Andrew Burgess

Suzanne Vigneron

Estelle Brioudes

Jean-Claude Labbé

Thierry Lorca
email: thierry.lorca@crbm.cnrs.fr

Anna Castro
email: anna.castro@crbm.cnrs.fr

PP2A Greatwall

Loss of human Greatwall results in G2 arrest and multiple

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INTRODUCTION

In eukaryotic cells, the mitotic state is maintained by the mitotic kinase cyclin B-Cdc2.

Historically, mitotic entry and exit was thought to be the direct consequence of cyclin B-Cdc2 activation and inactivation respectively [START_REF] Jackman | Cyclins and the G2/M transition[END_REF]. Recent results have expanded this model to include phosphatases [START_REF] Bollen | Mitotic phosphatases: from entry guards to exit guides[END_REF]. Specifically, recent evidence indicates that PP2A is responsible for dephosphorylation of cyclin B-Cdc2 substrates and that the regulation of this dephosphorylation is required in mitotic entry and exit [START_REF] Mochida | Regulated activity of PP2A-B55 delta is crucial for controlling entry into and exit from mitosis in Xenopus egg extracts[END_REF]. This suggests a new model in which the balance between cyclin B-Cdc2 and PP2A controls mitotic entry and exit. Thus, during G2 PP2A activity is high and cyclin B-Cdc2 activity low, thereby preventing phosphorylation of mitotic substrates, whereas, at mitotic entry the balance flips allowing entry into mitosis. The mechanisms controlling cyclin B-Cdc2 activity have been largely described [START_REF] Morgan | Cyclin-dependent kinases: engines, clocks, and microprocessors[END_REF]. Briefly, cyclin B-Cdc2 is inhibited during G2 by the inhibitory phosphorylation of Cdc2 on threonine 14 and tyrosine 15 by Wee1 and Myt1 kinases. Upon mitotic entry these are removed by the Cdc25 phosphatase [START_REF] Morgan | Cyclin-dependent kinases: engines, clocks, and microprocessors[END_REF].

Finally, at mitotic exit cyclin B-Cdc2 is inhibited by the ubiquitin-dependent degradation of its regulatory subunit cyclin B [START_REF] Glotzer | Cyclin is degraded by the ubiquitin pathway[END_REF]. Unlike cyclin B-Cdc2 regulation, very little is known about the mechanisms controlling PP2A activity during mitosis, and therefore our understanding of G2 and mitosis is incomplete.

Recently, Greatwall (Gwl) a new critical mitotic regulator has been discovered in Drosophila [START_REF] Bettencourt-Dias | Genome-wide survey of protein kinases required for cell cycle progression[END_REF][START_REF] Yu | Greatwall kinase: a nuclear protein required for proper chromosome condensation and mitotic progression in Drosophila[END_REF]. It is a member of the AGC family of serine/threonine kinases that phosphorylates substrates on S/T residues encircled by basic amino acids. [START_REF] Yu | Greatwall kinase: a nuclear protein required for proper chromosome condensation and mitotic progression in Drosophila[END_REF]. Work done in Xenopus egg extracts suggested that Gwl promoted mitotic entry by controlling the auto-amplification loop of cyclin B/Cdc2 [START_REF] Yu | Greatwall kinase participates in the Cdc2 autoregulatory loop in Xenopus egg extracts[END_REF][START_REF] Zhao | Roles of Greatwall Kinase in the Regulation of Cdc25 Phosphatase[END_REF]. However it has been recently demonstrated that the main role of this kinase is not the regulation of cyclin B-Cdc2 activity but the inhibition of PP2A, the phosphatase that dephosphorylates cyclin B-Cdc2 substrates [START_REF] Castilho | The M phase kinase Greatwall (Gwl) promotes inactivation of PP2A/B55delta, a phosphatase directed against CDK phosphosites[END_REF][START_REF] Vigneron | Greatwall maintains mitosis through regulation of PP2A[END_REF].

