Self-Assembly and Sorting of Acentrosomal Microtubules by TACC3 Facilitate Kinetochore Capture During the Mitotic Spindle Assembly

Self-assembly and sorting of acentrosomal microtubules by TACC3 facilitate kinetochore capture during the mitotic spindle assembly

Wenxiang Fu, Hao Chen1, Gang Wang1, Jia Luo1, Zhaoxuan Deng, Guangwei Xin, Nan Xu, Xiao Guo, Jun Lei, Qing Jiang, and Chuanmao Zhang2

Ministry of Education Key Laboratory of Cell Proliferation and Differentiation and State Key Laboratory of Biomembrane and Membrane Biotechnology, College of Life Sciences, Peking University, Beijing 100871, China

Edited by J. Richard McIntosh, University of Colorado at Boulder, Boulder, CO, and approved August 9, 2013 (received for review July 4, 2013)

Kinetochore capture by dynamic kinetochore microtubule fibers (K Previous studies have shown that transforming acidic coiled- fibers) is essential for proper chromosome alignment and accurate coil–containing protein 3 (TACC3) is essential for the mitotic distribution of the replicated genome during cell division. Although spindle assembly and chromosome alignment, but the mecha- this capture process has been extensively studied, the mechanisms nism remains largely unknown (14–18). Here we reveal that underlying the initiation of this process and the proper formation TACC3-dependent small microtubule aster formation and sort- of the K fibers remain largely unknown. Here we show that trans- ing near the kinetochores contribute to correct microtubule– forming acidic coiled-coil–containing protein 3 (TACC3) is essen- kinetochore connections. fi tial for kinetochore capture and proper K- ber formation in HeLa Results and Discussion cells. To observe the assembly of acentrosomal microtubules more TACC3 Regulates de Novo Assembly of Acentrosomal Microtubules. clearly, the cells were released from higher concentrations of noco- Several groups reported recently that TACC3 is essential for dazole into zero or lower concentrations. We find that small acen- chromosome alignment and spindle assembly in mitosis, but the trosomal TACC3–microtubule aster formation near the kinetochores clear mechanism remains unknown (14–18). To test how TACC3

and binding of the asters with the kinetochores are the initial steps CELL BIOLOGY regulates the spindle formation, we firstly knocked down TACC3 of the kinetochore capture by the acentrosomal microtubules, and in HeLa cells using small interfering RNA against TACC3 that the sorting of kinetochore-captured acentrosomal microtu- (siTACC3) (>95% efficiency) (Fig. 1A). Then we treated bules with centrosomal microtubules leads to the capture of kinet- TACC3-knockdown and the irrelevant-knockdown control cells ochore by centrosomal microtubules from both spindle poles. We with 1 μg/mL nocodazole for 5–8 h to abolish microtubule nu- demonstrate that the sorting of the TACC3-associated microtubules cleation and mitotic spindle assembly, followed by releasing with the centrosomal microtubules is a crucial process for spindle these cells into medium without nocodazole to allow them to fi assembly and chromosome movement. These ndings, which are reassemble their microtubules and spindles. As shown (Fig. 1B), also supported in the unperturbed mitosis without nocodazole, re- in control cells, microtubules were quickly nucleated from both veal a critical TACC3-dependent acentrosomal microtubule nucleation the separating centrosomes to form two big centrosomal asters; and sorting process to regulate kinetochore–microtubule connections meanwhile, other microtubules were also quickly (within 1.5 and provide deep insight into the mechanisms of mitotic spindle min) nucleated in the cytoplasm to form many small acen- assembly and chromosome alignment. trosomal asters. Then, these small asters quickly “fused” with each other and sorted into the big centrosomal asters, and finally centrosome | noncentrosomal | cell cycle Significance o ensure proper segregation of the chromosomes into its two Tdaughter cells during proliferation, the chromosomes of a Mitosis is a highly regulated cell division process in eukaryotes. mother cell must be captured by its assembling mitotic spindle Assembly of the spindle and segregation of chromosomes in through attachment of the chromosome kinetochores and the mitosis enable the mother cells to distribute the genetic dynamic spindle microtubules (1). A “search-and-capture” model materials equally to their daughter cells. Before chromosome was proposed long ago, in which the dynamic spindle micro- segregation, the kinetochores at the chromosomes must be tubules nucleated from the centrosomes search for and capture correctly captured by the microtubules. The mechanisms un- the chromosome kinetochores (2). Previous studies showed that derlying the initiation of this process and the proper formation the kinetochores are initially captured by the spindle-pole– of the kinetochore fibers remain largely unknown. This study nucleated microtubules with their lateral side (3, 4). Once captured, shows that transforming acidic coiled-coil–containing protein 3 the kinetochores with their chromosomes are transported along (TACC3) is essential for proper kinetochore capture and kinet- the microtubules toward a spindle pole, and the microtubules ochore fiber formation. Our findings reveal a critical TACC3- shrink at their plus ends until the establishment of the end-on dependent acentrosomal microtubule nucleation and sorting attachment (4, 5). However, this model is insufficient to explain process to regulate kinetochore–microtubule connections. the initial connection of the kinetochore and the spindle microtubules in the centrosome-independent spindle assembly process. Author contributions: W.F., Q.J., and C.Z. designed research; W.F., H.C., G.W., J. Luo, Z.D., Recent studies in Xenopus extracts indicated that microtubules G.X., N.X., X.G., and J. Lei performed research; C.Z. contributed new reagents/analytic are nucleated near the chromosomes and self-organize into a tools; W.F., Q.J., and C.Z. analyzed data; and W.F., Q.J., and C.Z. wrote the paper. spindle (6). A new model for acentrosomal spindle assembly has The authors declare no conflict of interest. been raised in mouse oocytes, in which self-organized microtubule This article is a PNAS Direct Submission. organizing centers (MTOCs) replace the centrosome function (7). Freely available online through the PNAS open access option. The somatic cells may also use the centrosome-independent 1H.C., G.W., and J. Luo contributed equally to this work. pathway for their spindle assembly (8–10). In Drosophila cells, 2To whom correspondence should be addressed. E-mail: [email protected]. fi the centrosome-independent assembled kinetochore bers can This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. be captured by centrosomal microtubules (11–13). 1073/pnas.1312382110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1312382110 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 AB Merge (Tubulin/TACC3/DNA) Merge (Tubulin/TACC3/DNA) Tubulin Merge Tubulin Merge Tubulin Merge Tubulin Merge Tubulin Merge C Tubulin Merge Tubulin Merge Tubulin Merge Tubulin Merge Tubulin Merge 0.5 min 1.5 min 3.5 min 7.5 min 15 min 0.5 min 1.5 min 3.5 min 7.5 min 15 min si-Con Si-Con

