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• W1936657395 abstract "•The spindle regulator BuGZ undergoes temperature-dependent phase transition•Phase transition of BuGZ relies on the aromatic and hydrophobic Phe and Tyr•BuGZ droplets promote microtubule polymerization•Phase transition of BuGZ promotes assembly of spindle and its matrix Spindle assembly required during mitosis depends on microtubule polymerization. We demonstrate that the evolutionarily conserved low-complexity protein, BuGZ, undergoes phase transition or coacervation to promote assembly of both spindles and their associated components. BuGZ forms temperature-dependent liquid droplets alone or on microtubules in physiological buffers. Coacervation in vitro or in spindle and spindle matrix depends on hydrophobic residues in BuGZ. BuGZ coacervation and its binding to microtubules and tubulin are required to promote assembly of spindle and spindle matrix in Xenopus egg extract and in mammalian cells. Since several previously identified spindle-associated components also contain low-complexity regions, we propose that coacervating proteins may be a hallmark of proteins that comprise a spindle matrix that functions to promote assembly of spindles by concentrating its building blocks. Spindle assembly required during mitosis depends on microtubule polymerization. We demonstrate that the evolutionarily conserved low-complexity protein, BuGZ, undergoes phase transition or coacervation to promote assembly of both spindles and their associated components. BuGZ forms temperature-dependent liquid droplets alone or on microtubules in physiological buffers. Coacervation in vitro or in spindle and spindle matrix depends on hydrophobic residues in BuGZ. BuGZ coacervation and its binding to microtubules and tubulin are required to promote assembly of spindle and spindle matrix in Xenopus egg extract and in mammalian cells. Since several previously identified spindle-associated components also contain low-complexity regions, we propose that coacervating proteins may be a hallmark of proteins that comprise a spindle matrix that functions to promote assembly of spindles by concentrating its building blocks. Since the discovery of spindle apparatus in the 1800s (Lukács, 1981Lukács D. [Walter Flemming, discoverer of chromatin and mitotic cell division].Orv. Hetil. 1981; 122: 349-350PubMed Google Scholar), much attention has focused on how microtubules (MT) interact with chromosomes to ensure equal partitioning of chromosomes into daughter cells. Investigation of the mechanisms by which MTs and MT-associated proteins regulate mitosis (Walczak et al., 2010Walczak C.E. Cai S. Khodjakov A. Mechanisms of chromosome behaviour during mitosis.Nat. Rev. Mol. Cell Biol. 2010; 11: 91-102Crossref PubMed Google Scholar) is fueled by the ease of visualizing the spindle-shaped MT fibers, the disruption of chromosome segregation and cell division upon MT perturbation, and the discovery of tubulin (Oakley, 2000Oakley B.R. An abundance of tubulins.Trends Cell Biol. 2000; 10: 537-542Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). In addition to spindle MTs, a set of material that surrounds and permeates spindle MTs have periodically drawn attention (Goldman and Rebhun, 1969Goldman R.D. Rebhun L.I. The structure and some properties of the isolated mitotic apparatus.J. Cell Sci. 1969; 4: 179-209PubMed Google Scholar, Schibler and Pickett-Heaps, 1980Schibler M.J. Pickett-Heaps J.D. Mitosis in Oedogonium: spindle microfilaments and the origin of the kinetochore fiber.Eur. J. Cell Biol. 1980; 22: 687-698PubMed Google Scholar, Scholey et al., 2001Scholey J.M. Rogers G.C. Sharp D.J. Mitosis, microtubules, and the matrix.J. Cell Biol. 2001; 154: 261-266Crossref PubMed Scopus (68) Google Scholar, Johansen and Johansen, 2007Johansen K.M. Johansen J. Cell and molecular biology of the spindle matrix.Int. Rev. Cytol. 