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• W2898895966 abstract "•Formin mDia2 is required for nuclear actin polymerization at G1 centromeres•Nuclear actin polymerization is required to maintain centromeric CENP-A levels•mDia2 and nuclear actin restrict centromere movement during CENP-A loading•Nuclear actin and MgcRacGAP are required for timely turnover of centromeric HJURP Centromeres are specialized chromosomal regions epigenetically defined by the histone H3 variant centromere protein A (CENP-A). CENP-A needs to be replenished in every cell cycle, but how new CENP-A is stably incorporated into centromeric chromatin remains unclear. We have discovered that a cytoskeletal protein, diaphanous formin mDia2, is essential for the stable incorporation of new CENP-A proteins into centromeric nucleosomes. Here we report that mDia2-mediated formation of dynamic and short nuclear actin filaments in G1 nucleus is required to maintain CENP-A levels at the centromere. Importantly, mDia2 and nuclear actin are required for constrained centromere movement during CENP-A loading, and depleting nuclear actin or MgcRacGAP, which lies upstream of mDia2, extends centromeric association of the CENP-A loading chaperone Holliday junction recognition protein (HJURP). Our findings thus suggest that nuclear actin polymerized by mDia2 contributes to the physical confinement of G1 centromeres so that HJURP-mediated CENP-A loading reactions can be productive, and centromere's epigenetic identity can be stably maintained. Centromeres are specialized chromosomal regions epigenetically defined by the histone H3 variant centromere protein A (CENP-A). CENP-A needs to be replenished in every cell cycle, but how new CENP-A is stably incorporated into centromeric chromatin remains unclear. We have discovered that a cytoskeletal protein, diaphanous formin mDia2, is essential for the stable incorporation of new CENP-A proteins into centromeric nucleosomes. Here we report that mDia2-mediated formation of dynamic and short nuclear actin filaments in G1 nucleus is required to maintain CENP-A levels at the centromere. Importantly, mDia2 and nuclear actin are required for constrained centromere movement during CENP-A loading, and depleting nuclear actin or MgcRacGAP, which lies upstream of mDia2, extends centromeric association of the CENP-A loading chaperone Holliday junction recognition protein (HJURP). Our findings thus suggest that nuclear actin polymerized by mDia2 contributes to the physical confinement of G1 centromeres so that HJURP-mediated CENP-A loading reactions can be productive, and centromere's epigenetic identity can be stably maintained. Accurate segregation of chromosomes during mitosis relies on the existence and integrity of centromeres, chromosomal regions that are epigenetically determined by nucleosomes containing the histone H3 variant centromere protein A (CENP-A) (Cleveland et al., 2003Cleveland D.W. Mao Y. Sullivan K.F. Centromeres and kinetochores: from epigenetics to mitotic checkpoint signaling.Cell. 2003; 112: 407-421Abstract Full Text Full Text PDF PubMed Scopus (821) Google Scholar). After the genome replicates in S phase, all CENP-A molecules redistribute to two sister chromatids, thus the total number of CENP-A molecules per centromere is reduced by half. It is therefore necessary to replenish the amount of CENP-A molecules at each centromere in every cell cycle, to ensure the stable inheritance of centromere identity over many generations of cell divisions. In mammals, new CENP-A proteins synthesized in the previous cell cycle are loaded at each centromere during the early G1 phase of the next cell cycle (Jansen et al., 2007Jansen L.E.T. Black B.E. Foltz D.R. Cleveland D.W. Propagation of centromeric