Control siTACC3-1 siTACC3-2 TACC3 siTACC3 siTACC3 Noc: 1 μg/ml to 0 α-tub Noc: 1 μg/ml to 15 ng/ml TACC3 Hec1 Merge TACC3 DNA Merge Tubulin DNA Merge D 20 E F G J No Noc 16 15 ng/ml Noc Control Hec1 12 Si-Con 8

4 Noc AurB Number of (8 min) acentrosomal microtubule foci

0 si-TACC3 chromosome kinetochore kinetochore fiber

Control Cold unattached acentrosomal MT TACC3 (10 min) siTACC3-1siTACC3-2 Clathrin si-AurA

H 02:15 04:15 08:45 11:15 15:30 17:15 22:00 29:3030:45 49:15 I 02:20 04:00 07:20 12:4016:40 20:00 24:20 29:00 34:20 38:40

01:30 03:15 05:30 11:00 15:45 24:3030:00 34:45 45:15 49:45 02:40 04:00 06:20 09:20 12:40 17:20 22:00 26:00 30:00 37:40 si-Co si-Con

02:00 04:20 08:2015:20 22:20 25:40 30:40 35:20 42:00 51:20 01:40 04:00 06:20 08:40 11:20 14:40 20:40 22:20 24:40 29:00

02:00 04:00 09:20 12:40 15:40 23:00 28:20 34:00 40:20 44:00 02:40 04:20 05:40 09:00 12:2015:40 21:00 23:00 27:00 30:00 si-TACC3

02:30 04:30 11:30 16:30 19:00 24:00 29:00 33:00 36:30 40:30 02:20 05:20 08:40 11:00 14:4018:00 22:20 26:40 31:20 39:40 Noc release: 500 ng/ml to 0 02:30 05:30 09:30 13:30 18:00 22:30 26:30 30:00 35:00 40:00 03:00 04:20 08:00 10:40 15:0019:20 24:00 27:20 33:40 39:20 Noc release: 500 ng/ml to 15 MLN8237 MLN8237 si-TACC3

Fig. 1. TACC3 is required for de novo assembly of acentrosomal microtubules in mitosis. (A) Detection of TACC3 RNAi depletion efﬁciency by Western blotting. HeLa cells were transfected with control and TACC3 siRNAs, respectively. The blots were probed with anti-TACC3 (Upper) and anti–α-tubulin (Lower). (B and C) Representative images of 1 μg/mL nocodazole-arrested normal control or TACC3-knockdown HeLa cells by siRNA followed by release into medium without nocodazole (B) or with 15 ng/mL nocodazole (C) at different time points (0.5, 1.5, 3.5, 7.5, and 15 min). The data are shown as maximum intensity projections of different z sections. TACC3 is in red, tubulin in green, and DNA in blue. (D) Statistics of numbers of acentrosomal microtubule seeds in control and TACC3 knockdown cells after nocodazole treatment and release for 1.5 min. More than 50 cells for each treatment were counted. (E) Control, nocodazole-treated (50 ng/mL for 8 min), and cold-treated (10 min on ice) HeLa cells were stained with anti-TACC3 (green) and anti-Hec1 (red) antibodies. (F) Staining of HeLa cells with TACC3 (green) and indicated proteins (Hec1, Aurora B, and clathrin) (red) after nocodazole treatment for 16 h. (G) HeLa cells were transfected with different siRNAs (control, siTACC3, siAurora A) as indicated. Cells were treated with 50 ng/mL nocodazole for 16 h before ﬁxation. (H and I) Live imaging of HeLa cells expressing GFP–tubulin under indicated different treatments (siRNA control, siTACC3, and Aurora A inhibitor MLN8237). The mitotic cells were arrested with 500 ng/mL nocodazole and released into medium without nocodazole (H) or with 15 ng/mL nocodazole (I). (J) Illustration of TACC3-dependent acentrosomal microtubule assembly and clustering. During mitosis, TACC3 containing small microtubule seeds/asters were assembled around the chromosomes and kinetochores, and these small microtubule asters were further assembled and clustered into a complete bipolar spindle structure. (Scale bar, 10 μm.)