2007; 263: 155-206Crossref PubMed Scopus (51) Google Scholar, Johansen et al., 2011Johansen K.M. Forer A. Yao C. Girton J. Johansen J. Do nuclear envelope and intranuclear proteins reorganize during mitosis to form an elastic, hydrogel-like spindle matrix?.Chromosome research. 2011; 19: 345-365Crossref PubMed Scopus (44) Google Scholar, Leslie et al., 1987Leslie R.J. Hird R.B. Wilson L. McIntosh J.R. Scholey J.M. Kinesin is associated with a nonmicrotubule component of sea urchin mitotic spindles.Proc. Natl. Acad. Sci. USA. 1987; 84: 2771-2775Crossref PubMed Scopus (43) Google Scholar, Pickett-Heaps et al., 1984Pickett-Heaps J. Spurck T. Tippit D. Chromosome motion and the spindle matrix.J. Cell Biol. 1984; 99: 137s-143sCrossref PubMed Scopus (52) Google Scholar, Pickett-Heaps and Forer, 2009Pickett-Heaps J. Forer A. Mitosis: spindle evolution and the matrix model.Protoplasma. 2009; 235: 91-99Crossref PubMed Scopus (25) Google Scholar, Schweizer et al., 2014Schweizer N. Weiss M. Maiato H. The dynamic spindle matrix.Curr. Opin. Cell Biol. 2014; 28: 1-7Crossref PubMed Scopus (25) Google Scholar, Wein et al., 1998Wein H. Bass H.W. Cande W.Z. DSK1, a kinesin-related protein involved in anaphase spindle elongation, is a component of a mitotic spindle matrix.Cell Motil. Cytoskeleton. 1998; 41: 214-224Crossref PubMed Scopus (11) Google Scholar, Zheng, 2010Zheng Y. A membranous spindle matrix orchestrates cell division.Nat. Rev. Mol. Cell Biol. 2010; 11: 529-535Crossref PubMed Scopus (68) Google Scholar, Zheng and Tsai, 2006Zheng Y. Tsai M.Y. The mitotic spindle matrix: a fibro-membranous lamin connection.Cell Cycle. 2006; 5: 2345-2347Crossref PubMed Scopus (27) Google Scholar). Historically, this ill-defined spindle-associated material has been referred to as spindle matrix. One vague but generally accepted feature of spindle matrix is that it retains some integrity upon MT disassembly. Based on this criterion, several spindle matrix proteins have been identified and studied in the context of spindle assembly and chromosome segregation. For example, among the Drosophilia spindle matrix proteins (Fabian et al., 2007Fabian L. Xia X. Venkitaramani D.V. Johansen K.M. Johansen J. Andrew D.J. Forer A. Titin in insect spermatocyte spindle fibers associates with microtubules, actin, myosin and the matrix proteins skeletor, megator and chromator.J. Cell Sci. 2007; 120: 2190-2204Crossref PubMed Scopus (40) Google Scholar, Johansen et al., 2011Johansen K.M. Forer A. Yao C. Girton J. Johansen J. Do nuclear envelope and intranuclear proteins reorganize during mitosis to form an elastic, hydrogel-like spindle matrix?.Chromosome research. 2011; 19: 345-365Crossref PubMed Scopus (44) Google Scholar, Qi et al., 2004Qi H. Rath U. Wang D. Xu Y.Z. Ding Y. Zhang W. Blacketer M.J. Paddy M.R. Girton J. Johansen J. Johansen K.M. Megator, an essential coiled-coil protein that localizes to the putative spindle matrix during mitosis in Drosophila.Mol. Biol. Cell. 2004; 15: 4854-4865Crossref PubMed Scopus (71) Google Scholar, Qi et al., 2005Qi H. Rath U. Ding Y. Ji Y. Blacketer M.J. Girton J. Johansen J. Johansen K.M. EAST interacts with Megator and localizes to the putative spindle matrix during mitosis in Drosophila.J. Cell. Biochem. 2005; 95: 1284-1291Crossref PubMed Scopus (30) Google Scholar, Rath et al., 2004Rath U. Wang D. Ding Y. Xu Y.Z. Qi H. Blacketer M.J. Girton J. Johansen J. Johansen K.M. Chromator, a novel and essential chromodomain protein interacts directly with the putative spindle matrix protein skeletor.J. Cell. Biochem. 