Greatwall is essential to prevent mitotic collapse after nuclear envelope breakdown in mammals

Abstract

Greatwall is a protein kinase involved in the inhibition of protein phosphatase 2 (PP2A)-B55 complexes to maintain the mitotic state. Although its biochemical activity has been deeply characterized in Xenopus, its specific relevance during the progression of mitosis is not fully understood. By using a conditional knockout of the mouse ortholog, Mastl, we show here that mammalian Greatwall is essential for mouse embryonic development and cell cycle progression. Yet, Greatwall-null cells enter into mitosis with normal kinetics. However, these cells display mitotic collapse after nuclear envelope breakdown (NEB) characterized by defective chromosome condensation and prometaphase arrest. Intriguingly, Greatwall is exported from the nucleus to the cytoplasm in a CRM1-dependent manner before NEB. This export occurs after the nuclear import of cyclin B-Cdk1 complexes, requires the kinase activity of Greatwall, and is mediated by Cdk-, but not Polo-like kinase 1-dependent phosphorylation. The mitotic collapse observed in Greatwall-deficient cells is partially rescued after concomitant depletion of B55 regulatory subunits, which are mostly cytoplasmic before NEB. These data suggest that Greatwall is an essential protein in mammals required to prevent mitotic collapse after NEB.

Keywords: cell cycle regulation; cell division; mitotic kinases; mitotic phosphatases; nuclear export.

Fig. 1.

Greatwall mutant mice and cells. (A) The exon 4 of the murine gene encoding Greatwall, Mastl, was flanked by loxP sites (green triangles) and a selection cassette [Mastl(loxfrt) allele]. Site-specific recombination by Flp results in the Mastl(lox) allele, which expresses normal levels of Greatwall. Cre-mediated recombination results in the germ-line [Mastl(–)] or conditionally induced [Mastl(Δ)] null alleles. (B) Representative PCR products showing the indicated alleles after amplification using oligonucleotides indicated in A. (C) The number (and percentage) of mice or embryos with the indicated genotype is shown, indicating that Mastl(−/−) embryos develop normally until blastocyte stage. (D) Immunohistochemical analysis of E14.5 embryos showing an increase in mitotic (M) figures (pH3, phosphohistone H3 signal, brown) versus interphasic (I) cells in the subventricular zone in Mastl(Δ/Δ) vs. Mastl(+/Δ). PM figures are indicated by arrows, whereas AT are indicated by arrowheads. **P < 0.01. (E) Transduction with Cre-expressing adenoviruses (AdCre) results in the depletion of Greatwall in primary MEFs compared with control cells transduced with adenoviral vectors expressing GFP. β-actin was used as a loading control. Genetic ablation of Greatwall impairs proliferation of primary MEFs. Mean ± SD for each time point is shown in the plot. PDL, population doubling levels. (F) Representative immunofluorescence images of mitotic cells stained with DAPI (blue), α-tubulin (green), and pH3 (red). (Scale bars: 10 μm.)

Fig. 2.

Lack of Greatwall does not inhibit mitotic entry but prevents chromosome segregation. (A) Schematic representation of the protocol used for genetic ablation of Greatwall. Confluent cultures of immortalized Mastl(lox/lox) MEFs expressing a mRFP-tagged histone H2B (H2B-mRFP) were serum deprived and transduced with Flp (AdFlp)- or Cre (AdCre)-expressing adenoviruses, giving rise to Mastl(lox/lox) or Mastl(Δ/Δ) cells, respectively. These cells were stimulated with serum and monitored 5 h later by using time-lapse microscopy during an additional 48 h. (B) Representative time-lapse images of Mastl(lox/lox) and Mastl(Δ/Δ) cells. H2B-mRFP is in red. (C) Quantification of mitotic entry (as scored by cell rounding and chromosome condensation) in these cultures after visual analysis of these time-lapse images. (D) Quantification of the cumulative percentage of cells positive for the mitotic marker MPM2 (Left) or levels of MPM2 per mitotic cell (Right) in these cultures. (E) Level of the indicated proteins in these cultures after the addition of serum to quiescent cells (t = 0). (F) Levels of the indicated antigens in Greatwall-null or control mitotic cells after treatment with taxol and mitotic shake-off. (G) Quantification of mitotic aberrations in Mastl(lox/lox) and Mastl(Δ/Δ) cells in the absence or presence of short interfering RNAs against Mad2 (siMad2) or luciferase (siLuc). (H) Duration of mitosis (from NEB until mitotic exit, based on DNA decondensation and loss of rounded morphology) in Greatwall-deficient and control cells in the absence or presence of siMad2 or siLuc. Error bars indicate SD. The depletion of Greatwall or Mad2 in these cultures is shown after immunoblot. n.s., not significant differences (P > 0.05; Fisher's exact test); *P < 0.05; ***P < 0.001 (Student t test).

