Htpx2 Is Required for Normal Spindle Morphology and Centrosome Integrity During Vertebrate Cell Division

hTPX2 Is Required for Normal Spindle Morphology and Centrosome Integrity during Vertebrate Cell Division

Sarah Garrett,²Kristi Auer,¹Duane A. Compton,¹and Tarun M. Kapoor^2,3hTPX2 Knockdown by siRNA Results in Multipolar Spindles

¹Department of Biochemistry Dartmouth Medical School
To examine hTPX2 function in vertebrate cells, we sought to disrupt the expression of human TPX2 by using siRNA [4]. An RNA duplex corresponding to bases 74–94 of the human TPX2 open reading frame was transfected into HeLa cells. Thirty hours after transfection, we consistently found hTPX2 protein levels to be reduced below 1% of those in untreated cells (Figure 2D). Knockdown of hTPX2 resulted in two effects. First, loss of hTPX2 arrested cells in mitosis. In three independent experiments, cells treated with hTPX2 siRNA showed a mitotic index of 30%, whereas control cells showed a mitotic index of 3% (n ϭ at least 300 cells; Figure 2E). Second, more than 40% of the mitotic cells after siRNA treatment had spindles with more than two poles (Figures 2A–2C and 2E). Each pole in these multipolar spindles had several microtubule bundles that interacted with at least one other pole. In cells arrested in mitosis, condensed chromosomes associated with spindle microtubules. However, chromosomes of- ten failed to align at a single metaphase plate, and lag- ging chromosomes not positioned between any two poles were observed in both bipolar and multipolar mito- ses. NuMA (Figures 2A–2C), which is a protein that local- izes to spindle poles and is required for their formation [5–7], and both ␥-tubulin (Figure 3A–2C) and centrin (our unpublished data), which are components of centrosomes and peri-centrosomal material [8, 9], targeted to each spindle pole in siRNA-treated cells. This suggested that each supernumerary pole formed in the absence of hTPX2 recruits essential structural components required for functional spindle poles. Interphase TPX2- depleted cells exhibited normal microtubule organization and centrosome number, as detected by ␣-tubulin and ␥-tubulin staining (our unpublished data).
Hanover, New Hampshire 03755 ²Laboratory of Chemistry and Cell Biology Rockefeller University New York, New York 10021

Summary Bipolar spindle formation is essential for the accurate segregation of genetic material during cell division. Although centrosomes influence the number of spindle poles during mitosis, motor and non-motor microtubule-associated proteins (MAPs) also play key roles in determining spindle morphology [1]. TPX2 is a novel MAP also characterized in Xenopus cell-free extracts [2, 3]. To examine hTPX2 (human TPX2) function in human cells, we used siRNA to knock-down its expression and found that cells lacking hTPX2 arrest in mitosis with multipolar spindles. NuMA, ␥-tubulin, and centrin localize to each pole, and nocodazole treatment of cells lacking hTPX2 demonstrates that the localization of ␥-tubulin to multiple spindle poles requires intact microtubules. Furthermore, we show that the formation of monopolar microtubule arrays in human cell extracts does not require hTPX2, demonstra- ting that the mechanism by which hTPX2 promotes spindle bipolarity is independent of activities focusing microtubule minus ends at spindle poles. Finally, inhibition of the kinesin Eg5 in hTPX2-depleted cells leads to monopolar spindles, indicating that Eg5 function is necessary for multipolar spindle formation in the absence of hTPX2. Our observations reveal a structural role for hTPX2 in spindles and provide evidence
Microtubule-Dependent Fragmentation of Centrosomal Material in hTPX2-Depleted Cells

for

a

balance between microtubule-based motor

forces and structural spindle components.

To test whether the surprising localization of centrosomal components to each supernumerary pole in the absence of hTPX2 was due to microtubule-dependent fragmentation of centrosomal material, we examined ␥-tubulin localization in hTPX2-depleted cells that en- tered mitosis in the absence of microtubules. To ensure that microtubules were depolymerized before hTPX2 levels were significantly reduced, we treated cells with 33 ␮M nocodazole (or DMSO) 12 hr after transfection with hTPX2 siRNA. After an additional 18 hr in nocodazole, cells were fixed, and the organization of DNA, ␣-tubulin, and ␥-tubulin was examined. At this time point, in control experiments 48% (n ϭ 100, two independent experiments) of the mitotic cells had multipolar spindles with ␥-tubulin at each pole. The knockdown of hTPX2 in siRNA-treated cells in the presence or absence of nocodazole was confirmed by Western blot analysis (our unpublished data). In hTPX2-depleted cells treated with nocodazole, 94% (n ϭ 100, two independent experi-

Results and Discussion Characterization of hTPX2 Distribution in Human Cells

To examine hTPX2 function in vertebrate cells, we raised a polyclonal antibody to the full-length human TPX2 (expressed-sequence-tag [EST] accession number BG389502) protein expressed in bacteria. This antibody recognized a single protein of approximately 98 kDa in Western blots of total HeLa cell protein (Figure 1A). Consistent with the properties of TPX2 in amphibian cells and extracts [2, 3], human TPX2 colocalized with spindle microtubules in mitotic cells (Figures 1B–D) and targeted to nuclei in interphase cells (Figures 1E–G).