2004; 93: 1033-1047Crossref PubMed Scopus (63) Google Scholar, Walker et al., 2000Walker D.L. Wang D. Jin Y. Rath U. Wang Y. Johansen J. Johansen K.M. Skeletor, a novel chromosomal protein that redistributes during mitosis provides evidence for the formation of a spindle matrix.J. Cell Biol. 2000; 151: 1401-1412Crossref PubMed Scopus (82) Google Scholar, Yao et al., 2012Yao C. Rath U. Maiato H. Sharp D. Girton J. Johansen K.M. Johansen J. A nuclear-derived proteinaceous matrix embeds the microtubule spindle apparatus during mitosis.Mol. Biol. Cell. 2012; 23: 3532-3541Crossref PubMed Scopus (24) Google Scholar, Yao et al., 2014Yao C. Wang C. Li Y. Ding Y. Rath U. Sengupta S. Girton J. Johansen K.M. Johansen J. The spindle matrix protein, Chromator, is a novel tubulin binding protein that can interact with both microtubules and free tubulin.PLoS ONE. 2014; 9: e103855Crossref PubMed Scopus (3) Google Scholar), Megator regulates spindle assembly checkpoints (SAC) (Lince-Faria et al., 2009Lince-Faria M. Maffini S. Orr B. Ding Y. Cláudia Florindo Sunkel C.E. Tavares A. Johansen J. Johansen K.M. Maiato H. Spatiotemporal control of mitosis by the conserved spindle matrix protein Megator.J. Cell Biol. 2009; 184: 647-657Crossref PubMed Scopus (96) Google Scholar). A conserved protein, BuGZ, which was identified as part of the lamin-B (LB) spindle matrix in Xenopus (Tsai et al., 2006Tsai M.Y. Wang S. Heidinger J.M. Shumaker D.K. Adam S.A. Goldman R.D. Zheng Y. A mitotic lamin B matrix induced by RanGTP required for spindle assembly.Science. 2006; 311: 1887-1893Crossref PubMed Scopus (236) Google Scholar, Ma et al., 2009Ma L. Tsai M.Y. Wang S. Lu B. Chen R. Iii J.R. Zhu X. Zheng Y. Requirement for Nudel and dynein for assembly of the lamin B spindle matrix.Nat. Cell Biol. 2009; 11: 247-256Crossref PubMed Scopus (86) Google Scholar), has recently been shown to facilitate chromosome alignment by controlling both stability and kinetochore loading of the SAC component Bub3 (Jiang et al., 2014Jiang H. He X. Wang S. Jia J. Wan Y. Wang Y. Zeng R. Yates 3rd, J. Zhu X. Zheng Y. A microtubule-associated zinc finger protein, BuGZ, regulates mitotic chromosome alignment by ensuring Bub3 stability and kinetochore targeting.Dev. Cell. 2014; 28: 268-281Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, Toledo et al., 2014Toledo C.M. Herman J.A. Olsen J.B. Ding Y. Corrin P. Girard E.J. Olson J.M. Emili A. DeLuca J.G. Paddison P.J. BuGZ is required for Bub3 stability, Bub1 kinetochore function, and chromosome alignment.Dev. Cell. 2014; 28: 282-294Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Additionally, LB (Tsai et al., 2006Tsai M.Y. Wang S. Heidinger J.M. Shumaker D.K. Adam S.A. Goldman R.D. Zheng Y. A mitotic lamin B matrix induced by RanGTP required for spindle assembly.Science. 2006; 311: 1887-1893Crossref PubMed Scopus (236) Google Scholar) and poly ADP-ribose (Chang et al., 2004Chang P. Jacobson M.K. Mitchison T.J. Poly(ADP-ribose) is required for spindle assembly and structure.Nature. 2004; 432: 645-649Crossref PubMed Scopus (191) Google Scholar), along with other spindle assembly factors (SAFs), such as dynein, Nudel, NuMA, and kinesin Eg5 (Civelekoglu-Scholey et al., 2010Civelekoglu-Scholey G. Tao L. Brust-Mascher I. Wollman R. Scholey J.M. Prometaphase spindle maintenance by an antagonistic motor-dependent force balance made robust by a disassembling lamin-B envelope.J. Cell Biol. 2010; 188: 49-68Crossref PubMed Scopus (57) Google Scholar, Goodman et al., 2010Goodman B. Channels W. Qiu M. Iglesias P. Yang G. Zheng Y. Lamin B counteracts the kinesin Eg5 to restrain spindle pole separation during spindle assembly.J. Biol. Chem. 2010; 285: 35238-35244Crossref PubMed Scopus (34) Google Scholar, Ma et al., 2009Ma L. Tsai M.Y. Wang S. Lu B. Chen R. Iii J.R. Zhu X. Zheng Y. Requirement for Nudel and dynein for assembly of the lamin B spindle matrix.Nat. Cell Biol. 2009; 11: 247-256Crossref PubMed Scopus (86) Google Scholar, Tsai et al., 2006Tsai M.Y. Wang S. Heidinger J.M. Shumaker D.K. Adam S.A. Goldman R.D. Zheng Y. A mitotic lamin B matrix induced by RanGTP required for spindle assembly.Science. 