Fig. 3.

Greatwall is exported to the cytoplasm before NEB in a Cdk-dependent manner. (A) Micrographs represent mitotic entry in U2OS cells stably expressing GFP-Gwl (green) and CFP-tagged lamin A (blue). Quantification of the cytoplasmic Greatwall signal before NEB. (B) Mitotic entry in cells stably expressing GFP-Gwl (green) and mCherry-tagged cyclin B1 (red). Quantification of cytoplasmic Greatwall and nuclear cyclin B1. Fluorescence mean intensity was set as 100% at NEB (time = 0). (C) Greatwall displays a normal export in the presence of the Plk1 inhibitor BI2536 (100 nM). (D) Quantification of cytoplasmic export (as indicated in B) of a Greatwall mutant harboring the T194A and T207A mutations (Cdk phosphorylation sites). (E) Quantification of cytoplasmic export of a Greatwall kinase-dead mutant harboring a mutation in the ATP-binding site (D156A). (F) Comparison of the dynamic changes in the subcellular localization of Greatwall in the different mutants or treatments. All graphs show means ± SD of at least 10 cells per condition.

Fig. 4.

Greatwall prevents PP2A–B55-dependent defects in chromosome condensation after nuclear envelope breakdown. (A) Immunodetection of the indicated antigens in Mastl(lox/lox) and Mastl(Δ/Δ) cells at the indicated time points after serum stimulation following the scheme represented in Fig. 2. Cells were treated with short interfering RNAs against the B55 subunits α, β, γ, and δ (siB55) or luciferase (siLuc) sequences as indicated. (B) Relative levels of phospho-Cdk substrates at 24 h, normalized versus Mastl(lox/lox) control cultures. (C) Quantification of the depletion of the most abundant B55 isoforms in MEFs (B55α and δ) by real-time RT-PCR after transfection with siLuc or siB55 oligonucleotides. (D) Representative images of metaphase spread from the indicated cultures. Condensation defects were determined based on the abnormal length of mitotic chromosomes and classified based on the number of affected chromosomes per metaphase, as mild (5–10 chromosomes) or severe (more than 10 chromosomes). At least 50 metaphases were counted per condition. (Scale bars: 10 µm; Insets, 1 µm.) (E) Mastl depletion and phosphorylation of cyclin B1 at S126 residue (pS126) was monitored by immunoblot. The level of depletion of the different B55 isoforms was analyzed by real-time RT-PCR 24 h after transfection with siLuc or siB55 oligonucleotides.

Fig. 5.

Greatwall nuclear export prevents mitotic defects. (A) Greatwall-knockout cells were complemented with Greatwall variants (NLS or H2B-fusions) or the wild-type protein (wt). Cells were synchronized as indicated in Fig. 2A, and expression levels were monitored by immunoblot. Micrographs represent the localization of Gwl-H2B during mitosis (time 0 = metaphase). Analysis of duration of mitosis (B) and mitotic cell fate in those cultures showing a partial rescue by the wild-type (wt) and NLS fusion forms of Greatwall, but not in the H2B-fusion (C). *P < 0.05; ***P < 0.001; n.s., not significant. (D) In normal cell cycles, activation and nuclear import of CycB/Cdk1 complexes leads to the phosphorylation of substrates (change from blue to reddish colors), thus triggering Greatwall export to the cytoplasm, where it inhibits PP2A–B55 complexes just before NEB (Upper). A defective inhibition of PP2A in early mitosis would cause a defective phosphorylation of Cdk substrates upon NEB, leading to the mitotic collapse observed in Greatwall-null cells (Lower).