these big and small microtubule asters assembled into a bi- fibers, supporting the notion that clathrin and TACC3 co- polar mitotic spindle within 15 min (3.5–15 min). In contrast, function as a complex in mitosis (Fig. 1F)(15–18). Moreover, in TACC3-knockdown cells, although microtubules were nucle- knockdown of Aurora A, which functions upstream of ated around the separating centrosomes to form the two big TACC3 (19), clearly abolished the acentrosomal microtubule centrosomal microtubule asters and finally assemble an imma- aster formation (Fig. 1G). ture bipolar mitotic spindle, there was nearly no other microtubule To further examine the roles of TACC3 in regulating acen- nucleation to form the small acentrosomal microtubule asters in trosomal microtubule assembly, we performed live-imaging the cytoplasm (Fig. 1 B–D and Fig. S1 A and B). These results assays in HeLa cells expressing GFP–tubulin to analyze the dy- suggested that TACC3 might contribute to the acentrosomal namic assembly of spindle microtubules. The cells were released microtubule aster assembly and the microtubule–kinetochore from 500 ng/mL nocodazole into medium with 0 (Fig. 1H and connection. To further confirm these, we released these cells into Movies S1–S6) or 15 ng/mL nocodazole (Fig. 1I and Movies the medium containing 15 ng/mL nocodazole and also verified S7–S12). As shown (Fig. 1H and Movies S1 and S2), in control that TACC3 is required for acentrosomal microtubule assembly cells, microtubules were assembled both around the centrosomes (Fig. 1 C and D and Fig. S1 C and D). and in the acentrosomal regions. In contrast, TACC3 knockdown To understand why TACC3 is required for the acentrosomal had little effect on centrosomal microtubule nucleation while microtubule aster assembly, we treated the mitotic HeLa cells strongly inhibiting the formation of acentrosomal microtubules with nocodazole or cold temperatures and observed that (Fig. 1H and Movies S3 and S4). Moreover, we treated the cells TACC3 stayed with the stable kinetochore-connected fibers with MLN8237, a small-molecule inhibitor of Aurora A, and (K fibers) (Fig. 1E and Fig. S1E). We treated HeLa cells with found it suppressed both the centrosomal and acentrosomal nocodazole (50 ng/mL) for 16 h to allow the cells to enter microtubule assembly (Fig. 1H and Movies S5 and S6). To into mitosis without centrosomal microtubule nucleation and specifically analyze the formation of acentrosomal micro- verified that TACC3 connected to kinetochore markers, i.e., tubules, the cells were released into 15 ng/mL nocodazole. Hec1 and Aurora B (Fig. 1F). Meanwhile, a fraction of cla- Similar as what we observed in fixed samples (Fig. 1 C and G), thrin heavy chain also colocalized with TACC3 on their the live tracking of microtubules also revealed that TACC3

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1312382110 Fu et al. Downloaded by guest on October 1, 2021 and Aurora A is crucial for acentrosomal microtubule for- microtubules (Fig. 2 A and B and Fig. S2 A and D). As shown mation (Fig. 1I and Movies S7–S12). Together, we propose that (Fig. 2 C and D and Fig. S2B), TACC3 knockdown resulted in TACC3-dependent acentrosomal microtubule nucleation is an increase of lagging chromosomes and metaphase plate width. regulated by Aurora A, and TACC3-containing acentrosomal Meanwhile, ablation of TACC3 led to abundant centrosome- microtubule small aster assembly contributes to microtubule– nucleated astral microtubules outside the “spindle frame” as in- kinetochore connections (Fig. 1J). dicated by arrows, suggesting deﬁciency of the search-and-capture process (Fig. 2A). The microtubule intensity around the chromo- TACC3-Dependent Acentrosomal Microtubule Nucleation Is Required for somes in TACC3-depleted cells was much less than control (Fig. E C ﬁ Proper Kinetochore–Microtubule Capture and Correct Mitosis. Because 2 and Fig. S2 ), suggesting insuf cient assembly of acentrosomal the previous conclusions were mainly based on nocodazole-treated microtubules. Furthermore, we found that TACC3 depletion resulted in marked decrease of distances between paired kinet- cells, we next investigated the function of TACC3-dependent F G acentrosomal microtubule nucleation in kinetochore–microtubule ochores (Fig. 2 and ), indicating that TACC3 is required capture during unperturbed mitosis in HeLa cells without noco- for maintenance of proper interkinetochore tension. More- over, TACC3 knockdown led to increased BubR1 levels at the dazole (Fig. 2). In normal metaphase cells, proper end-on attach- kinetochores, suggesting the activation of spindle assembly check- ments between kinetochores and microtubules were formed, point (Fig. 2 H and I). Because TACC3 localizes to acentrosomal whereas in TACC3-knockdown cells, many kinetochores were microtubules and is not essential for centrosomal microtubule not connected by microtubules or only bound laterally with few nucleation as discussed above (Fig. 1), we proposed that TACC3- dependent acentrosomal microtubule nucleation and small aster formation mainly contribute to the regulation of kinetochore function, including kinetochore–microtubule attachment and ABTubulin/ TubulinCRESTTACC3 DNA Merge CREST 100 End-on attachment maintenance of interkinetochore tension. Lateral binding 80 TACC3-dependent acentrosomal microtubule nucleation must Unattached be regulated by other mitotic regulators in performing its func-