2006; 311: 1887-1893Crossref PubMed Scopus (236) Google Scholar), may regulate spindle morphogenesis. Despite these studies, the structural nature of the spindle matrix remains undefined and whether it constitutes a cohesive functional unit is unclear. In fact, some modeling and biophysical probing of spindle apparatus have not provided evidence for the existence of spindle matrix (Brugués and Needleman, 2014Brugués J. Needleman D. Physical basis of spindle self-organization.Proc. Natl. Acad. Sci. USA. 2014; 111: 18496-18500Crossref PubMed Scopus (100) Google Scholar, Gatlin et al., 2010Gatlin J.C. Matov A. Danuser G. Mitchison T.J. Salmon E.D. Directly probing the mechanical properties of the spindle and its matrix.J. Cell Biol. 2010; 188: 481-489Crossref PubMed Scopus (33) Google Scholar, Shimamoto et al., 2011Shimamoto Y. Maeda Y.T. Ishiwata S. Libchaber A.J. Kapoor T.M. Insights into the micromechanical properties of the metaphase spindle.Cell. 2011; 145: 1062-1074Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Thus, whether spindle matrix is a real structural element of spindle apparatus or a mere artifact induced upon depolymerization of spindle MTs remains an open question. Unlike membranous organelles, the spindle apparatus is not surrounded by membrane barrier during vertebrate mitosis. However, spindles may need to concentrate many components in order to support spatially and temporally diverse reactions. Consistently, tubulin and some SAFs are shown to be concentrated in the region where nascent spindle begins to assemble in Caenorhabditis elegans embryos (Hayashi et al., 2012Hayashi H. Kimura K. Kimura A. Localized accumulation of tubulin during semi-open mitosis in the Caenorhabditis elegans embryo.Mol. Biol. Cell. 2012; 23: 1688-1699Crossref PubMed Scopus (24) Google Scholar). This concentration process is independent of MTs but it requires nuclear envelope permeabilization and RanGTPase, which stimulates spindle assembly (Kalab et al., 1999Kalab P. Pu R.T. Dasso M. The ran GTPase regulates mitotic spindle assembly.Curr. Biol. 1999; 9: 481-484Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, Ohba et al., 1999Ohba T. Nakamura M. Nishitani H. Nishimoto T. Self-organization of microtubule asters induced in Xenopus egg extracts by GTP-bound Ran.Science. 1999; 284: 1356-1358Crossref PubMed Scopus (251) Google Scholar, Wilde and Zheng, 1999Wilde A. Zheng Y. Stimulation of microtubule aster formation and spindle assembly by the small GTPase Ran.Science. 1999; 284: 1359-1362Crossref PubMed Scopus (324) Google Scholar). Proteins, such as elastin and elastin-like peptides, can undergo liquid-liquid phase transition or coacervation to form liquid droplets (Yeo et al., 2011Yeo G.C. Keeley F.W. Weiss A.S. Coacervation of tropoelastin.Adv. Colloid Interface Sci. 2011; 167: 94-103Crossref PubMed Scopus (170) Google Scholar). The phase separation has been proposed to promote concentration of molecules into the liquid droplets, which can then facilitate biochemical reactions (Hyman et al., 2014Hyman A.A. Weber C.A. Jülicher F. Liquid-liquid phase separation in biology.Annu. Rev. Cell Dev. Biol. 2014; 30: 39-58Crossref PubMed Scopus (1451) Google Scholar). Indeed, the liquid droplet feature of P granules and nucleoli is consistent with the idea that assembly and function of these non-membranous organelles could be driven by the phase transition of some of their structural components (Brangwynne et al., 2009Brangwynne C.P. Eckmann C.R. Courson D.S. Rybarska A. Hoege C. Gharakhani J. Jülicher F. Hyman A.A. Germline P granules are liquid droplets that localize by controlled dissolution/condensation.Science. 