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Greatwall Is Essential to Prevent Mitotic Collapse After Nuclear Envelope Breakdown in Mammals

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Greatwall Is Essential to Prevent Mitotic Collapse After Nuclear Envelope Breakdown in Mammals Greatwall is essential to prevent mitotic collapse after nuclear envelope breakdown in mammals Mónica Álvarez-Fernándeza, Ruth Sánchez-Martíneza,1, Belén Sanz-Castilloa, Pei Pei Gana, María Sanz-Floresa, Marianna Trakalaa, Miguel Ruiz-Torresa, Thierry Lorcab, Anna Castrob, and Marcos Malumbresa,2 aCell Division and Cancer Group, Spanish National Cancer Research Centre, Centro Nacional de Investigaciones Oncológicas (CNIO), 28029 Madrid, Spain; and bUniversités Montpellier 2 et 1, Centre de Recherches de Biochimie Macromoléculaire, Centre National de la Recherche Scientifique Unité Mixte de Recherche 5237, 34293 Montpellier, France Edited* by Tim Hunt, Cancer Research UK, South Mimms, United Kingdom, and approved September 19, 2013 (received for review June 6, 2013) Greatwall is a protein kinase involved in the inhibition of protein cyclin B–Cdk1. The lack of Greatwall activity results in defects in phosphatase 2 (PP2A)-B55 complexes to maintain the mitotic state. chromosome condensation after NEB, and these defects can be Although its biochemical activity has been deeply characterized in rescued by concomitant ablation of B55 proteins. Our results Xenopus, its specific relevance during the progression of mitosis imply that cells are subjected to a mitotic stress resulting from is not fully understood. By using a conditional knockout of the NEB, a moment in which nuclear chromatin becomes exposed to mouse ortholog, Mastl, we show here that mammalian Greatwall cytoplasmic phosphatases. We therefore propose that Greatwall is essential for mouse embryonic development and cell cycle pro- shuttles to the cytoplasm before NEB and prevents mitotic col- gression. Yet, Greatwall-null cells enter into mitosis with normal lapse by inhibiting the PP2A–B55-dependent dephosphorylation kinetics. However, these cells display mitotic collapse after nuclear of Cdk substrates. envelope breakdown (NEB) characterized by defective chromo- some condensation and prometaphase arrest. Intriguingly, Great- Results wall is exported from the nucleus to the cytoplasm in a CRM1- Genetic Ablation of Mastl in the Mouse. Mastl(+/−) mice (Fig. 1 A dependent manner before NEB. This export occurs after the nuclear and B) develop normally and are fertile. However, no viable importofcyclinB–Cdk1 complexes, requires the kinase activity homozygous mutants were found after intercrosses between of Greatwall, and is mediated by Cdk-, but not Polo-like kinase these mice, suggesting embryonic lethality in the absence of CELL BIOLOGY 1-dependent phosphorylation. The mitotic collapse observed in Greatwall. Mastl(−/−) embryos developed normally in vitro Greatwall-deficient cells is partially rescued after concomitant de- during a few days and generate normal blastocysts (Fig. S1). pletion of B55 regulatory subunits, which are mostly cytoplasmic However, these mutant embryos were not detected in the utero before NEB. These data suggest that Greatwall is an essential pro- by embryonic day (E) 10.5, suggesting lethality at periimplanta- tein in mammals required to prevent mitotic collapse after NEB. tional stages (Fig. 1C). Because the germ-line null mutation in Greatwall resulted in cell cycle regulation | cell division | mitotic kinases | mitotic phosphatases | early embryonic lethality, we intercrossed Mastl(+/lox) mice nuclear export harboring an additional allele that expresses the Cre-estrogen receptor (ERT2) recombinase in the ubiquitous RNA polymerase reatwall was originally identified in Drosophila as a modu- II locus (13). We treated pregnant females at E12.5 with ta- Glator of Polo activity and a protein required for DNA con- moxifen to induce Cre activity, and embryos were analyzed at densation and normal progression through mitosis (1–4). E14.5. Genetic ablation of Greatwall resulted in increased mitotic fi Mastl Δ Δ Biochemical assays in Xenopus extracts have demonstrated that gures in the neuroepithelium of ( / ) embryos compared Mastl + Δ D fi Greatwall is able to inhibit PP2A–B55 phosphatase complexes by with ( / ) littermates (Fig. 1 ). These mitotic gures dis- phosphorylating the cAMP-regulated phosphoprotein Arpp19 played an accumulation of prometaphases/metaphases (PM) in and α-endosulfine, thus participating in the maintenance of the the absence of Greatwall compared with more abundant ana- D mitotic state (5–8). The control of PP2A through the Greatwall- phases/telophases (AT) in control sections (Fig. 1 ), thus sug- dependent phosphorylation of Arpp19/Ensa proteins has also gesting that Greatwall is essential for proper mitotic progression been supported by genetic studies in Drosophila (9, 10). The in vivo in mammals. mammalian ortholog of Greatwall, also known as microtubule- associated serine/threonine kinase-like protein (Mastl), also Significance participates in the maintenance of the mitotic state by inhibiting PP2A phosphatases (11, 12). Inhibition of Greatwall is required Nuclear envelope breakdown (NEB) leads