chromatin requires exit from mitosis.J. Cell Biol. 2007; 176: 795-805Crossref PubMed Scopus (457) Google Scholar). Many factors have been identified to be responsible for the initiation and execution of recruiting newly synthesized CENP-A molecules to the centromeres (Dunleavy et al., 2009Dunleavy E.M. Roche D. Tagami H. Lacoste N. Ray-Gallet D. Nakamura Y. Daigo Y. Nakatani Y. Almouzni-Pettinotti G. HJURP is a cell-cycle-dependent maintenance and deposition factor of CENP-A at centromeres.Cell. 2009; 137: 485-497Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, Foltz et al., 2009Foltz D.R. Jansen L.E.T. Bailey A.O. Yates III, J.R. Bassett E.A. Wood S. Black B.E. Cleveland D.W. Centromere-specific assembly of CENP-A nucleosomes is mediated by HJURP.Cell. 2009; 137: 472-484Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar, Fujita et al., 2007Fujita Y. Hayashi T. Kiyomitsu T. Toyoda Y. Kokubu A. Obuse C. Yanagida M. Priming of centromere for CENP-A recruitment by human hMis18α, hMis18β, and M18BP1.Dev. Cell. 2007; 12: 17-30Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar, Maddox et al., 2007Maddox P.S. Hyndman F. Monen J. Oegema K. Desai A. Functional genomics identifies a Myb domain–containing protein family required for assembly of CENP-A chromatin.J. Cell Biol. 2007; 176: 757-763Crossref PubMed Scopus (166) Google Scholar, McKinley and Cheeseman, 2014McKinley K.L. Cheeseman I.M. Polo-like kinase 1 licenses CENP-A deposition at centromeres.Cell. 2014; 158: 397-411Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, Moree et al., 2011Moree B. Meyer C.B. Fuller C.J. Straight A.F. CENP-C recruits M18BP1 to centromeres to promote CENP-A chromatin assembly.J. Cell Biol. 2011; 194: 855-871Crossref PubMed Scopus (149) Google Scholar, Silva et al., 2012Silva M.C. Bodor D.L. Stellfox M.E. Martins N.M. Hochegger H. Foltz D.R. Jansen L.E. Cdk activity couples epigenetic centromere inheritance to cell cycle progression.Dev. Cell. 2012; 22: 52-63Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar); among them is the Holliday junction recognition protein (HJURP) that functions as a chaperone to assemble new CENP-A molecules into nucleosomes (Barnhart et al., 2011Barnhart M.C. Kuich P.H.J.L. Stellfox M.E. Ward J.A. Bassett E.A. Black B.E. Foltz D.R. HJURP is a CENP-A chromatin assembly factor sufficient to form a functional de novo kinetochore.J. Cell Biol. 2011; 194: 229-243Crossref PubMed Scopus (232) Google Scholar). However, it remains unclear how new CENP-A molecules become stably incorporated into centromeric nucleosomes. The male germ cell Rac GTPase-activating protein (MgcRacGAP), as well the small Rho GTPases under its regulation, Cdc42 and Rac1, have been shown to be essential for stabilizing newly loaded CENP-A at centromeres (Lagana et al., 2010Lagana A. Dorn J.F. De Rop V. Ladouceur A.-M. Maddox A.S. Maddox P.S. A small GTPase molecular switch regulates epigenetic centromere maintenance by stabilizing newly incorporated CENP-A.Nat. Cell Biol. 2010; 12: 1186-1193Crossref PubMed Scopus (93) Google Scholar). The diaphanous formin (mDia) proteins are important small Rho GTPase effectors and can regulate cytoskeletal dynamics by stabilizing microtubules and nucleating filamentous actin in a linear fashion (Chesarone et al., 2010Chesarone M.A. DuPage A.G. Goode B.L. Unleashing formins to remodel the actin and microtubule cytoskeletons.Nat. Rev. Mol. Cell Biol. 2010; 11: 62-74Crossref PubMed Scopus (379) Google Scholar). Previously we have reported that formin mDia2 is required for maintaining CENP-A levels at the centromere (Liu and Mao, 2016Liu C. Mao Y. Diaphanous formin mDia2 regulates CENP-A levels at centromeres.J. Cell Biol. 2016; 213: 415-424Crossref PubMed Scopus (16) Google Scholar, Liu and Mao, 2017Liu C. Mao Y. Formin-mediated epigenetic maintenance of centromere identity.Small GTPases. 