Despite the critical roles of Gwl in mitosis, the functional human orthologue of this kinase is currently unknown. The human protein with the closest homology to Drosophila and Xenopus Gwl is the Microtubule Associated Serine/Threonine kinase Like (MAST-L) (50,2% and 65.7% of sequence homology respectively) (Figure S1). In addition to the high homology of MAST-L with the other members of the Gwl family, it also contains a very long T-loop (>500 amino acids) that separates the kinase subdomains VII and VIII, a particular feature exclusive to Gwl kinases. In contrast, although MAST-L was first classified as a member of the MAST family, it contains very little homology to any of the MAST kinases. All the members of the MAST family are large enzymes (1309-2444 amino acids) with a short T-loop (31 amino acids for MAST1), and contain a PDZ domain in the C terminus. However, MAST-L has minimal homology to MAST proteins (10,4% with MAST1), and no PDZ domain, suggesting that it is not a true member of the MAST family.

Very little is known about the role of MAST-L in human cells, with only two publications to date.

Both of these publications focus on the role of MAST-L in Autosomal Dominant

Thrombocytopenia showing that a single point mutation (E167D) in the N-terminal kinase domain correlates with this syndrome [START_REF] Gandhi | FLJ14813 missense mutation: a candidate for autosomal dominant thrombocytopenia on human chromosome 10[END_REF], while transient knockdown in zebrafish results in a reduction of circulating thrombocytes [START_REF] Johnson | In vivo inactivation of MASTL kinase results in thrombocytopenia[END_REF].

In the present study, we verified that MAST-L is the functional human orthologue of Gwl. Using siRNA knockdown of hGwl we show in human cells that this kinase mediates mitotic entry and maintains the mitotic state by inhibiting PP2A and thus, keeping the correct equilibrium between cyclin B-Cdc2 and PP2A.

RESULTS

MAST-L is the functional human homologue of Xenopus Greatwall

To analyse the role of Greatwall in human cells, we cloned the closest related human protein to the Drosophila and Xenopus Greatwall, the Microtubule Associated Serine/Threonine Kinase-Like protein (MAST-L) [START_REF] Gandhi | FLJ14813 missense mutation: a candidate for autosomal dominant thrombocytopenia on human chromosome 10[END_REF]. Our previous results demonstrated that the depletion of Greatwall from mitotic egg extracts induced the loss of the mitotic state. To check whether MAST-L corresponds to the Greatwall orthologue, we translated MAST-L in "M-phase frog egg extracts" (CSF extracts) and tested its capacity to rescue the loss of endogenous Greatwall. The expression, at endogenous levels, of wild type MAST-L in these CSF extracts completely rescued the mitotic state (Fig. S2A). However, expression of a kinase-dead mutant of MAST-L failed to rescue, indicating that MAST-L is the functional ortologue of Greatwall. Thus, from now, it will be referred to as human Greatwall (hGwl) in this manuscript.

We next analysed by western blot the expression of the endogenous human protein in Hela cells.

Our antibodies clearly recognised and specifically immunoprecipitated a band around 110 KDa, the expected molecular weight of hGwl (Fig. S2B). The levels of hGwl appeared to be constant throughout the cell cycle (Fig. 1A and Fig. S2C andD). However a lower electrophoretic mobility of this protein appeared as cells entered mitosis, was maximal in prometaphase and disappeared by anaphase (Fig. 1A). This lower mobility was induced by phosphorylation as λ-Phosphatase treatment reversed the shift (Fig. S2E).