to the exposure of for the activation of PP2A–B55α,δ complexes during mitotic exit nuclear structures to cytoplasmic activities. Greatwall is a ki- (13), thus suggesting the relevance of this pathway in maintaining nase able to inhibit PP2A phosphatases that counteract Cdk- the mitotic state (4). How Greatwall activity and function is dependent phosphorylation required for mitosis. Here we show regulated is not well established. Several evidences support a role that Greatwall, an essential protein in mammals, is exported to for cyclin-dependent kinase (Cdk)-dependent phosphorylation in the cytoplasm in a Cdk-dependent manner before NEB, thus the activation of Greatwall, and several phosphorylation sites for protecting mitotic phosphates from phosphatase activity. multiple kinases have been mapped (14, 15). In addition, although Greatwall is mostly nuclear in interphase (3, 11), the cellular and Author contributions: M.M. designed research; M.A.-F., R.S.-M., B.S.-C., P.P.G., M.S.-F., molecular basis of the control of its dynamic intracellular M.T., and M.R.-T. performed research; M.T., T.L., and A.C. contributed new reagents/analytic fi tools; M.A.-F., R.S.-M., B.S.-C., P.P.G., M.S.-F., M.T., M.R.-T., and M.M. analyzed data; and traf cking and its activity remains largely unknown. M.A.-F. and M.M. wrote the paper. We show in this work that the murine Greatwall ortholog, fl encoded by the Mastl gene, is essential for mouse development The authors declare no con ict of interest. and cell cycle progression. Greatwall-null cultures, however, *This Direct Submission article had a prearranged editor. 1Present address: Madrid Institute for Advanced Studies (IMDEA)-Food, 28049 Madrid, Spain. display normal kinetics during the G2/M transition, suggesting that this protein is not required for mitotic entry. Greatwall is 2To whom correspondence should be addressed. E-mail: [email protected]. exported to the cytoplasm before nuclear envelope breakdown This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (NEB) but, interestingly, this export follows nuclear import of 1073/pnas.1310745110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1310745110 PNAS Early Edition | 1of6 Downloaded by guest on October 2, 2021 A B serum, cells were monitored by using time-lapse microscopy Mastl(+) Wild-type during 2 d (Fig. 2A). No differences were observed in the kinetics 1234 56789 101112 Mastl(+/+) (–/–) (+/–) of mitotic entry (determined by cell rounding and chromosome F1 F2 R1 Mastl(+) Mastl(loxfrt) Hypomorphic? B C 34frt-neo-frt condensation) in these cultures (Fig. 2 and ). In addition, no Flp Mastl(–) differences were detected in the accumulation of MPM2-positive Mastl(lox) Conditional 34 F1/R1 cells by immunofluorescence (Fig. 2D) or in the accumulation of Mastl(–) Cre Mastl(+) Null E Mastl( ) cyclin B1 or phospho-histone H3 (Fig. 2 ). However, Greatwall- F2/R1 3 loxP frt C Developmental stage Mastl(+/+) Mastl(+/–) Mastl(–/–) Total Live births 22 (27%) 60 (73%) 0 (0%) 82 Serum addition/ Embryos E13.5-18.5 15 (37%) 23 (56%) 0 (0%) 38 A Mastl(lox/lox) nucleofection H2B-mRFP Start video Embryos E10.5-11.5 14 (21%) 35 (51%) 0 (0%) 49 Serum deprivation (72h) 5h 48h Embryos E1.5 + 4 days i.v.c 5 (25%) 8 (40%) 7 (35%) 20 Adeno infection (48h) t=0 60h 84h post infection D pH3 ** ** E GFP Cre GFP Cre B ) 120 min 100 Greatwall (lox/lox) (20-min ( frames) 80 Actin Mastl Mastl 60 Mastl(lox/lox) 4 ) Mastl( ) 40 360 min ) 3 ( (60-min Percentage of cells Percentage ( 20 2 frames) Mastl Mastl 0 I MIM PM AT PM AT 1 PDL (rel to day 0) Mastl( ) ( )( )( ) 0 0246 C D 100 Mastl 25 3 * Days (lox/lox) Mastl(lox/lox) DAPI -tub pH3 merged Mastl( ) Mastl n.s. F 80 20 ( ) 2 60 15 (lox/lox) n.s. 40 10 Mastl 1 n.s. control) (rel. to 20 MPM2+ cells (%) 5 MPM2 levels per cell MPM2 levels per Cumulative mitotic index ) Cumulative mitotic entry (%) 0 0 0 ( 0 10 20 30 h 0 20 24 36 h Mastl (lox/lox) ( ) Time Time Mastl E Mastl(lox/lox) Mastl( ) F Mastl G Normal division Binucleated (lox/lox)( ) No segregation Death in mitosis 02024 36020 24 36 h Fig. 1. Greatwall mutant mice and cells. (A) The exon 4 of the murine gene Greatwall 100 fl Greatwall encoding Greatwall, Mastl, was anked by loxP sites (green triangles) and 75 fi P-Cdk a selection cassette [Mastl(loxfrt) allele]. Site-speci c recombination by Flp CyclinB1 substrates results in the Mastl(lox) allele, which expresses normal levels of Greatwall. 50 pH3 Cre-mediated recombination results in the germ-line [Mastl(–)] or conditionally 25 CyclinB1 of Percentage cells induced [Mastl(Δ)] null alleles. (B) Representative PCR products showing the -actin Securin 0 indicated alleles after amplification using oligonucleotides indicated in A. (lox/lox) ( )((lox/lox) ) (C) The number (and percentage) of mice or embryos with the indicated + siMad2 *** *** genotype is shown, indicating that Mastl(−/−) embryos develop normally H 600 Mastl(lox/lox) Mastl( until blastocyte stage. (D) Immunohistochemical analysis of E14.5 embryos siLuc siMad2 siLuc siMad2 fi showing an increase in mitotic (M) gures (pH3, phosphohistone H3 signal, 400 0 28000 28 28 82 h brown) versus interphasic (I) cells in the subventricular zone in Mastl(Δ/Δ) vs.
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