2017; 8: 245-250Crossref PubMed Scopus (4) Google Scholar). Importantly, overexpressing a constitutively active form of mDia2 can rescue defective centromeric CENP-A levels caused by depleting MgcRacGAP. Nevertheless, the mechanisms by which mDia2 functions to promote stable CENP-A loading remains elusive. Among all three members of the mammalian diaphanous formin family, only mDia2, but not mDia1 or 3, can redistribute extensively from the cytoplasm to the nucleus and can biochemically associate with a number of nuclear proteins including histones and topoisomerases via its formin homology (FH) 2 domain (Daou et al., 2014Daou P. Hasan S. Breitsprecher D. Baudelet E. Camoin L. Audebert S. Goode B.L. Badache A. Essential and nonredundant roles for Diaphanous formins in cortical microtubule capture and directed cell migration.Mol. Biol. Cell. 2014; 25: 658-668Crossref PubMed Scopus (0) Google Scholar, Miki et al., 2009Miki T. Okawa K. Sekimoto T. Yoneda Y. Watanabe S. Ishizaki T. Narumiya S. mDia2 shuttles between the nucleus and the cytoplasm through the importin-α/β- and CRM1-mediated nuclear transport mechanism.J. Biol. Chem. 2009; 284: 5753-5762Crossref PubMed Scopus (45) Google Scholar). Inside the nucleus, mDia2 can effectively nucleate filamentous actin polymers (Baarlink et al., 2013Baarlink C. Wang H. Grosse R. Nuclear actin network assembly by formins regulates the SRF coactivator MAL.Science. 2013; 340: 864-867Crossref PubMed Scopus (247) Google Scholar). Accumulating evidence demonstrates that filamentous actin polymerized inside the nucleus plays important roles in regulating chromosome dynamics, including repositioning of chromosomal loci (Dundr et al., 2007Dundr M. Ospina J.K. Sung M.-H. John S. Upender M. Ried T. Hager G.L. Matera A.G. Actin-dependent intranuclear repositioning of an active gene locus in vivo.J. Cell Biol. 2007; 179: 1095-1103Crossref PubMed Scopus (238) Google Scholar), initiation of DNA replication (Parisis et al., 2017Parisis N. Krasinska L. Harker B. Urbach S. Rossignol M. Camasses A. Dewar J. Morin N. Fisher D. Initiation of DNA replication requires actin dynamics and formin activity.EMBO J. 2017; 36: 3212-3231Crossref PubMed Scopus (51) Google Scholar), response to and repair of DNA double-strand breaks (DSBs) (Belin et al., 2015Belin B.J. Lee T. Mullins R.D. DNA damage induces nuclear actin filament assembly by Formin-2 and Spire-1/2 that promotes efficient DNA repair.Elife. 2015; 4: e07735PubMed Google Scholar, Wang et al., 2017Wang Y.-H. Hariharan A. Bastianello G. Toyama Y. Shivashankar G.V. Foiani M. Sheetz M.P. DNA damage causes rapid accumulation of phosphoinositides for ATR signaling.Nat. Commun. 2017; 8: 2118Crossref PubMed Scopus (40) Google Scholar), interactions with chromatin remodeling complexes (Andrin and Hendzel, 2004Andrin C. Hendzel M.J. F-actin-dependent insolubility of chromatin-modifying components.J. Biol. Chem. 2004; 279: 25017-25023Crossref PubMed Scopus (37) Google Scholar, Rando et al., 2002Rando O.J. Zhao K. Janmey P. Crabtree G.R. Phosphatidylinositol-dependent actin filament binding by the SWI/SNF-like BAF chromatin remodeling complex.Proc. Natl. Acad. Sci. U S A. 2002; 99: 2824-2829Crossref PubMed Scopus (195) Google Scholar), and cross talk with important epigenetic enzymes (Serebryannyy et al., 2016Serebryannyy L.A. Cruz C.M. de Lanerolle P. A role for nuclear actin in HDAC 1 and 2 regulation.Sci. Rep. 2016; 6: 28460Crossref PubMed Scopus (43) Google Scholar). Of particular interest is the recent observation of filamentous nuclear actin with chromobodies right after mitotic exit during nuclear volume expansion and chromatin decondensation (Baarlink et al., 2017Baarlink C. Plessner M. Sherrard A. Morita K. Misu S. Virant D. Kleinschnitz E.