Previously in Drosophila, Gwl was identified as a nuclear protein [START_REF] Yu | Greatwall kinase: a nuclear protein required for proper chromosome condensation and mitotic progression in Drosophila[END_REF]. However, while hGwl is mostly present in the nucleus of interphase human cells, we also noted a consistent partial localization in the cytoplasm and at the centrosomes during G1, S and G2 phases (Fig. 1B). The centrosomal hGwl appeared to surround γ-tubulin, indicating that hGW is localized around the pericentriolar material (Fig. 1C). As cells entered mitosis, hGwl concentrated at the poles of the spindle and emanated out along the microtubule spindle fibres (Fig. 1B andC). At anaphase the protein started to leave the mitotic spindle and becomes more diffuse in the cytoplasm. Finally, from telophase to cytokinesis, once the nuclear membrane reformed, most of the hGwl is localised in the nucleus, but a small fraction stayed at the cleavage furrow where it remained on the midzone microtubules although occasionally it was also observed at the mid-body (Fig. 1C). We detected a similar localization of this protein in in U2OS cells and by using two different antibodies, one against the full-length protein and the other against the last 12 C-terminal amino acids of hGwl.

hGwl knockdown promotes G2 arrest and mitotic defects in Hela cells that can be rescued by the knockdown or the inhibition of PP2A by OA It has been previously shown that depletion of Gwl in Xenopus egg extracts both prevents mitotic entry and promotes mitotic exit [START_REF] Yu | Greatwall kinase participates in the Cdc2 autoregulatory loop in Xenopus egg extracts[END_REF][START_REF] Castilho | The M phase kinase Greatwall (Gwl) promotes inactivation of PP2A/B55delta, a phosphatase directed against CDK phosphosites[END_REF][START_REF] Vigneron | Greatwall maintains mitosis through regulation of PP2A[END_REF].

To investigate the role of hGwl in human cells we induced its knockdown by siRNA in Hela cells.

The levels of hGwl decreased in a dose-dependent manner when increasing doses of the hGW siRNA were used, with an almost complete knockdown of this protein after 48h with 100 nM dose of this siRNA, both by western blot (Fig. 1D) and immunofluorescence (Fig. 1E).

We next analysed if knockdown of hGwl in cells resulted in any alteration in mitotic entry or in the maintenance of the mitotic state. Flow Cytometry analysis (FACS) after a 48 h siRNA treatment of asynchronous cells produced an accumulation (40%) of cells with a 4n DNA content and a notable increase of sub-diploid cells, indicating that the knockdown of hGwl induced G2/M cell cycle dependent defects and an increase in cell death (Fig. 1F).

Immunofluorescence analysis revealed that the accumulation of cells with 4n DNA content was not primarily due to an increase in the mitotic index (6,9% of mitotic cells from 40% of knockdown cells containing 4n DNA content), but due most likely to an increase in multinuclear cells (Fig. S3A and Fig. 1G, vii), although some of these 4n cells could be arrested in G2.

The multinuclear cells are likely the result of an improper cytokinesis, as there were numerous signs of incorrect mitotic divisions with many cells displaying DNA bridges connecting daughter cells (Fig. S3A and Fig. 1G,vi). Finally, in some cases we noticed a more severe cytokinesis phenotype with one daughter cell containing the complete DNA content whereas the other was completely devoid of chromatin (Fig. 1G,viii).

In addition to multinuclear cells, we also observed significant chromosome abnormalities in mitotic cells. Metaphase cells showed either a mass of undercondensed chromatin that was scattered along the spindle (Fig. 1G, iii) or condensed chromosomes that did not correctly congress to the metaphase plate (Fig. 1G,v). A very low number of anaphases were observed, but those that were present showed multiple lagging chromosomes and chromosome bridges (Fig. 1G, iv). No major defects were observed in the spindle although some were longer and appeared to have a lower number of microtubules (Fig. 1G, iii). These phenotypes were specific to Gwl knockdown as similar phenotypes were observed with two other siRNAs targeting different regions of hGwl mRNA (Fig. S3B andC).