Si-Con 60 40 tion, such as clathrin and Aurora A (20). So, here we checked the roles of other mitotic regulators in HeLa cells. Knockdown of

kinetochore 20 Percentage of 0 TPX2, which activates Aurora A (21), reduced TACC3 targeting siTACC3 attachement type (%) si-Control siTACC3-1 siTACC3-2 to the spindle, whereas inhibition of Aurora B by small molecule CDEG I ZM447439 (22) or ablation of centromere protein E had no or CELL BIOLOGY 16 2.0 7 little effect on the localization of TACC3 to the spindle (Fig. 100 0.5 6 A 80 0.4 1.5 S3 ). Moreover, unlike eg5 inhibition by monastral (23), in- 12 5 hibition of Plk1 by small molecule BI2536 (24) and knockdown 60 0.3 4 8 1.0 of Nedd1 (25) both inhibited the targeting of TACC3 to the 40 0.2 3 0.5 B width (μm) 2 spindles (Fig. S3 ). Overexpression of HSET, which mainly 20 4 0.1 Inter-k distance (μm)

Metaphase plate 1

0 0.0 functions on centrosomal microtubules (26), had no effect on the Relative MT intensity BubR1 intensity (a.u.)

cells with lagging chromosomes (%) 0.0 0 around chromosomes 0

Percentage of mitotic spindle localization of TACC3 (Fig. S3C). We also knocked

si-ControlsiTACC3-1siTACC3-2 si-ControlsiTACC3-1siTACC3-2 si-ControlsiTACC3-1siTACC3-2 si-ControlsiTACC3-1siTACC3-2 si-ControlsiTACC3-1siTACC3-2 down Hec1 and found the location of TACC3 to the spindles was partially reduced (Fig. S3D). Taken together, these results in- F Hec1/ H BubR1/ Hec1 CREST TACC3 DNA Merge CREST BubR1CRESTTACC3 DNA Merge CREST dicate that inhibiting or knocking down upstream regulators for TACC3 (i.e., TPX2) or interfering with the kinetochore micro-

Si-Con tubule assembly activity (i.e., Plk1, Nedd1, and Hec1) can down- Si-Con regulate the targeting of TACC3 to the spindles and lead to the defects in bipolar spindle assembly and chromosome alignment due to kinetochore–microtubule attachment failure. siTACC3 siTACC3