-M. Harniman R. Alibhai D. Baumeister S. et al.A transient pool of nuclear F-actin at mitotic exit controls chromatin organization.Nat. Cell Biol. 2017; 19: 1389-1399Crossref PubMed Scopus (99) Google Scholar), which shares a similar time window of CENP-A loading in early G1. It is therefore intriguing to ask whether nuclear actin polymerized by mDia2 directly contributes to the stable loading of CENP-A at G1 centromeres. Using a utrophin-based probe that visualizes polymeric nuclear actin, here we provide direct evidence that mDia2 is required for polymerizing nuclear actin in G1 and that polymerization-competent nuclear actin is required for maintaining CENP-A levels at the centromere. Importantly, both mDia2 and nuclear actin are required to restrict centromere movement during CENP-A loading, and the absence of nuclear actin or MgcRacGAP results in prolonged centromere association of HJURP, the molecular chaperone that transiently localizes to the centromere to assemble new CENP-A nucleosomes. We have shown that the formin mDia2 and its nuclear localization are required to maintain CENP-A levels at centromeres (Liu and Mao, 2016Liu C. Mao Y. Diaphanous formin mDia2 regulates CENP-A levels at centromeres.J. Cell Biol. 2016; 213: 415-424Crossref PubMed Scopus (16) Google Scholar). However, whether this novel role for mDia2 requires mDia2-mediated nuclear actin polymerization remains unresolved. Although latrunculin A or cytochalasin D treatment does not affect CENP-A levels at centromeres (Lagana et al., 2010Lagana A. Dorn J.F. De Rop V. Ladouceur A.-M. Maddox A.S. Maddox P.S. A small GTPase molecular switch regulates epigenetic centromere maintenance by stabilizing newly incorporated CENP-A.Nat. Cell Biol. 2010; 12: 1186-1193Crossref PubMed Scopus (93) Google Scholar), actin in the nucleus can form short oligomers or other forms of structures that are less sensitive to drug treatment (Belin et al., 2013Belin B.J. Cimini B.A. Blackburn E.H. Mullins R.D. Visualization of actin filaments and monomers in somatic cell nuclei.Mol. Biol. Cell. 2013; 24: 982-994Crossref PubMed Scopus (115) Google Scholar, Gonsior et al., 1999Gonsior S.M. Platz S. Buchmeier S. Scheer U. Jockusch B.M. Hinssen H. Conformational difference between nuclear and cytoplasmic actin as detected by a monoclonal antibody.J. Cell Sci. 1999; 112: 797-809Crossref PubMed Google Scholar, McDonald et al., 2006McDonald D. Carrero G. Andrin C. de Vries G. Hendzel M.J. Nucleoplasmic β-actin exists in a dynamic equilibrium between low-mobility polymeric species and rapidly diffusing populations.J. Cell Biol. 2006; 172: 541-552Crossref PubMed Scopus (214) Google Scholar, Schoenenberger et al., 2005Schoenenberger C.A. Buchmeier S. Boerries M. Sütterlin R. Aebi U. Jockusch B.M. Conformation-specific antibodies reveal distinct actin structures in the nucleus and the cytoplasm.J. Struct. Biol. 2005; 152: 157-168Crossref PubMed Scopus (107) Google Scholar). To examine if there are actin filaments inside the nucleus concurrent with CENP-A loading, a utrophin-based nuclear actin probe, Utr230-EGFP-NLS (nuclear localization signal) (Belin et al., 2013Belin B.J. Cimini B.A. Blackburn E.H. Mullins R.D. Visualization of actin filaments and monomers in somatic cell nuclei.Mol. Biol. Cell. 2013; 24: 982-994Crossref PubMed Scopus (115) Google Scholar; Table S1), was transiently expressed in synchronized HeLa cells transfected with mDia2 or GAPDH (control) small interfering RNA (Figure 1A; Table S2). Short-term live cell imaging revealed that about 40% of control G1 cells showed dynamic and short nuclear actin filaments with a typical “nuclear