The mitotic defects observed after hGwl knockdown are indicative of a premature exit from mitosis. To assess whether this was the case, time-lapse videos were performed on synchronised HeLa cells stably expressing EB3-GFP and H2B-CherryFP. Scramble siRNA treated cells progressed through mitosis normally (Fig. 2A, video S1). One of the major phenotypes observed at a 50 nM-dose of hGwl siRNA was a decrease in the total number of cells that were able to perform mitosis during the experiment (65% versus 80% in controls), suggesting that some cells were arrested at G2, similar to what has been described in Xenopus egg extracts [START_REF] Vigneron | Greatwall maintains mitosis through regulation of PP2A[END_REF]. However, most cells were still able to enter into mitosis, albeit with numerous mitotic defects. Although the mitotic spindle structure did not contain any observable defects, in some cases the spindle rotated dynamically within the cell performing several rotations (Fig. 2A, video S3, Fig. S4). Moreover, some knockdown cells showed normal formation of the metaphase plate, however chromosomes segregated incorrectly resulting in the formation of DNA bridges (Fig. 2A, "Segregation Defects" and video S2). Other cells displayed clear defects in chromosome congression showing a high number of lagging chromosomes that were constantly present at the spindle poles and never correctly congressed to the metaphase plate. Many of these cells arrested during metaphase for long periods and finally died (Fig. 2A, "Metaphase Arrest", video S3), whereas, others first delayed during metaphase and subsequently performed an aberrant anaphase with significant chromosome segregation and cytokinesis defects (Fig. 2A, "Metaphase Delay", video S4). This delay/arrest in metaphase appeared to be mediated by the SAC as the SAC proteins BubR1 and Aurora B were clearly localized at the kinetochores in hGwl depleted cells (Fig. 2B). Finally, a subpopulation of cells displayed the most severe mitotic phenotype, in which cells entered and exited mitosis prematurely without forming a metaphase plate and with severe chromosome segregation defects (Fig. 2A, "No Metaphase", video S5). In these cells a cytokinesis furrow was formed and attempted to pinch through the DNA (Fig. S5), promoting the formation of multiple DNA bridges that were resolved by the cell rejoining and the formation of a multinuclear cell (Fig. 2A, "No Metaphase", video S5).

Comparing the different phenotypes ("Segregation Defects", "Metaphase Arrest", "Metaphase Delay" and "No Metaphase") against the time spent in mitosis, we noticed that all hGwl knockdown cells had an increased transit time through mitosis. Mitotic length in the "Metaphase Arrest" and "Metaphase Delay" phenotypes was four times longer compared to those of control cells. However, mitotic length in the "Segregation Defects" and "No Metaphase Plate" phenotypes was only twice that of controls (Fig. 2C), suggesting that despite the total absence of chromosome alignment, cells displaying a "No Metaphase Plate" phenotype were unable to maintain the mitotic state. No difference was observed in spindle rotation between the various subpopulation of hGwldepleted cells, although, cells that maintained prolonged SAC activity were more likely to rotate. Thus, similar to Xenopus extracts, one of the major phenotypes of hGwl knockdown is a premature exit from mitosis. The knockdown of hGwl at a 50 nM-dose induced a variety of phenotypes probably due to the intrinsic heterogeneity of the siRNA transfection procedure. To try to separate the various phenotypes we repeated the experiment using various concentrations of hGwl siRNA (25, 50 and 100 nM). As the concentration of siRNA increased, there was a shift from cells that maintained the mitotic state (cells displaying the phenotypes "Segregation Defects", "Metaphase Arrest" and the "Metaphase Delay") towards cells incapable of maintaining mitosis (cells displaying the "No Metaphase Plate" phenotype) (Fig. 3A). Accordingly mitotic length decreased concomitantly with the increasing siRNA concentration (Fig. 3B).

Furthermore, as the dose of siRNA increased, there was a significant delay in the time of mitotic entry (Fig 3C ), suggesting that cells were arrested in G2. To assess this issue in more detail, synchronized cells were analyzed by 2 dimensional FACS (Fig. 3D). Both scramble and hGwl siRNA treated cells transited S and entered G2 phase with similar kinetics, indicating that hGwl is not critical for S phase progression. Entry into mitosis in scramble cells began at 8 h peaking at 9 h post-thymidine release. In contrast, there was a clear impairment in the ability of hGwl depleted cells to enter mitosis, with almost 3 times more (7.8% vs 20.9%) mitotic cells present in the scramble treated mitotic captured (nocodazole) 10 h time-point. Thus, at the highest dose of hGwl siRNA, cells arrest in G2.