³Correspondence: [email protected]
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Figure 1. Human TPX2 Localization during Mitosis and Interphase (A) A Western blot of HeLa cell extracts probed with an antibody raised against amino acids 1–747 of hTPX2. The antibody specifically recognizes a 98 kDa band in total HeLa cell protein. Immunolocalization of ␣-tubulin (B and E) and hTPX2 (C and F) in a mitotic and an interphase HeLa cell, respectively. Overlays of ␣-tubulin (green), DNA (blue), and hTPX2 (red) are also shown (D and G). Scale bars, (B–D) 2.5 ␮m and (E–G) 20 ␮m.

ments) of mitotic cells contained two distinct ␥-tubulin foci, and only one cell was observed with more than two foci (Figures 3D–G). Thus, the appearance of the peri-centrosomal component ␥-tubulin at supernumerary spindle poles in cells lacking hTPX2 is microtubule dependent. It has previously been demonstrated that peri-centrosomal material does not fragment during a prolonged mitotic arrest in nocodazole [10] and that ␥-tubulin targeting to centrosomes does not depend on microtubules [11]. Taken together, our data are consistent with the idea that the loss of hTPX2 in mitotic cells results in microtubule-dependent fragmentation of peri- centrosomal material and not changes in centrosome number. between hTPX2-depleted and control extracts (Figure 4B). Western blotting demonstrated that the efficiency of hTPX2 depletion was essentially 100%, and no hTPX2 was detectable in the subsequent soluble and insoluble fractions. In the absence of hTPX2, we observed no detectable change in the efficiencies with which any other aster-associated components, including human KLP2, were recruited to microtubule asters in this system (Figure 4C). These data demonstrate that human TPX2 does not play an essential role in focusing microtubule minus ends at spindle poles.

The Mitotic Kinesin Eg5 Is Required for Multipolar Spindle Formation in hTPX2-Depleted Cells

The kinesin Eg5 is required for the assembly of bipolar spindles in vertebrate cells [15, 16]. To determine if Eg5 was also required for multipolar spindle formation in mitotic cells lacking hTPX2, we inhibited Eg5 activity with monastrol, a cell-permeable small-molecule inhibitor of Eg5 [17, 18]. Thirty hours after transfection with hTPX2 siRNA, cells were either fixed and stained to confirm the appearance of arrested mitotic cells with multipolar spindles or treated for an additional 4 hr with 100 ␮M monastrol and then stained for microtubules and DNA (Figures 5A–C). Knockdown of hTPX2 was also confirmed by Western blot analysis (data not shown). Both untreated and monastrol-treated TPX2-depleted cells had similar mitotic indices. However, unlike cells lacking hTPX2 alone, 99% of hTPX2-depleted monastrol-treated cells contained monopolar spindles (Fig- ure 5D).

hTPX2 Is Not Required to Focus Microtubule Minus Ends to Form Spindle Poles

It is possible that multipolar spindles form because of the loss of activities required for focusing microtubule minus ends at each spindle pole. To test whether hTPX2 participates in focusing microtubule minus ends at spindle poles, we examined microtubule organization in a HeLa cell mitotic extract after immunodepletion of hTPX2 (Figure 4; [12–14]). Under control conditions (untreated or preimmune antibody depletion), microtubules organized into astral arrays with NuMA concentrated at the core of each microtubule aster (Figure 4A). hTPX2 was efficiently recruited to the asters and was found exclusively in the aster-containing pellet fraction (Figure 4C). Microtubule organization into asters and NuMA concentration at the aster cores were indistinguishable

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Figure 2. Knockdown of hTPX2 by siRNA Arrests Vertebrate Cells in Mitosis with Multipolar Spindles Immunolocalization of (A) ␣-tubulin and (B) NuMA in HeLa cells 36 hr after transfection with 0.2 ␮M hTPX2 siRNA. (C) An overlay of ␣-tubulin (green), DNA (blue), and NuMA for the same cell is shown. (D) Immunblots of HeLa cells transfected with buffer (1) or hTPX2 siRNA (2). Loading controls (anti-dynamitin) are shown below. (B) Quantitation of the mitotic index (purple) and of the percentage of multipolar cells within the mitotic population (blue) in control or siRNA-treated cells (n ϭ greater than 300, three independent experiments). The scale bar represents 2.5 ␮m.

To test whether the release of Eg5 inhibition would result in the reformation of multipolar spindles from monopolar arrays in hTPX2-depleted cells, we utilized the fact that monastrol inhibition is reversible [17, 18]. Cells were again transfected with siRNA, and after 30 hr they were treated with 100 ␮M monastrol for 4 hr. As above, we took samples at each time point to confirm the phenotype. In this case, however, after the incubation in monastrol, cells were placed in monastrol-free cell culture medium. One hour after washout of monastrol, 39% of hTPX2-depleted mitotic cells contained multipolar spindles (Figure 5D). Together, these observations indicate that Eg5 function is capable of inducing multipolar spindle formation in the absence of hTPX2.