The Sorting of the Small Acentrosomal Microtubule Seeds/Asters into – Fig. 2. TACC3 facilitates kinetochore microtubule capture, interkinetochore Big Centrosomal Asters Is a General Mechanism for Spindle Assembly tension, and spindle assembly checkpoint. Micrographs are presented as and Kinetochore Movement. maximum intensity projections of different z sections. (A) Ablation of TACC3 To better illustrate the roles for led to defects in chromosome alignment and kinetochore–microtubule at- TACC3 in chromosome alignment and mitotic spindle assembly, we analyzed the microtubule behaviors by live imaging of HeLa tachment. (Right) Representative paired kinetochores in single z sections. The –α oval lines indicate the spindle frame and the arrows indicate the microtubules cells expressing GFP -tubulin. We found that the sorting of the outside the oval frame. Kinetochores stained with serum from patients with small acentrosomal asters into the big centrosomal asters is one CREST syndrome are in red (CREST), tubulin in green, TACC3 in magenta, and key process for the mitotic spindle assembly (Fig. 3 A and B, DNA in blue. (B) Quantification of kinetochore attachment types in different Figs. S4 and S5, and Movies S13 and S14). During mitosis, both groups. Percentages of end-on attachment, lateral binding status, and the centrosomal microtubules and the small acentrosomal aster unattached kinetochores are shown. (C) Quantification of the percentages of microtubules kept growing in the cytoplasm, and when they en- metaphase cells with lagging chromosomes. (D) Measurement of the meta- countered, these acentrosomal microtubules were immediately phase plate width in control and TACC3-depleted cells. (E) Quantification of sorted into the microtubule arrays of the centrosomal asters. Five the relative microtubule intensity around chromosomes. (F) HeLa cells were examples of microtubule sorting from the two movies mentioned stained with anti-Hec1 (green), CREST (red), and TACC3 (magenta) antibodies. above (Movies S13 and S14) are shown in magnified images Representative images are shown. (Right) Micrographs in single z sections. (G) (Fig. 3C, boxes a–e). As indicated, during mitosis, acentrosomal Statistics of interkinetochore distances in control and TACC3-ablated cells. microtubules can be captured by and sorted into microtubules The interkinetochore distances were measured according to paired Hec1 and from one centrosome (Fig. 3C, boxes a, d, and e) or both of the CREST fluorescent signals. The distances between the centers of two paired C b c Hec1 dots were measured with ImageJ. More than 150 kinetochore pairs in two centrosomes (Fig. 3 , boxes and ). The sorting of acentrosomal microtubules into centrosomal asters was also con- the same z sections in each group were analyzed. Data are presented as fi D E A–C means plus SEs. (H) Cells were stained with anti-BubR1 (green), CREST (red), rmed in other cases (Fig. 3 and , Fig. S6 ,andMovies and TACC3 (magenta) antibodies. DNA is in blue. (I) Statistics of BubR1 S15 and S16). Together, these data indicate that the microtubule fluorescence intensities in control and TACC3-ablated cells. The BubR1 in- sorting of the small acentrosomal asters with the big centrosomal tensities were quantified by ImageJ software, and the average cytoplasmic asters is a general mechanism for spindle assembly. immunofluorescence intensity was subtracted as background. Data are pre- To further characterize the acentrosomal microtubule sorting sented as means plus SEs. (Scale bar, 10 μm.) process, we analyzed the microtubule behaviors when the HeLa

Fu et al. PNAS Early Edition | 3of6 Downloaded by guest on October 1, 2021 cells were released from 500 ng/mL nocodazole to medium acentrosomal microtubules and kinetochores (Fig. S7 A–D), we without nocodazole (Fig. 1H and Movies S1 and S2) or with 15 propose that the TACC3-dependent acentrosomal microtubule ng/mL nocodazole (Fig. 1I and Movies S7 and S8). It can be assembly and sorting process facilitate kinetochore movement easily seen in the movies that multiple large microtubule asters and capture. and spindle poles were assembled and fused with each other A cell normally assembles its mitotic spindle into a bipolar (also see Fig. S6D showing sorting of two spindle poles, derived structure, which is regulated by multiple and complex mechani- from Movie S7). Fig. 3 F and G are two cases showing the cal strengths (27). A mathematical model has been proposed for assembly and sorting of individual acentrosomal foci when anastral spindle assembly in Drosophila oocytes (28). To illus- nocodazole was removed to observe the de novo assembly of trate the role of the microtubule sorting in establishing the microtubules. With 25 ng/mL nocodazole, the sorting process spindle bipolarity, here we hypothesized a simpliﬁed mathematic was also observed and reorganizations of spindle poles are shown model based on our abovementioned results (Fig. S8). For (Fig. S6E and Movie S17). These data further demonstrate that sorting of microtubules or asters, parallel microtubule motors acentrosomal microtubule sorting is a basic process for con- mainly function to bundle these microtubules into paralleled structing the mitotic spindle and spindle poles during mitosis. bundles and clusters, whereas antiparallel microtubule motors Furthermore, by using live cell imaging, we analyzed the usually make these microtubules slide and separate (Fig. S8 A behaviors of acentrosomal microtubules and kinetochores when and B). To simply address this, we hypothesized that there exists the cells were released from 500 ng/mL nocodazole into 0 ng/mL. a minus-end–directed pulling force (F) for generating microtu- As shown (Fig. 3H and Movie S18), acentrosomal micro- bule sorting power (Fig. S8C). For sorting of two microtubule tubules were preferentially assembled around and attached to the structures (m, n): If 0° ≤θ <90° (where θ indicates the angle kinetochores, and sorting of these microtubule structures fur- between Fm and Fn), fsorting (m,n) function results in the formation ther resulted in kinetochore capture and movement. Similarly, of one clustered force: F(m,n) (new) = (Fm + Fn)(jFmj+jFnj)/jFm + when the cells were released into 15 ng/mL nocodazole, the Fnj. If 90° ≤θ ≤180°, fsorting (m,n) results in formation of two an- acentrosomal microtubule assembly and sorting process was ac- tiparallel forces: Fm (new) = (Fm − Fn)jFmj/jFm − Fnj;Fn (new) = companied by chromosome kinetochore movement (Fig. 3I and (Fn − Fm)jFnj/jFm − FnPj. For sorting of all of the microtubule = Movie S19). Considering that TACC3 is initially associated with structures: ftotal sorting ðm;nÞfsortingðm;nÞ. During spindle assembly without centrosomes, the bipolarity is formed through sorting of multiple acentrosomal microtubules (Fig. S8D) (7, 29). In spindle assembly with centrosomes, A b c centrosomes function as the main MTOCs with high sorting a Fa Fb 00:00 00:50 05:00 06:20 07:20 08:00 09:10 10:40 12:40 16:00 forces ( and ) to generate the bipolarity. When centrosomes are artiﬁcially removed in some situations, spindle bipolarity B 00:00 00:20 01:20 01:50 02:30 03:10 03:50 05:47 07:17 08:41 d can still be established through acentrosomal MTOCs (8, 27, e 30). In nocodazole-treated cells, due to the reduced sorting abcde (m:s) f (m:s) g (m:s) (m:s) force, some acentrosomal forces cannot be sorted, resulting in C DE02:40 02:13 F 0:45 G4:30 02:50 02:33 1:00 4:45 extra acentrosomal MTOCs, i.e., formation of multipolar mi- 03:00 02:53 1:15 5:00 03:10 03:13 5:15 crotubule structures (Fig. S8 C and D). 03:20 03:43 1:30 5:30 03:30 03:53 1:45 5:45 03:40 04:13 2:00 6:00 Loading of Preformed Acentrosomal TACC3–Microtubule Seeds/ 03:50 04:33 6:15 04:00 04:53 2:15 6:30 Asters to Kinetochores Followed by Sorting Are Crucial Steps for 04:10 05:13 2:30 6:45 01:30 03:00 07:30 12:00 14:15 18:00 24:4534:30 40:30 50:15 Kinetochore Capture. As indicated above, TACC3-dependent H acentrosomal microtubule assembly and sorting facilitate proper