puncta” pattern. In contrast, the percentage of cells with nuclear puncta pattern was significantly reduced upon mDia2 depletion (Figures 1B and 1C; Video S1). Longer term live cell imaging from mitotic exit all the way into G1 was also carried out with cells stably expressing Utr230-EGFP-NLS. Apparent nuclear punctate signals of Utr230-EGFP-NLS were observed after cells enter G1 (approximately 46 min after anaphase onset on average), and once they appeared the punctate signals inside the nucleus remain visible for 176 min on average, although individual punctum did not necessarily persist throughout the whole time (Figures 1D, 1E, and S1; Videos S2 and S3). However, the occurrence and duration of nuclear punctate Utr230-EGFP-NLS were significantly reduced upon mDia2 depletion (Figures 1D, 1E, and S1; Video S2). To further examine at higher resolution the localization of these nuclear puncta in relation to centromeres, synchronized early G1 cells stably expressing Utr230-EGFP-NLS were fixed and stained for centromere marker (ACA). Remarkably, nuclear actin polymers visualized by the Utr230-EGFP-NLS probe, although exhibit punctate morphology throughout the nucleoplasm, have apparent enrichment in the vicinity of some but not all centromeres in the nucleus (Figure 1F; Video S4), and both the intensity enrichment of Utr230-EGFP-NLS adjacent to centromeres and the percentage of centromeres with adjacent Utr230-EGFP-NLS puncta per nucleus were significantly reduced upon mDia2 depletion (Figures 1F–1H). These results support the existence of short and dynamic nuclear actin polymers in early G1 cells concurrent with CENP-A loading, dependent on mDia2. To examine whether actin polymerization activity is required for mDia2 function in stable CENP-A assembly we used quantitative imaging and the integrated nuclear CENP-A (INCA) algorithm that we developed for automated measurement (Liu and Mao, 2016Liu C. Mao Y. Diaphanous formin mDia2 regulates CENP-A levels at centromeres.J. Cell Biol. 2016; 213: 415-424Crossref PubMed Scopus (16) Google Scholar). K853A mutation was introduced to the FH2 domain of full-length mDia2, as this mutant is known to be defective in actin polymerization (Bartolini et al., 2008Bartolini F. Moseley J.B. Schmoranzer J. Cassimeris L. Goode B.L. Gundersen G.G. The formin mDia2 stabilizes microtubules independently of its actin nucleation activity.J. Cell Biol. 2008; 181: 523-536Crossref PubMed Scopus (179) Google Scholar). Similar to the actin mutations in the constitutively active mDia2-FH1FH2 fragment (Bartolini et al., 2008Bartolini F. Moseley J.B. Schmoranzer J. Cassimeris L. Goode B.L. Gundersen G.G. The formin mDia2 stabilizes microtubules independently of its actin nucleation activity.J. Cell Biol. 2008; 181: 523-536Crossref PubMed Scopus (179) Google Scholar, Liu and Mao, 2016Liu C. Mao Y. Diaphanous formin mDia2 regulates CENP-A levels at centromeres.J. Cell Biol. 2016; 213: 415-424Crossref PubMed Scopus (16) Google Scholar), K853A mutation in full-length mDia2 failed to restore the decreased CENP-A levels at centromeres upon depleting endogenous mDia2 (Figures S2A and S2B). This further supports that the actin nucleation activity of mDia2 is important for its role in CENP-A loading. To test the hypothesis that filamentous nuclear actin polymerized by mDia2 directly participates in stable CENP-A incorporation at the centromere, we set out altering the pool of actin inside the nucleus. Importin 9 (IPO9) has been shown to be responsible for shuttling actin monomers into the nucleus (Dopie et al., 2012Dopie J. Skarp K.