It has been recently shown that the inhibition of the PP2A phosphastase activity is required to allow the phosphorylation of cyclin B-Cdc2 substrates, which promotes entry and maintains mitosis [START_REF] Mochida | Regulated activity of PP2A-B55 delta is crucial for controlling entry into and exit from mitosis in Xenopus egg extracts[END_REF][START_REF] Castilho | The M phase kinase Greatwall (Gwl) promotes inactivation of PP2A/B55delta, a phosphatase directed against CDK phosphosites[END_REF][START_REF] Vigneron | Greatwall maintains mitosis through regulation of PP2A[END_REF]. Therefore, we next analysed the capacity of these cells to phosphorylate the different cyclin B-Cdc2 substrates at the different doses of siRNA (Fig. 3E). As most cells treated with a dose of 25 nM of siRNA arrest in mitosis after nocodazole treatment, we only observed a slight decrease in the levels of phosphorylation of cyclin B-Cdc2 substrates. However, at the higher doses of 50 and 100 nM, the phosphorylation levels of cyclin B-Cdc2 substrates were significantly reduced. This is most likely a result of the G2 arrest, although a partial degradation of cyclin B was also observed, probably as a result of cells that entered and subsequently slipped out of mitosis. In agreement with these results, we observed only a slight decrease in the immunostaining of cyclin B-Cdc2 substrates in mitotic hGwl-depleted cells (Fig. 3F), whereas a significant decrease in this signal was observed when cells prematurely exited mitosis (note decondensed DNA in the presence of a mitotic spindle). This decrease in the phosphorylation pattern of cyclin B-Cdc2 substrates was specific to hGwl knockdown as it was mostly rescued by the concomitant transfection of a siRNA-resistant plasmid encoding Xenopus Gwl (Fig. S6).

To assess if the decrease in cyclin B-Cdc2 substrate phosphorylation after hGW knockdown could be the consequence of an increase in PP2A activity due to a loss of hGW, we performed a double hGwl-PP2A siRNA knockdown (Fig. 3G). As expected, hGwl knockdown decreased the cyclin B-Cdc2 substrate phosphorylation by 80% in nocodazole captured cells. When PP2A siRNA knockdown was performed, we noticed that the general substrate phosphorylation pattern was partially decreased compared to the scramble siRNA. This decrease is probably due to the 15% increase in cell death induced by PP2A depletion. Importantly, the double hGwl/PP2A knockdown rescued the general phosphorylation pattern of cyclin B-Cdc2 substrates from 20% to 64%. These results indicate that the hGwl knockdown phenotype is likely mediated by increased PP2A activity. However, it is possible that the knockdown of PP2A could promote a pleiotropic effect in other phases of the cell cycle [START_REF] Janssens | Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling[END_REF]. Thus, to more specifically investigate the role of Gwl in the regulation of PP2A activity at mitotic entry, we analysed if the inhibition of this phosphatase by OA could rescue the G2 arrest, the most severe phenotype observed in hGwl knockdown cells. Cells transfected with 100 nM of hGwl siRNA, were synchronised in G1/S phase, treated with OA and followed by time-lapse microscopy. The addition of OA at the end of S phase promoted a small but consistent advance in mitotic entry in scramble (SC) transfected cells (SC cells entered at an average time of 10 h 20 min after release versus 9 h 35min in OA treated cells) (Fig. 3H). As previously observed, hGwl knockdown cells delayed or arrested for a significant time in G2 (mean time of mitotic entry 12 h 12 min with significant number of cells arrested in G2 for as long as 20 hours post-release) (video S6). Importantly, OA treatment rescued the G2 delay/arrest induced by hGwl knockdown and cells entered into mitosis with similar kinetics as control cells (mean-time of mitotic entry 9 h 45 min versus 9 h 35 min in OA treated SC control cells) indicating that G2 arrest was mediated by an increased PP2A activity (video S7).