Merge kinetochore capture. However, how TACC3 regulates microtubule and kinetochore behaviors remains unknown. To address this, we analyzed the behaviors of TACC3, microtubules, and Tubulin kinetochores in mitotic HeLa cells in the presence of different – μ

Hec1 concentrations (0 1 g/mL) of nocodazole. With the decrease of nocodazole concentration, the microtubule nucleation activity

Noc: 500 ng/ml to 0 and kinetochore capture by the nucleated microtubules were

Magnify remarkably increased (Fig. 4A and Fig. S9 A and B). Data I 00:45 06:45 08:15 11:15 13:00 18:00 24:00 29:15 37:30 49:30 showed that microtubule plus-end tracking protein EB1 spread

Merge through the kinetochore-targeted TACC3-containing microtubule asters to spindle poles in the presence of nocodazole (Fig. S9C). Next, we conﬁrmed TACC3 dynamically associated with Tubulin microtubules and kinetochores in various treatments using nocodazole (Fig. S10 A–G). To understand how these TACC3– Hec1 microtubule seeds/asters and kinetochores get connected, we detected the behaviors of TACC3–microtubule complexes dur- Noc: 500 ng/ml to 15

Magnify ing early mitosis in the presence of 100 ng/mL nocodazole (Fig. 4B). Before nuclear envelope breakdown, TACC3 and tubulin Fig. 3. Sorting of TACC3-associated acentrosomal microtubules is a general stayed outside the nucleus and almost no TACC3 or tubulin mechanism for spindle assembly and kinetochore movement. (A–E) Live – located on the kinetochores. With nuclear envelope break- imaging of HeLa cells expressing GFP tubulin. Red arrows indicate cen- down in early mitosis, partial TACC3 was loaded onto the short trosomal asters and green arrows are acentrosomal microtubules. (A and B) microtubules to form TACC3–microtubule seeds/asters, and the Sorting of acentrosomal microtubules into centrosomal asters. (C) Magniﬁed – seeds/asters quickly connected with the kinetochores (Fig. 4 microtubule sorting regions (boxes a e) as shown in A and B.(D and E) A–C – Another two examples of microtubule sorting existed in normal mitosis. (F ). We also analyzed the TACC3 microtubule behaviors in and G) Assembly and sorting of multiple acentrosomal microtubule seeds HeLa cells with 25 ng/mL nocodazole. There were at least four when HeLa cells expressing GFP–tubulin were released from 500 ng/mL kinds of microtubule structures in early mitosis, the microtubules nocodazole into medium without nocodazole. (H and I) Live imaging of without TACC3, the dissociative TACC3–microtubules, the lat- HeLa cells coexpressing mcherry–tubulin and GFP–Hec1. The cells were re- erally kinetochore-bound TACC–microtubules, and the kineto- leased from 500 ng/mL nocodazole into 0 (H) or 15 ng/mL (I) nocodazole as chore-attached TACC3–microtubule asters (Fig. 4D). In contrast, indicated. (Scale bars in A, B, H, and I,10μm and in C–G,2μm.) most TACC3–microtubule structures attached to kinetochores

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1312382110 Fu et al. Downloaded by guest on October 1, 2021 kinetochores facilitate the progression of mitosis and spindle A Tubulin CREST TACC3 DNA Merge C Tubulin/TACC3/ACA Early mitosis formation. In summary, mainly based on the results observed in noco- 1000 Late mitosis dazole-treated cells, our present work leads us to hypothesize a unique model to illustrate the mechanism of the kinetochore 100 100 ng/ml Noc capture by microtubules (Fig. 5 C and D). First, once the cell enters mitosis and the nuclear envelope breaks down, TACC3 50 D Tubulin TACC3 CREST DNA Merge – a c and the short microtubules bind together to form small TACC3 d b microtubule seeds near the kinetochores along with the assembly 25 of two big centrosomal asters, and the seeds grow gradually into small TACC3–microtubule asters through the nucleation of the Early mitosis