2009; 324: 1729-1732Crossref PubMed Scopus (1516) Google Scholar, Brangwynne et al., 2011Brangwynne C.P. Mitchison T.J. Hyman A.A. Active liquid-like behavior of nucleoli determines their size and shape in Xenopus laevis oocytes.Proc. Natl. Acad. Sci. USA. 2011; 108: 4334-4339Crossref PubMed Scopus (732) Google Scholar). No proteins of these organelles, however, have yet been shown to undergo functionally relevant phase transition. Interestingly, when engineered as multiple tandem repeats, SRC homology 3 (SH3) domains of NCK and proline-rich motif (PRM) of N-WASP form multivalent interactions, which allow the protein mixture to undergo phase transition to form liquid droplets. These droplets concentrate actin to promote F-actin assembly in vitro (Li et al., 2012Li P. Banjade S. Cheng H.C. Kim S. Chen B. Guo L. Llaguno M. Hollingsworth J.V. King D.S. Banani S.F. et al.Phase transitions in the assembly of multivalent signalling proteins.Nature. 2012; 483: 336-340Crossref PubMed Scopus (1282) Google Scholar). Despite the observed in vitro phase transition into liquid droplets, proteins have been shown to function in vivo via phase transition. Here, we examine the spindle regulatory protein BuGZ, which we noted contains evolutionarily conserved low complexity sequence, and demonstrate that it forms a MT-independent structure through temperature- and hydrophobic residue-dependent coacervation. This phase transition property allows the concentration of tubulin along MTs and supports assembly of spindle MTs and of the biochemically defined spindle matrix structure. Based on these results, we propose a model and line of investigation for further developing our understanding of observed properties and possible functions of spindle matrix. Our previous studies show that BuGZ binds MTs to promote kinetochore loading of Bub3 and chromosome alignment (Jiang et al., 2014Jiang H. He X. Wang S. Jia J. Wan Y. Wang Y. Zeng R. Yates 3rd, J. Zhu X. Zheng Y. A microtubule-associated zinc finger protein, BuGZ, regulates mitotic chromosome alignment by ensuring Bub3 stability and kinetochore targeting.Dev. Cell. 2014; 28: 268-281Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). We noticed that human BuGZ (hBuGZ) depletion in HeLa cells resulted in a more severe disruption of spindle morphology and reduction of MT intensity than those depleted of Bub3, especially when RNAi treatment was extended to 72 hr (Figures S1A and S1B). The more severe spindle defects in hBuGZ-depleted cells were consistent with a stronger chromosome misalignment than those depleted of hBub3 (Figure S1C). This suggests that BuGZ could directly regulate spindle assembly independent of Bub3′s kinetochore function. Previously, we developed a bead-based spindle assembly assay (Tsai and Zheng, 2005Tsai M.Y. Zheng Y. Aurora A kinase-coated beads function as microtubule-organizing centers and enhance RanGTP-induced spindle assembly.Curr. Biol. 2005; 15: 2156-2163Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) by tethering the mitotic kinase Aurora A to 2.8-μm magnetic beads via antibodies. These beads function as MT organizing centers to induce efficient spindle assembly in the cytostatic factor (CSF) arrested Xenopus egg extract (referred to as extract below) in the presence of RanGTP. Since spindles induced by Aurora A beads and RanGTP do not have chromosomes and kinetochores, we can test the kinetochore-independent function of BuGZ in spindle assembly. Immunodepletion of Xenopus BuGZ (xBuGZ) by ∼90% (Figure 1A) resulted in a significant reduction of astral MT length and bipolar spindle numbers (Figures 1B, 1C, and 1E). Most bipolar spindles