-P. Kaisa Rajakylä E. Tanhuanpää K. Vartiainen M.K. Active maintenance of nuclear actin by importin 9 supports transcription.Proc. Natl. Acad. Sci. U S A. 2012; 109: E544-E552Crossref PubMed Scopus (162) Google Scholar). Knocking down IPO9 depletes the pool of actin proteins inside the nucleus, but neither does it change the total amount of YFP-CENP-A or untagged CENP-A proteins nor does it alter the relative distribution of CENP-A proteins in the cytoplasm or nucleus upon cellular fractionation (Figures 2A , 2B, and S2C). To evaluate only the “loading” machineries of CENP-A at centromeres, we expressed NLS-tagged actin constructs that accumulate in the nucleus independent of IPO9 (Belin et al., 2015Belin B.J. Lee T. Mullins R.D. DNA damage induces nuclear actin filament assembly by Formin-2 and Spire-1/2 that promotes efficient DNA repair.Elife. 2015; 4: e07735PubMed Google Scholar) (Figure S2D). Like nuclear actin polymers visualized by Utr230-EGFP-NLS, wild-type NLS-FLAG-actin shows short punctate filaments inside the nucleus, whereas the polymerization-incompetent R62D mutant remains mostly diffusive in cells depleted of IPO9 (Figure S2D). IPO9 depletion resulted in a CENP-A loading phenotype comparable to that caused by mDia2 depletion. Significantly, the wild-type polymerizable actin tagged with NLS can effectively restore the reduced CENP-A levels upon IPO9 depletion, whereas the nonpolymerizable (Posern et al., 2002Posern G. Sotiropoulos A. Treisman R. Mutant actins demonstrate a role for unpolymerized actin in control of transcription by serum response factor.Mol. Biol. Cell. 2002; 13: 4167-4178Crossref PubMed Scopus (193) Google Scholar) R62D mutant of actin cannot (Figures 2C and 2D). These results collectively suggest that actin polymerization inside the nucleus is essential for the stable maintenance of CENP-A levels at centromeres. Dynamic and short nuclear actin filaments can potentially regulate nuclear events by providing mechanical inputs that either actively transport cargos or help organize nuclear contents. Upon telomere damage, it has been shown that telomere movement is enhanced, which is likely to facilitate DNA repair (Chen et al., 2013Chen B. Gilbert L.A. Cimini B.A. Schnitzbauer J. Zhang W. Li G.-W. Park J. Blackburn E.H. Weissman J.S. Qi L.S. et al.Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system.Cell. 2013; 155: 1479-1491Abstract Full Text Full Text PDF PubMed Scopus (1259) Google Scholar, Dimitrova et al., 2008Dimitrova N. Chen Y.C. Spector D.L. de Lange T. 53BP1 promotes non-homologous end joining of telomeres by increasing chromatin mobility.Nature. 2008; 456: 524-528Crossref PubMed Scopus (442) Google Scholar, Wang et al., 2008Wang X. Kam Z. Carlton P.M. Xu L. Sedat J.W. Blackburn E.H. Rapid telomere motions in live human cells analyzed by highly time-resolved microscopy.Epigenetics Chromatin. 2008; 1: 4Crossref PubMed Google Scholar). To test whether mDia2-mediated nuclear actin polymers affect centromere movement during new CENP-A loading, single-particle tracking of centromere movement was performed in early G1 nuclei by filming HeLa cells stably expressing YFP-CENP-A at relatively high sampling frequency during initial CENP-A loading (Figure 3A and Video S5). Trajectories of these loci in control cells displayed relatively confined movement (Figure 3B), with anomalous diffusion over the timescale of initial CENP-A loading in early G1, about 25–200 min post anaphase onset. The confined centromere motion, however, is significantly impaired upon mDia2 or IPO9 knockdown (Figures 3A, 3B, and S3), with relative apparent diffusion coefficients elevated by 22.1% and 22.6%, respectively (Figure 3D), implying that either mDia2 or IPO9 depletion could be perturbing the same pathway. In particular, long-range centromere movements over the scale of several micrometers were occasionally observed in mDia2 or IPO9 knockdowns but not