10 a

Nocodazole treatment (ng/ml) short microtubules bound on the seeds. Second, the small TACC3–microtubule seeds/asters get connected with different 0 b parts of the kinetochores; and meanwhile, the TACC3–micro- Tubulin CREST TACC3 DNA Merge tubule seeds/asters keep growing. Third, continuous nucleation B – c and sorting of the TACC3 microtubule seeds/asters enable the

d 100 ng/ml Noc A Microtubule assembly in the presence of 15 ng/ml nocodazole E Tubulin TACC3 CREST DNA Merge F 00:08:00 00:25:00 00:33:00 00:40:00 00:47:00 01:00:00 01:15:00 01:32:00 e (+) (-) (-) (-) (-)

(+) Merge (+) (+)

Late mitosis e MT kinetochore TACC3 Tubulin Fig. 4. Targeting of preassembled acentrosomal TACC3–microtubule seeds/ asters to kinetochore facilitates kinetochore capture by centrosomal micro- – Hec1

tubules. (A) TACC3 microtubule complex dynamically associated with CELL BIOLOGY kinetochores upon nocodazole treatment. Staining of mitotic HeLa cells B Microtubule assembly in the presence of 50 ng/ml nocodazole arrested by different concentrations (1 μg/mL, 100 ng/mL, 50 ng/mL, 25 ng/ 00:00:00 00:09:00 00:15:15 00:19:30 00:35:15 00:49:30 01:09:45 01:23:15 mL, 10 ng/mL, and 0 ng/mL) of nocodazole. Tubulin is in magenta, CREST in

red, TACC3 in green, and DNA in blue. (B) The total of 100 ng/mL nocoda- Merge zole-arrested HeLa cells in early mitosis were stained with tubulin (magenta), CREST (red), TACC3 (green), and DNA (blue). The white dashed lines indicate

the cell boundary. (C) Illustration of acentrosomal TACC3–microtubule (MT) Tubulin seeds/asters in 100 ng/mL nocodazole-arrested early and late mitotic HeLa cells. (D and E) Different types of acentrosomal microtubules assemble the mitotic spindle. HeLa cells were treated with 25 ng/mL nocodazole for 5 h. Hec1 The representative early (D) and late (E) mitotic cells are shown. The maxi- (1) (-) (2) (3) (4) C (-)(-) mum intensity projections of 3D images are shown. In early mitosis (D), four (-) (+) (-) (+) (+) (-) (-) – (+) (+) (+)(+) different types of acentrosomal microtubule structures (a d) are indicated (-) (+)(+) (-) (-) (-) (+) (+) (+) by projection of selected z stacks followed by magnification. In contrast, only (-) (+) (+) (+) (+) (-) (-) one type of acentrosomal microtubules (e in E) was in late mitosis. The kinetochore pair TACC3 MT TACC3-MT seed square e in E is magnified and illustrated (Below). (F) Illustration of the acentrosomal microtubule nucleation, TACC3–microtubule seeds/asters as- D TACC3-dependent kinetochore (-) capture by centrosomal MT sembly and the end-on capture of the kinetochores by the small aster tubules Syntelic (-) through MTOC sorting μ during transition from early mitosis to late mitosis. (Scale bar, 10 m.) (-) Centrosomal MTs (-) Amphitelic Acentrosomal MTs (-) (-) kinetochore pair in late mitosis (Fig. 4E), suggesting that the TACC3–microtubule Amphitelic Syntelic TACC3 seeds/asters had been sorted into the kinetochore-attached K fi Fig. 5. A unique model for kinetochore capture by microtubules during bers. Thus, we propose that the capture of the kinetochores by mitotic spindle assembly in somatic cells with centrosomes. (A and B) Live spindle microtubules is facilitated through stepwise assembly of imaging of HeLa cells coexpressing mcherry–tubulin and GFP–Hec1 in the TACC3–microtubule seeds/asters near the kinetochores (Fig. presence of 15 ng/mL (A) or 50 ng/mL (B) nocodazole. Magnified images are 4F) and sorting of the TACC3–microtubule seeds/asters into the shown. Tubulin is in red and Hec1 is in green. (Scale bar, 5 μm.) (C) Illus- big centrosomal asters during the mitotic spindle assembly. tration of initial kinetochore capture process by microtubules in a TACC3- As indicated previously (Fig. 3 H and I and Movies S18 and dependent way. The small green dots represent TACC3 proteins, the ma- S19), the de novo assembly and sorting of microtubules in the genta lines represent the microtubules (MT), and the large red dots stand for cytoplasm facilitate kinetochore association. To further in- the kinetochore pair. The initial kinetochore capture by adjacent acentrosomal microtubules can be divided into four steps: (i) microtubule as- vestigate the initial kinetochore capture process, we analyzed sembly followed by formation of TACC3–microtubule seeds; (ii) binding of the dynamic behaviors of kinetochores and microtubules during TACC3–microtubule seeds with kinetochores and further nucleation of the the progression from late G2 to mitosis in the presence of 15 ng/mL microtubules; (iii) further assembly and sorting of the TACC3–microtubule (Fig. 5A, Fig. S11A,andMovie S20) and 50 ng/mL nocodazole seeds lead to the formation of small TACC3–microtubule seeds/asters; and (Fig. 5B, Fig. S11B,andMovie S21) in HeLa cells. In late G2 (iv) clustering of TACC3–microtubule seeds/asters and further nucleation of or early mitosis (e.g., before 00:30:00 in Fig. 5A), there were a the acentrosomal microtubules produces the initial capture of the kinet- few polymerized microtubules that were not attached with kinetochores by the acentrosomal aster microtubules. (D) Illustration of TACC3- dependent kinetochore capture in establishing bipolar spindle assembly. The ochores. With the time increase, the microtubule seeds/asters acentrosomal microtubule-captured kinetochores are further captured by began to assemble and sort around the kinetochores. In agreement centrosomal microtubules through sorting the small acentrosomal asters with what we saw in fixed samples (Fig. 4C),thesedatain- into the big centrosomal microtubule asters to generate amphitelic and dicated that loading and self-assembly of microtubules at the synthelic attachments before final chromosome biorientation.