formed in the absence of xBuGZ were also significantly shorter than those of controls (Figure 1D). These defects were fully rescued by purified xBuGZ (Figures 1A–1E). xBuGZ depletion also disrupted spindle assembly induced by sperm chromatin. Major phenotypes included spindles with MT aggregates surrounding sperm chromatin or spindles with reduced MTs, followed by asters, half spindles, or abnormal spindle shapes with normal MT density (Figures 1F–1H), and all of the defects were also rescued by purified xBuGZ (Figures 1F–1H). Thus BuGZ promotes spindle assembly independent of its kinetochore function. To understand how BuGZ promotes spindle assembly, we treated HeLa cells with control or hBuGZ siRNA and then depolymerized MTs in the cold. MT regrowth was examined after returning cells to 37°C. hBuGZ depletion greatly reduced astral MT regrowth, which was rescued by expressing the RNAi-insensitive wild-type mouse BuGZ (mBuGZ, Figures 1I and 1J). The N-terminal 92 amino acids of BuGZ bind directly to MTs, while the Gle2-binding sequence (GLEBS) within the C terminus of BuGZ directly binds and stabilizes Bub3 (Jiang et al., 2014Jiang H. He X. Wang S. Jia J. Wan Y. Wang Y. Zeng R. Yates 3rd, J. Zhu X. Zheng Y. A microtubule-associated zinc finger protein, BuGZ, regulates mitotic chromosome alignment by ensuring Bub3 stability and kinetochore targeting.Dev. Cell. 2014; 28: 268-281Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Replacing the two highly conserved glutamic acids (E) in GLEBS with alanine (A) results in a mutant (mBuGZAA) that fails to bind and stabilize Bub3, while mBuGZΔN lacking the N-terminal 92 amino acids does not bind to spindles in vivo and MTs in vitro (Jiang et al., 2014Jiang H. He X. Wang S. Jia J. Wan Y. Wang Y. Zeng R. Yates 3rd, J. Zhu X. Zheng Y. A microtubule-associated zinc finger protein, BuGZ, regulates mitotic chromosome alignment by ensuring Bub3 stability and kinetochore targeting.Dev. Cell. 2014; 28: 268-281Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The wild-type mBuGZ and mBuGZAA bound to spindle MTs and MTs assembled from pure tubulin (Figures S1D–S1F). To analyze which of these two known domains in BuGZ promotes assembly of spindle, we depleted endogenous hBuGZ from HeLa or U2OS cells by RNAi. BuGZ RNAi-induced spindle defects, judged by spindle MT intensity, were rescued fully by wild-type mBuGZ and partially by mBuGZAA, but not by mBuGZΔN (Figures 1J–1L). hBuGZ depletion did not alter interphase MT densities (Figure S1G). Thus MT binding of BuGZ promotes spindle MT assembly. Since BuGZ was identified as a spindle matrix component, we assayed for spindle matrix by assembling Aurora A-bead spindles in extract and then depolymerizing MTs using nocodazole at room temperature (RT) (Ma et al., 2009Ma L. Tsai M.Y. Wang S. Lu B. Chen R. Iii J.R. Zhu X. Zheng Y. Requirement for Nudel and dynein for assembly of the lamin B spindle matrix.Nat. Cell Biol. 2009; 11: 247-256Crossref PubMed Scopus (86) Google Scholar, Tsai et al., 2006Tsai M.Y. Wang S. Heidinger J.M. Shumaker D.K. Adam S.A. Goldman R.D. Zheng Y. A mitotic lamin B matrix induced by RanGTP required for spindle assembly.Science. 2006; 311: 1887-1893Crossref PubMed Scopus (236) Google Scholar). The nocodazole-insensitive material that remains on the Aurora A beads, i.e., the spindle matrix, was isolated using a magnet and analyzed by western blotting or immunostaining probing known spindle matrix markers, lamin-B3 (LB3, the major lamin in extracts), dynein, Eg5, NuMA, and XMAP215 (Ma et al., 2009Ma L. Tsai M.Y. Wang S. Lu B. Chen R. Iii J.R. Zhu X. Zheng Y. Requirement for Nudel and dynein for assembly of the lamin B spindle matrix.Nat. Cell Biol. 2009; 11: 247-256Crossref PubMed Scopus (86) Google Scholar, Tsai et al., 2006Tsai M.Y. Wang S. Heidinger J.M. Shumaker D.K. Adam S.A. Goldman R.D. Zheng Y. A mitotic lamin B matrix induced by RanGTP required for spindle assembly.Science. 