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

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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 micro- tubules 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, noco- dazole-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.
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