Fu et al. PNAS Early Edition | 5of6 Downloaded by guest on October 1, 2021 kinetochores to be captured by the small TACC3–microtubule the kinetochore-bound TACC3–microtubule asters also pro- asters through lateral binding. Fourth, by skating along the mote the cluster formation and separation of the acentrosomal microtubules of the TACC3–microtubule asters, the kinet- MTOCs as well as the movement and rotation of chromosome ochores are firmly captured through end-on connection of the kinetochores. In agreement with the previous study that TACC3 microtubules and the kinetochores. Simultaneously, the small depletion delays the mitotic progression (18), our study further acentrosomal asters are sorted and join the two big cen- highlights the role of the acentrosomal TACC3 in establishing trosomal asters. And finally, depending on the transport of kinetochore capture by microtubules in a fast and accurate way. acentrosomal microtubule asters toward the centrosomes by the In mammalian somatic cells, Ran GTPase activity promotes microtubule sorting mechanism, the acentrosomal asters are microtubule nucleation at kinetochores (33), and Ran effectors totally fused in the centrosomal asters, and a bipolar spindle is such as microspherule protein 1 and γ-TuRC also function in K eventually established. Noticeably, although the abovementioned fibers (34, 35). As TACC3 is also a target of the Ran GTPase results are mainly based on observations in nocodazole-treated system (20), it may coordinate with Ran to regulate acen- cells, TACC3 is also required for kinetochore capture in un- trosomal microtubules for kinetochore capture. Together these perturbed mitosis without nocodazole treatment (Fig. 2). Be- provide deep insights into the key events during the kinetochore cause acentrosomal microtubule seeds in normal mitosis act capture process by the microtubules. However, further analyses similarly as in nocodazole-treated samples (Fig. 3 A–E), and at a higher resolution level to elucidate the kinetochore behav- TACC3 targets to acentrosomal microtubules (Fig. 1E and Fig. S7) and may regulate acentrosomal microtubule assembly in iors and functions are still needed in the future. normal mitosis (Fig. 2E), the acentrosomal TACC3–microtubule Materials and Methods seeds/asters mechanisms may be true in normal mitosis. Efficient chromosome capture requires a bias to complete the All experiments were done in HeLa cells. Living cell imaging data was acquired by a DeltaVision system (Applied Precision). For microscopy of the capture process during mitotic spindle assembly (31). Meanwhile, fixed samples, the images were acquired by the DeltaVision or Zeiss LSM710 chromosome movements and rotations are required to acceler- microscope. Details of cell culture and drug treatment, RNA interference, ate mitotic spindle assembly according to computer simulations small-molecule inhibitors, antibodies, live cell imaging and microscopy, (32). How does a cell speed up the process of kinetochore cap- Western blotting, immunofluorescence, and statistical analyses are provided ture? The lateral surface area of a microtubule is larger than that in SI Materials and Methods. of its tip and enables the fast kinetochore capture (3, 4). The – formation of TACC3 microtubule seeds/asters near the kineto- ACKNOWLEDGMENTS. We thank Xuebiao Yao for HeLa cells stably express- chore may speed up the kinetochore capture process through ing mcherry-tubulin and plasmids and Jennifer Deluca, Jun Zhou, Xueliang forming acentrosomal microtubule-attached kinetochores, thus Zhu, Michael Lampson, and Wen-Hwa Lee for plasmids and reagents. This work increasing the microtubule capture surface of the kinetochores was supported by funds from the State Key Basic Research and Development and accelerating the movement of kinetochores/chromosomes Plan (2010CB833705) and the National Natural Science Foundation of China along the microtubules. Meanwhile, the growth and sorting of (31071188, 31030044, and 90913021).

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