291988/0003125.pdf by guest on 10 June 2022 Figure 8. D epletion of TPX2 affects spindle pole structure. (A ) Immunoblot probed with anti-TPX2N demonstrating the depletion efficiency. 1-, 0.2-, and 0.05-ml samples of mitotic X enopus egg extract are compared with 1 ml of control- and TPX2-depleted extract supplemented with bacterially expressed TPX2. The molecular mass of marker proteins is indicated on the left. (B) Q uantification of structures observed in control- and TPX2-depleted extract 1 h after cycling the extract back into mitosis. The number of quantified structures is indicated on top. (C) R epresentative bipolar structures observed in control- and TPX2-depleted extract supplemented with recombinant TPX2. Cycled spindles were assembled in the presence of rhodamine-labeled tubulin, fixed, centrifuged onto coverslips, and stained with anti-TPX2N and H oechst 33258. (D ) Q uantification of the microtubule density in bipolar structures assembled in control- and TPX2-depleted extract supplemented with recombinant TPX2 from two independent experiments. The red boxes indicate representative areas in which the rhodamine fluorescence intensity was measured. The number of structures analyzed is shown on top of the columns. E rror bars indicate the standard deviation. Bars, 10 mm. An Excess of TPX2 Affects Spindle Pole Structure and Disrupts Bipolarity The lack of effect of Xklp2 depletion on spindle bipolarity did not agree with the inhibition of spindle pole separation produced by the addition of G ST-Xklp2-Tail (Boleti et al., 1996). Thus, we wondered whether the addition of TPX2 would have an effect similar to the addition of G STXklp2-Tail. Indeed, the addition of a 5–10-fold molar excess of TPX2 to cycled spindle assembly reactions resulted in a dramatic decrease of bipolar spindle formation. 60 min after reentry of the extract into mitosis, we observed a mixture of aggregated nuclei and monopolar microtubule structures that were very strongly stained by anti-TPX2 antibodies (Fig. 10, A and B). These monopolar structures were reminiscent of half-spindles observed at early time points during spindle assembly in CSF-extracts (Sawin and Mitchison, 1991a) and were comparable in length (24 6 3.8 mm, n 5 67). H owever, the microtubule density in these structures appeared to be extraordinarily high and they had enlarged ring-like poles. Q uantification of the rhodamine-tubulin fluorescence between pole and chromatin showed that the microtubule density in the presence of excess TPX2 was increased by z30% compared with bipolar spindles in the control reaction (Fig. 10 C). NuMA was still localized in a tight, crescent-shaped area at the poles of these monopolar structures and did not seem to Wittmann et al. TPX2, A Novel Spindle Pole Component overlap with the majority of TPX2 (Fig. 10 D ). TPX2 addition also induced the formation of numerous ectopic asters with NuMA concentrated in the center (data not shown). Discussion TPX2 Is a Novel Vertebrate Spindle Pole Protein TPX2 was identified and purified on the basis of its ability to bind to microtubules and to mediate the binding of the CO O H -terminal domain of Xklp2 to microtubules in mitotic X enopus egg extract (Wittmann et al., 1998). Native and recombinant TPX2 binds to microtubules with high affinity, but TPX2 has no sequence homology with other MA Ps and it does not contain a tau- or CA P-G ly–like microtubule-binding motif (D e Z eeuw et al., 1997; D rewes et al., 1998). H owever, we found that TPX2 shares homologies with predicted proteins from different vertebrate species and is highly homologous to a human protein of unknown function, fls353 or D IL-2, that is differentially expressed in different types of cancerous cells (H ufton et al., 1999; Manda et al., 1999). We did not find any homology to D rosophila, C. elegans, or yeast proteins. Thus, TPX2 defines a novel family of vertebrate MA Ps. It is interesting to note that D IL-2 is predominantly expressed in 1415 Downloaded from http://rupress.org/jcb/article-pdf/149/7/1405/1291988/0003125.pdf by guest on 10 June 2022 Figure 9. Cycled spindle assembly in Xklp2- and TPX2-depleted extract. (A ) Immunoblot of 1-ml samples of control-, TPX2-, Xklp2-, and double-depleted extract compared with 1, 0.2, 0.05, and 0.02 ml of mitotic egg extract and probed for Xklp2 and TPX2 as indicated. (B) Q uantification of structures observed under the various depletion conditions. The number of quantified structures is indicated on top. (C) R epresentative bipolar structures observed in Xklp2and