DISCUSSION

Historically, mitotic entry and exit was thought to be directly equivalent to cyclin B-Cdc2 activation and inactivation respectively, while the activity of the phosphatases required for cyclin B/Cdc2 substrate dephosphorylation during mitosis was thought to be constant. According to this hypothesis the large increase in cyclin B/cdc2 activity was sufficient to over-come the background dephosphorylation of mitotic substrates, thereby promoting mitotic entry. Similarly, inactivation of the cyclin B-Cdc2 complex by cyclin B degradation was sufficient to trigger mitotic exit.

However, recent data from our and other laboratories has demonstrated that PP2A is highly regulated during mitotic entry and exit [START_REF] Mochida | Regulated activity of PP2A-B55 delta is crucial for controlling entry into and exit from mitosis in Xenopus egg extracts[END_REF][START_REF] Castilho | The M phase kinase Greatwall (Gwl) promotes inactivation of PP2A/B55delta, a phosphatase directed against CDK phosphosites[END_REF][START_REF] Vigneron | Greatwall maintains mitosis through regulation of PP2A[END_REF]. Therefore the old cell cycle model now must be update to include a new regulator of mitosis that modulates PP2A during this phase of the cell cycle. In this work we demonstrate that Gwl is this critical and evolutionally conserved new regulator of PP2A in human cells.

Greatwall is a novel kinase whose role is essential in Xenopus egg extracts to promote mitotic entry and to maintain the mitotic state [START_REF] Yu | Greatwall kinase participates in the Cdc2 autoregulatory loop in Xenopus egg extracts[END_REF][START_REF] Castilho | The M phase kinase Greatwall (Gwl) promotes inactivation of PP2A/B55delta, a phosphatase directed against CDK phosphosites[END_REF][START_REF] Vigneron | Greatwall maintains mitosis through regulation of PP2A[END_REF]. Here we identified MAST-L as the functional human orthologue of Gwl and demonstrated that its role is evolutionary conserved from Xenopus to human cells. During mitotic division, there is a highly regulated balance between cyclin B-Cdc2 and PP2A and this balance dictates the phosphorylation level of mitotic substrates. The regulation of this balance, performed by Gwl-dependent inhibition of PP2A, is essential to allow the correct entry into and exit from mitosis. Accordingly, the phenotypes observed after hGwl knockdown likely reflect the differential phosphorylation pattern of cyclin B-Cdc2 substrates. Thus, cells lacking hGwl have a fully active PP2A that will completely dephosphorylate cyclin B-Cdc2 substrates preventing cells from entering mitosis. Cells in which hGwl is partially present show an incomplete inactivation of PP2A. If this inactivation is sufficient, then cells can enter into mitosis, but display only a partial phosphorylation of the different mitotic substrates of cyclin B-Cdc2.

Depending on the level of cyclin B-Cdc2-substrate phosphorylation, cells will display different mitotic defects such as improper chromosome congression or spindle rotation, which in some cases leads to a permanent prometaphase arrest by the SAC while in others mitotic slippage. In this regard, other reports described the presence of similar mitotic defects due to a partial inhibition of cyclin-Cdk activity [START_REF] Beamish | Cyclin a/CDK2 regulates adenomatous poylposis coli dependent mitotic spindle anchoring[END_REF][START_REF] Burgess | Inhibition of S/G2 phase CDK4 reduces mitotic fidelity[END_REF][START_REF] Potapova | The reversibility of mitotic exit in vertebrate cells[END_REF]. For example, siRNA knockdown of cyclin A results in improper chromosome congression and spindle rotation [START_REF] Beamish | Cyclin a/CDK2 regulates adenomatous poylposis coli dependent mitotic spindle anchoring[END_REF]. Moreover, the inhibition of cyclin B-Cdc2 activity in mitotic cells also induced mitotic slippage resulting in severe cytokinesis defects [START_REF] Potapova | The reversibility of mitotic exit in vertebrate cells[END_REF][START_REF] Potapova | Fine tuning the cell cycle: activation of the Cdk1 inhibitory phosphorylation pathway during mitotic exit[END_REF][START_REF] Vassilev | Selective small-molecule inhibitor reveals critical mitotic functions of human CDK1[END_REF].