2006; 311: 1887-1893Crossref PubMed Scopus (236) Google Scholar). Depleting xBuGZ greatly diminished recovery of the spindle matrix, but this was rescued by purified xBuGZ (see the Noc, RT panels in Figures 2A–2C ). Although xBuGZ depletion diminished the recovery of LB3, depleting LB3 did not affect association of xBuGZ with the spindle matrix (Figure S2A). Thus, BuGZ may function upstream of LB3 to promote spindle matrix assembly.Figure S2Effects of BuGZ and Temperature on Spindle Matrix, Related to Figure 2Show full caption(A) Depletion of LB3 did not reduce xBuGZ amount in Aurora A bead-associated spindle matrix. xBuGZ depletion was used as a control. Western blots on the left show the depletion efficiency. The spindle matrix was isolated by depolymerizing MTs at room temperature (RT). Matrix associated with Aurora A beads was labeled with xBuGZ antibody (see images in the middle). To quantify xBuGZ immunostaining intensity in spindle matrix, only spindle matrix associated with one or two beads was selected as one structure and ∼30 of these structures were measured for the average intensity graph on the right. The bright Aurora A beads in xBuGZ stained samples appear bigger than the beads in rhodamine-tubulin channel because the secondary anti-rabbit antibody used for xBuGZ staining also recognized the rabbit Aurora A antibody on the beads. The beads were excluded from quantifications. Error bars, SEM. Student’s t test: ns, not significant, ∗∗∗p < 0.001 from three independent experiments. Scale bar, 5 μm.(B) Treatment of HeLa cells on ice for 5 min reduced spindle-associated BuGZ signal as compared to cells kept at RT for 5 min. Cells were immunostained with tubulin and BuGZ antibodies. Fluorescence signals for MTs and BuGZ were measured in ∼100 cells to determine the intensity ratio of BuGZ:tubulin (see the plot on the right). Error bars, SEM. Student’s t test: ∗∗p < 0.01 from three independent experiments. Scale bar, 5 μm.(C) hBuGZ was more extractable in the cold than at RT in mitotic HeLa cells as determined by Western blotting. Mitotic cells were incubated at RT or on ice for 5 min with (+E) or without (-E) detergent extraction. The cell pellets were analyzed by Western blotting using antibodies to hBuGZ, CENP-A, and tubulin. Quantifications were based on three independent experiments. Error bars, SEM. Student’s t test: ns, not significant, ∗∗∗p < 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Depletion of LB3 did not reduce xBuGZ amount in Aurora A bead-associated spindle matrix. xBuGZ depletion was used as a control. Western blots on the left show the depletion efficiency. The spindle matrix was isolated by depolymerizing MTs at room temperature (RT). Matrix associated with Aurora A beads was labeled with xBuGZ antibody (see images in the middle). To quantify xBuGZ immunostaining intensity in spindle matrix, only spindle matrix associated with one or two beads was selected as one structure and ∼30 of these structures were measured for the average intensity graph on the right. The bright Aurora A beads in xBuGZ stained samples appear bigger than the beads in rhodamine-tubulin channel because the secondary anti-rabbit antibody used for xBuGZ staining also recognized the rabbit Aurora A antibody on the beads. The beads were excluded from quantifications. Error bars, SEM. Student’s t test: ns, not significant, ∗∗∗p < 0.001 from three independent experiments. Scale bar, 5 μm. (B) Treatment of HeLa cells on ice for 5 min reduced spindle-associated BuGZ signal as compared to cells kept at RT for 5 min. Cells were immunostained with tubulin and BuGZ antibodies. Fluorescence