double-depleted extract. Cycled spindles were assembled in the presence of rhodaminelabeled tubulin, fixed, centrifuged onto coverslips and stained with anti-TPX2N and H oechst 33258. (D ) TPX2-depleted structure stained for NuMA . Bars, 10 mm. fetal tissues consistent with a function in cell division (Manda et al., 1999). TPX2 shows a dynamic localization during the cell cycle, accumulating at spindle poles during mitosis. In interphase, it is nuclear like other mitotic MA Ps or certain centrosomal proteins (O egema et al., 1995, 1997; Merdes et al., 1996; A ndersen and Karsenti, 1997) and in nuclei assembled in egg extracts, TPX2 associates with a branched intranuclear structure. Since in TPX2-depleted extracts, interphase nuclei form without any visible defect and the chromatin condenses normally upon entry into mitosis, TPX2 is unlikely to have a nuclear function. A lack of nuclear phenotype has also been observed in NuMA depletions (Merdes and Cleveland, 1998). This suggests that nuclear import of spindle pole components may be merely a way to sequester them from the cytoplasm during interphase. bacterially expressed protein bind to microtubules in biochemical assays. U sing mass spectrometry we have been able to detect that at least one of the cdc2 sites and all of the MA P kinase sites can be phosphorylated in egg extract (Wittmann, T., G . Neubauer, and M. Wilm, unpublished results). Furthermore, the conservation of some of the phosphorylation sites throughout a number of vertebrate TPX2 homologues (Fig. 1 C) suggests that these sites are functionally relevant. Since it has recently been shown that activated MA P kinase localizes to kinetochores and mitotic spindle poles (Shapiro et al., 1998; Z ecevic et al., 1998), it is possible that some aspects of TPX2 function are regulated by MA P kinase. Targeting of Motor Proteins The TPX2 sequence contains a number of potential phosphorylation sites for cdc2 and MA P kinase. TPX2 is indeed phosphorylated in mitotic versus interphase extract and mitotic phosphorylation is stimulated by polymerization of microtubules. NuMA is the only other protein where something similar has been observed (G aglio et al., 1995). While in many cases the affinity of MA Ps for microtubules is downregulated by phosphorylation (A ndersen et al., 1994; D rewes et al., 1997), phosphorylated TPX2 purified from egg extract as well as the unphosphorylated The mechanisms by which kinesin motor proteins are targeted to, anchored, and activated at specific cellular locations appear to be complex and are still largely unknown (for a recent review see G oldstein and Philp, 1999). In this paper, we have demonstrated that TPX2 is required for the localization of Xklp2 to microtubule minus ends during mitosis. We were also able to reconstitute the binding of the CO O H -terminal domain of Xklp2 to microtubules using purified components. Thus, the targeting of Xklp2 requires some sort of direct interaction between Xklp2 and TPX2. We do not yet understand the exact nature of this interaction but it probably involves the CO O H -terminal leucine zipper domain of Xklp2 (Wittmann et al., The Journal of Cell Biology, Volume 149, 2000 1416 TPX2 Is a Mitotic Phosphoprotein Downloaded from http://rupress.org/jcb/article-pdf/149/7/1405/1291988/0003125.pdf by guest on 10 June 2022 Figure 10. A n excess of TPX2 prevents the formation of bipolar spindles. (A ) R epresentative structures observed in control and TPX2supplemented reactions. Cycled spindles were assembled in the presence of rhodamine-labeled tubulin, fixed, centrifuged onto coverslips, and stained with anti-TPX2N and H oechst 33258. (B) Q uantification of structures observed in spindle assembly reactions supplemented with either control buffer or a 5–10-fold molar excess of TPX2. The number of quantified structures is indicated on top. (C) Q uantification of the microtubule density in bipolar control spindles and in monopolar structures in TPX2-supplemented reactions. E rror bars indicate the standard deviation (n 5 20). (D ) Monopolar structure in the presence of excess TPX2 stained for NuMA . Bars, 10 mm. in bipolar arrays with unfocused, frayed poles (Merdes et al., 1996; H eald et al., 1997). Interestingly, both NuMA and TPX2 require cytoplasmic dynein for their localization to spindle poles. Whereas NuMA binds directly to the dynein–dynactin complex (Merdes et al., 1996), we could not detect a direct interaction of TPX2 with either dynein– dynactin or NuMA , but we cannot rule out a transient interaction of TPX2 with these