The exact mechanism of how Gwl regulates PP2A activity is currently unknown, although we previously demonstrated that xGwl can bind with PP2A/A and /C [START_REF] Vigneron | Greatwall maintains mitosis through regulation of PP2A[END_REF]. PP2A itself is a complicated ternary complex comprising of a catalytic subunit (C), a scaffold subunit (subunit A) and a regulatory B subunit [START_REF] Janssens | Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling[END_REF]. The substrate specificity of the PP2A complex is thought to be conferred by the B subunit that include the B55 (B), B56 (B') and B72/130 (B''), with each also containing multiple isoforms. Recently PP2A-B55! was identified as a key phosphatase responsible for removing cyclin B/cdc2 phosphorylations (3). There are several possible phosphorylation sites in B55!, suggesting that this complex could be regulated by phosphorylation, perhaps by Gwl itself.

It is clear from our work and others that the classical cyclin/cdk cell cycle model, must be perfectly co-ordinated with the phosphatases (especially PP2A) for correct mitotic entry, progression and exit. In this regard, it has been shown that cyclin B-Cdc2 is capable of phosphorylating and activating Gwl kinase in vitro [START_REF] Yu | Greatwall kinase participates in the Cdc2 autoregulatory loop in Xenopus egg extracts[END_REF] suggesting that cyclin B-Cdc2 itself would induce PP2A inhibition through Gwl activation (Fig. 4). Thus, it is possible that at mitotic entry, cyclin B-Cdc2 is first activated and this activation would further induce Gwl phosphorylation and activation. Accordingly, our data identified a pool of Gwl localized at the centrosome during G2 phase, the same subcellular and temporal localization where cyclin B-Cdc2 is first activated [START_REF] Jackman | Active cyclin B1-Cdk1 first appears on centrosomes in prophase[END_REF]. Activated Gwl could subsequently induce PP2A inhibition permitting the correct phosphorylation of the different cyclin B-Cdc2 substrates. However, PP2A can also regulate cyclin B-Cdc2 activity, as the Wee1/Myt1 inhibitory kinases and the Cdc25 activatory phosphatase are themselves substrates of PP2A. Thus, the G2-arrest and the premature mitotic exit observed in Gwl knockdown cells are probably the result of both, a dephosphorylation of cyclin B-Cdc2 substrates and a subsequent inhibition of cyclin B-Cdc2 by Myt1/Wee1/Cdc25 dephosphorylation. Therefore, cyclin B-Cdc2 kinase cannot be activated at mitotic entry if there is not first a partial inhibition of PP2A. This inhibition of PP2A could be mediated by a partial activation of Gwl through a cyclin B-Cdc2-independent phosphorylation of Gwl. Once cyclin B-Cdc2 is activated, it could act as the starter promoting full activation of Gwl and mitotic phosphorylation. The fact that cyclin B-Cdc2 could be one of the starters that promotes mitotic entry would assure that cells enter into mitosis only when a complete activation of this kinase is achieved.

Once cells enter into mitosis, Gwl-dependent inhibition of PP2A is still required to maintain the mitotic state. We have previously demonstrated that even in the presence of a high cyclin B-Cdc2 activity the substrates of this kinase become dephosphorylated and Xenopus egg extracts slip out of mitosis when PP2A cannot be inactivated by Gwl [START_REF] Vigneron | Greatwall maintains mitosis through regulation of PP2A[END_REF]. Finally, at mitotic exit, cyclin B is degraded by the ubiquitin-dependent pathway promoting the inactivation of the cyclin B-Cdc2 complex. This inactivation results in the dephosphorylation of Gwl and the re-activation of PP2A.