signals for MTs and BuGZ were measured in ∼100 cells to determine the intensity ratio of BuGZ:tubulin (see the plot on the right). Error bars, SEM. Student’s t test: ∗∗p < 0.01 from three independent experiments. Scale bar, 5 μm. (C) hBuGZ was more extractable in the cold than at RT in mitotic HeLa cells as determined by Western blotting. Mitotic cells were incubated at RT or on ice for 5 min with (+E) or without (-E) detergent extraction. The cell pellets were analyzed by Western blotting using antibodies to hBuGZ, CENP-A, and tubulin. Quantifications were based on three independent experiments. Error bars, SEM. Student’s t test: ns, not significant, ∗∗∗p < 0.001. When spindle MTs were depolymerized by nocodazole on ice, less matrices were associated with Aurora A beads than those incubated at RT (compare IgG matrix panels in Figures 2A and 2B). Quantification of LB3 staining revealed a significant reduction of matrices around beads upon cold treatment or upon xBuGZ depletion (Figure 2C). xBuGZ depletion plus cold treatment caused an additional matrix reduction that could be rescued by purified xBuGZ (Figure 2C). Thus spindle matrix assembly and stability require BuGZ and a physiological temperature. Unlike many MT-associated SAFs that decorate MT fibers densely and brightly, BuGZ appears as a loose “haze” enriched on spindles (Figure S1E) (Jiang et al., 2014Jiang H. He X. Wang S. Jia J. Wan Y. Wang Y. Zeng R. Yates 3rd, J. Zhu X. Zheng Y. A microtubule-associated zinc finger protein, BuGZ, regulates mitotic chromosome alignment by ensuring Bub3 stability and kinetochore targeting.Dev. Cell. 2014; 28: 268-281Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). When HeLa cells were incubated at RT or on ice for 5 min followed by immunostaining, we found that cold treatment diminished BuGZ signal on spindles, whether or not the spindle MTs were stabilized with Taxol (Figures 2D and S2B). xBuGZ signal was also reduced on cold-treated Aurora A-bead spindles stabilized by Taxol (Figure 2E). We then treated HeLa cells by Taxol and collected mitotic cells by shake-off. Detergent extraction of these cells on ice or at RT in the presence of Taxol followed by immunoblotting showed that more hBuGZ was extracted in the cold as compared to tubulin or CENP-A controls (Figure S2C). Immunostaining further indicated that spindle-associated hBuGZ was more sensitive to the extraction on ice than at RT (Figure 2F). We analyzed vertebrate BuGZ protein sequences using PONDR and SEG, programs designed to predict the disordered (Xue et al., 2010Xue B. Dunbrack R.L. Williams R.W. Dunker A.K. Uversky V.N. PONDR-FIT: a meta-predictor of intrinsically disordered amino acids.Biochim. Biophys. Acta. 2010; 1804: 996-1010Crossref PubMed Scopus (824) Google Scholar) and low complexity regions (Wootton, 1994Wootton J.C. Non-globular domains in protein sequences: automated segmentation using complexity measures.Comput. Chem. 1994; 18: 269-285Crossref PubMed Scopus (386) Google Scholar) in proteins, respectively. The N terminus of BuGZ, containing the MT binding domain and zinc fingers, was pre" @default.
• W1936657395 created "2016-06-24" @default.
• W1936657395 creator A5001097950 @default.
• W1936657395 creator A5049949505 @default.
• W1936657395 creator A5063592070 @default.
• W1936657395 creator A5077586136 @default.
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• W1936657395 creator A5088041187 @default.
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• W1936657395 date "2015-09-01" @default.
• W1936657395 modified "2023-10-12" @default.
• W1936657395 title "Phase Transition of Spindle-Associated Protein Regulate Spindle Apparatus Assembly" @default.
• W1936657395 cites W1578773583 @default.
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