molecules. Thus, the two models that we proposed previously for the mechanism of localization of TPX2/Xklp2 to microtubule minus ends during mitosis, a direct interaction of TPX2 with dynein– dynactin versus a microtubule affinity-regulating gradient along the spindle axis (Wittmann et al., 1998), are still possible and the exact mechanism remains to be elucidated. It has become clear over the last few years that spindle poles are fundamentally different from centrosomes and that centrosomes are in fact not required for the formation of a bipolar spindle (H eald et al., 1996; Khodjakov et al., 1999). It has been suggested that NuMA provides a structural framework at the pole, cross-linking microtubules in a centrosome-independent way (Compton, 1998; D ionne et al., 1999). A lthough the primary structures of TPX2 and NuMA are completely unrelated, both proteins appear to have overlapping functions leading us to propose that TPX2 is essential for proper assembly of vertebrate mitotic spindle poles. Role of TPX2 in Spindle Pole Organization Submitted: 23 March 2000 R evised: 24 May 2000 A ccepted: 26 May 2000 We would like to thank all members of the Karsenti and H yman labs for helpful discussions, especially A ndrei Popov and Célia A ntonio for communal extracts, as well as Nathalie Le Bot and A rshad D esai for critical reading and comments on the manuscript. Special thanks go to H ironori Funabiki and A rshad D esai for the hint about the protein A dynabeads and to A ndreas Merdes for anti-NuMA antibodies. We also thank the members of the E MBL mass spectrometry and protein sequencing facility, G itte Neubauer, A ngela Bachi, and Keith A shman. TPX2 has other functions in addition to targeting Xklp2 to the poles. Immunodepletion of TPX2 results in the formation of bipolar spindles with abnormal morphologies and disintegrating poles often with bright foci of microtubules loosely attached. These foci still contain NuMA , however, it is unclear whether they represent intact centrosomes, fragments of nucleating material or centers of self-organized asters. In addition, TPX2-depleted structures have a reduced density of microtubules between poles and chromatin. These results indicate that TPX2 is involved in stabilizing the spindle and/or spindle poles. H owever, it is possible that the pole defects are an indirect consequence of a weakened spindle structure. A n excess of TPX2 blocks the formation of bipolar spindles and leads to an abnormally enlarged pole structure very similar to those formed after addition of G ST-Xklp2Tail (Boleti et al., 1996). This suggests that the addition of an excess of a pole component may stabilize the poles in such a way that bipolarity cannot be established anymore. Interestingly, in these monopolar structures, NuMA still localizes to a very tight area around the pole indicating that the overall organization of pole proteins may still be intact. NuMA and the dynein–dynactin complex are key players in spindle pole formation. Inhibition of any of these molecules severely affects spindle pole integrity resulting A ndersen, S.S.L. 2000. Spindle assembly and the art of regulating microtubule dynamics by MA Ps and stathmin/O p18. T rends Cell B iol. 10:261–267. A ndersen, S.S.L., and E . Karsenti. 1997. XMA P310: A X enopus rescue-promoting factor localized to the mitotic spindle. J. Cell B iol. 139:975–983. A ndersen, S.S.L., B. Buendia, J.E . D omínguez, A . Sawyer, and E . Karsenti. 1994. E ffect on microtubule dynamics of XMA P230, a microtubule-associated protein present in X enopus laevis eggs and dividing cells. J. Cell B iol. 127:1289–1299. A usubel, F., R . Brent, R .E . Kingston, D .D . Moore, J.G . Seidman, J.A . Smith, and K. Struhl. 1995. 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A ddition of antibodies against Xklp2 or the dominant negative G ST-Xklp2-Tail protein to egg extracts inhibited spindle assembly resulting in mainly monopolar structures (Boleti et al., 1996). Since TPX2 is required for the localization of Xklp2 to spindle poles we expected that delocalization of Xklp2 by immunodepletion of TPX2 would have a similar phenotype. H owever, TPX2 depletion does not have a significant effect on spindle bipolarity leading us to reexamine the previous interpretation of Xklp2 function. We now find that depletion of Xklp2 from egg extracts does not prevent the formation of bipolar spindles. Xklp2 is not essential either to organize a bipolar spindle around chromatin beads (Walczak et al., 1998). 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