Figure 3. In Cells Treated with hTPX2 siRNA, Centrosomal Material Fragments in a Microtubule-Dependent Manner Immunolocalization of (A) ␣-tubulin and (B) ␥-tubulin in a hTPX2- depleted cell. (C) An overlay of ␣-tubulin (green), DNA (blue), and ␥-tubulin (red) for the same cell is shown. Immunolocalization of (D) ␣-tubulin and (E) ␥-tubulin in a cell treated with nocodazole and TPX2 siRNA. Twelve hours after siRNA transfection, cells were incu- bated with 33 ␮M nocodazole, and after an additional 18 hr, cells were fixed and processed for immunofluorescence. (F) An overlay for the cell shows ␣-tubulin (green), ␥-tubulin (red), and DNA (blue). (G) Quantitation of the number of cells staining with two foci of ␥-tubulin (blue) or more than two foci of ␥-tubulin (purple) in cells treated with hTPX2 siRNA or both nocodazole and hTPX2 siRNA. The scale bars represent 5 ␮m.

Conclusions

The data presented here demonstrate that hTPX2 plays an essential role in spindle organization and centrosome integrity in cultured vertebrate cells. Loss of hTPX2 leads to multipolar spindles and fragmentation of peri- centrosomal material through a mechanism that is independent of microtubule minus-end focusing at spindle poles. The most likely explanation for these results is that hTPX2, perhaps relying on microtubule cross-link- ing activity, plays a structural role within the spindle to maintain cohesion among all spindle microtubules within each half spindle. Structural support provided by hTPX2 most likely maintains spindle integrity as various microtubule-dependent motors exert force on spindle microtubules, and we speculate that an appropriate bal-

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Figure 5. In hTPX2-Depleted Cells, the Mitotic Kinesin Eg5 Is Re- quired for Multipolar Spindle Formation Immunolocalization of (A) ␣-tubulin and (B) DNA in hTPX2-depleted cells after a 4 hr incubation in 100 ␮M monastrol. (C) An overlay of ␣-tubulin (green), DNA (blue), and Eg5 (red) for the same cell is shown. (D) The percentage of mitotic cells with monopolar (blue), bipolar (purple), or multipolar (white) spindles was quantified in hTPX2-depleted and control cells either just before monastrol washout or 1 hr after washout. The scale bar represents 2.5 ␮m.

for multipolar spindle formation in the absence of hTPX2 activity is entirely consistent with such an idea. The fact that somatic cells lacking hTPX2 display multipolar spindles but frog egg extracts depleted of TPX2 display bipolar spindles with “multiple asters loosely attached to the disintegrating spindle pole” [3] may merely reflect differences in how imbalances between motor force and structural support are manifested in each experimental system.

Figure 4. hTPX2 Is Not Required for Focusing Microtubule Minus Ends into Monopolar Arrays

Supplementary Material

Microtubule organization in control (A) and hTPX2-depleted (B) HeLa cell mitotic extracts was examined by indirect immunofluorescence analysis with antibodies against ␣-tubulin and NuMA, as indicated. (C) Western blots of the antibody pellet (P_Ab) and the resulting soluble (S) and aster-associated insoluble (P) fractions from the control and hTPX2-depleted (⌬TPX2) samples for the aster-associated proteins NuMA, KLP2, Eg5, hTPX2, HSET, and tubulin are as indicated. The scale bar represents 5 ␮m.
Supplementary Experimental Procedures are available with this arti- cle online at http://images.cellpress.com/supmat/supmatin.htm.

Acknowledgments

The authors thank Sandy Jablonski and Tim Yen (Fox Chase Cancer Center, Philadelphia, Pennsylvania) for providing human KLP2 antibodies and Jeffery Salisbury (Mayo Clinic Foundation, Rochester, Minnesota) for supplying centrin antibodies. In addition, we thank Aime Levesque and Janet Yang for technical assistance. T.M.K. is a Pew Scholar. S.G. is a Howard Hughes Medical Institute pre- doctoral fellow. This work is supported by National Institutes of Health grants GM65933 (T.M.K.) and GM51542 (D.A.C.).

ance of both motor force and structural support is required for optimal spindle organization. In this context, imbalances in the contributions of either motor force or structural support would be expected to have deleteri- ous effects on spindle structure. Consistent with this idea, Wittman et al. demonstrated that excess quantities of TPX2 impaired spindle organization in frog egg extracts [3]. Conversely, defects observed in spindle pole organization upon reduction of hTPX2 would occur through exaggeration of forces from microtubule motors, and our observation that Eg5 activity is required

Received: August 21, 2002 Revised: September 11, 2002 Accepted: September 25, 2002 Published: December 10, 2002

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