in control cells (Video S5). Thus the formin mDia2 and nuclear actin are required for the relatively confined movement of centromeres on the timescale of CENP-A loading in early G1. Furthermore, intensity profiling of individual tracks showed that the relative YFP-CENP-A loading ratio in control cells was significantly higher than that in mDia2 or IPO9 knockdown cells (Figures 3C and 3D), supporting that mDia2 and nuclear actin are essential for new CENP-A loading. It has been reported that a small GTPase signaling pathway utilizing the GTPase-activating protein (GAP) MgcRacGAP is essential for the stable maintenance of new CENP-A levels at centromeres (Lagana et al., 2010Lagana A. Dorn J.F. De Rop V. Ladouceur A.-M. Maddox A.S. Maddox P.S. A small GTPase molecular switch regulates epigenetic centromere maintenance by stabilizing newly incorporated CENP-A.Nat. Cell Biol. 2010; 12: 1186-1193Crossref PubMed Scopus (93) Google Scholar). We have previously shown that overexpressing a constitutively active fragment of mDia2 can rescue defective CENP-A levels at the centromere caused by MgcRacGAP depletion, suggesting an epistasis where mDia2 could work downstream of MgcRacGAP pathway (Liu and Mao, 2016Liu C. Mao Y. Diaphanous formin mDia2 regulates CENP-A levels at centromeres.J. Cell Biol. 2016; 213: 415-424Crossref PubMed Scopus (16) Google Scholar). However, MgcRacGAP has been observed to have apparent centromere enrichment during late G1 (Lagana et al., 2010Lagana A. Dorn J.F. De Rop V. Ladouceur A.-M. Maddox A.S. Maddox P.S. A small GTPase molecular switch regulates epigenetic centromere maintenance by stabilizing newly incorporated CENP-A.Nat. Cell Biol. 2010; 12: 1186-1193Crossref PubMed Scopus (93) Google Scholar), when active new CENP-A loading supposedly has finished. To address this apparent inconsistency, we performed high-resolution ratiometric live cell imaging after directly depleting MgcRacGAP, to examine the temporal requirement of the MgcRacGAP-dependent small GTPase pathway during CENP-A loading. Depleting MgcRacGAP caused apparent CENP-A loading defects in live cells going through early G1 phase: in the absence of MgcRacGAP, the increase in YFP-CENP-A levels at individual centromeres cannot be maintained despite an initial slight increase within 2 hr after anaphase onset (Figures 4A and 4B; Video S6). Non-linear regression using a first-order reaction model predicted a substantially lower plateau (maximum) loading amount that is approximately 50% of the normal loading amount, as well as higher rate constant, consistent with attempted yet failed loading behaviors (Figure 4C). These data agree with the phenotype caused by mDia2 depletion, thus by revealing the temporal requirement of the MgcRacGAP-dependent small GTPase pathway, they support the epistatic relationship between MgcRacGAP and mDia2 in early G1 phase. To further confirm that endogenous mDia2 indeed functions downstream of the MgcRacGAP-dependent small Rho GTPase molecular switch, we introduced exogenous fragment of the Dia autoinhibition domain (DAD) of mDia2 into cells depleted of MgcRacGAP. DAD fragment can bind to the Dia-interacting domain (DID) of endogenous mDia2 with high affinity, and thus ectopically opens up the otherwise auto-inhibited endogenous mDia2 due to a lack of small Rho GTPase binding to the GTPase-binding domain at the N terminus (Figure 4D) (Kovar, 2006Kovar D.R. Molecular details of formin-mediated actin assembly.Curr. Opin. Cell Biol. 2006; 18: 11-17Crossref PubMed Scopus (195) Google Scholar, Palazzo et al., 2001Palazzo A.F. Cook T.A. Alberts A.S. Gundersen G.G. mDia mediates Rho-regulated formation and orientation of stable microtubules.Nat. Cell Biol. 2001; 3: 723-729Crossref PubMed Scopus (478) Google Scholar). A point mutation (L1168G) was introduced in the nuclear exporting signal (NES)-like motif of DAD such that this fragment shifts its cytosolic distribution into the nucleus (Baarlink et al., 2013Baarlink C. Wang H. Grosse R. Nuclear actin network assembly by formins regulates the SRF coactivator MAL.Science. 2013; 340: 864-867Crossref PubMed Scopus (247) Google Scholar, Miki et al., 2009Miki T. Okawa K. Sekimoto T. Yoneda Y. Watanabe S. Ishizaki T. Narumiya S. mDia2 shuttles between the nucleus and the cytoplasm through the importin-α/β- and CRM1-mediated nuclear transport mechanism.J. Biol. Chem. 2009; 284: 5753-5762Crossref PubMed Scopus (45) Google Scholar). Another point mutation (M1141A) at the core of DAD abolishes its interaction with DID (Alberts, 2001Alberts A.S. Identification of a carboxyl-terminal diaphanous-related formin homology protein autoregulatory domain.J. Biol. Chem. 2001; 276: 2824-2830Crossref PubMed Scopus (277) Google Scholar), thus the combination of L1168G and M1141A serves as an “inactive” control (Figure 4E). As expected, centromeric CENP-A levels were significantly reduced upon knocking down MgcRacGAP. Importantly, the decrease of CENP-A levels can be rescued by co-expressing DAD-L1168G, but not the DAD-L1168G-inactive fragment (Figures 4F and 4G). Because the DAD fragment per se is not necessary for restoring centromeric CENP-A levels upon depleting endogenous mDia2 (Liu and Mao, 2016Liu C. Mao Y. Diaphanous formin mDia2 regulates CENP-A levels at centromeres.J. Cell Biol. 2016; 213: 415-424Crossref PubMed Scopus (16) Google Scholar), the results here suggest an epistatic relationship between the upstream MgcRacGAP-based small GTPase signaling and downstream endogenous mDia2 in maintaining CENP-A levels at the centromeres. To further understand how mDia2-mediated nuclear actin polymers, which lie downstream of the MgcRacGAP pathway, affect CENP-A loading, we looked at molecular machineries directly responsible for CENP-A assembly. New CENP-A loading during early G1 requires HJURP, the molecular chaperone with CENP-A nucleosome assembly activity (Barnhart et al., 2011Barnhart M.C. Kuich P.H.J.L. Stellfox M.E. Ward J.A. Bassett E.A. Black B.E. Foltz D.R. HJURP is a CENP-A chromatin assembly factor sufficient to form a functional de novo kinetochore.J. Cell Biol. 2011; 194: 229-243Crossref PubMed Scopus (232) Google Scholar). HJURP transiently localizes to the centromere, and thus only a subset of G1 nuclei have HJURP-positive centromeres (Dunleavy et al., 2009Dunleavy E.M. Roche D. Tagami H. Lacoste N. Ray-Gallet D. Nakamura Y. Daigo Y. Nakatani Y. Almouzni-Pettinotti G. HJURP is a cell-cycle-dependent maintenance and deposition factor of CENP-A at centromeres.Cell. 2009; 137: 485-497Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, Foltz et al., 2009Foltz D.R. Jansen L.E.T. Bailey A.O. Yates III, J.R. Bassett E.A. Wood S. Black B.E. Cleveland D.W. Centromere-specific assembly of CENP-A nucleosomes is mediated by HJURP.Cell. 2009; 137: 472-484Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar). We have previously shown that mDia2 depletion results in higher indices of G1 nuclei with HJURP-positive centromeres, which can be caused by reduced HJUPR turnover, and the prolonged association of HJURP molecules with the centromere could prevent further association of HJURP carrying new CENP-A, thus resulting in CENP-A loading defects (Liu and Mao, 2016Liu C. Mao Y. Diaphanous formin mDia2 regulates CENP-A levels at centromeres.J." @default.
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• W2898895966 title "Nuclear Actin Polymerized by mDia2 Confines Centromere Movement during CENP-A Loading" @default.
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