Once activated, PP2A induces dephosphorylation of mitotic substrates triggering an irreversible exit of mitosis until next G2 when cyclin B will accumulate again. Interestingly to date, mitotic slippage has been solely attributed to the slow continual degradation of cyclin B [START_REF] Brito | Mitotic checkpoint slippage in humans occurs via cyclin B destruction in the presence of an active checkpoint[END_REF]. However, from our results, premature activation of PP2A or loss of Gwl during mitosis is a new mechanism that could induce mitotic slippage independently of cyclin B degradation. This could have broad implication in defining new chemotherapies for cancer treatments that target the mitotic slippage pathway.

MATERIALS AND METHODS

Chemicals, Reagents and Antibodies

MG132 and Okadaic Acid were purchase from Calbiochem (La Jolla, CA). Anti-Cyclin B1 and Cdc2 (Santa Cruz, Santa Cruz, CA), anti-Phospho-(Ser) CDK Substrate and Phospho-Cdc2-Tyr15 (Cell signalling, Denver, MA), monoclonal 1D6 against PP2AC (Millipore Temecula, CA), anti-Aurora B and BUBR1 (Becton Dickinson Franklin Lakes, NJ), and monoclonal anti-Gamma and ß-Tubulin (Sigma-Aldrich) were used. Anti-Cdc25 and Cdc27 were obtained as previously described [START_REF] Yu | Greatwall kinase: a nuclear protein required for proper chromosome condensation and mitotic progression in Drosophila[END_REF][START_REF] Vassilev | Selective small-molecule inhibitor reveals critical mitotic functions of human CDK1[END_REF][START_REF] Jackman | Active cyclin B1-Cdk1 first appears on centrosomes in prophase[END_REF][START_REF] Brito | Mitotic checkpoint slippage in humans occurs via cyclin B destruction in the presence of an active checkpoint[END_REF](22). pCMVsport6-MAST-L was obtained from RZPD Deutsches Ressourcenzentrum für Genomforschung GmbH, amplified by PCR and subcloned in the pGEX4T2 plasmid. The fusion protein was expressed in Escherichia coli.

Inclusion bodies were prepared and used to immunize rabbits. Immune serum against the fulllength protein (anti-hGwl) was affinity purified on an immobilized GST-MAST-L column.

Anti-C-terminal hGwl antibodies were also generated against the last 12 aminoacids of the Cterminal sequence of this protein. Peptides were coupled to thyroglobulin for immunization and to immobilized bovine serum albumin for affinity purification as previously described (23).

Cell synchrony

Thymidine block/release synchronizations were performed as previously described (24). For Nocodazole synchrony, cells were first released from G1/S Thymidine block, and treated with 100 ng/ml of Nocodazole for 16 h. Floating cells were then removed, washed 3 times and seeded back in fresh media.

siRNA Design and Transfection

The Promega (Madison, WI) T7 Ribomax Express RNAi system was used to produce in vitro transcribed siRNAs against human Greatwall and PP2Ac. The following target sequences were identified and appropriate sense and anti-sense oligonucleotides were synthesized. For human Greatwall positions from #1=389-409, #2=840-860 and #3=1516-136. For PP2Ac, position #169-187, which targeted both " and ß isoforms. The following Scramble (SC) sequence was used SC=GGATTGTGCGGTCATTAAC, as a control. Transfection of siRNA was performed using Lipofectamine RNAiMax Reagent (Invitrogen) as per the manufacture's instructions. Rescue experiments were performed as per Figure S6. Briefly, synchronized HeLa cells were first transfected with hGwl siRNA sequence #2 and subsequently transfected with a plasmid (PCS2) encoding full length Xenopus Gwl. Cells were synchronized, captured in mitosis with nocodazole, and analyzed by western blot.

Immunofluorescent Staining

Cells were grown on poly-lysine coated glass coverslips and pre-permeabilised for 30 seconds in PHEM buffer (60 mM PIPES, 25 mM HEPES, 1 mM EGTA, 2 mM MgCl2) + 0.25% Triton X-100. Then cells were fixed for 10 min in PHEM buffer with 3.7% formaldehyde and 0.5% Triton X-100, washed and blocked (3% BSA, 0,1% tween 20 in PBS) for 30 min. Primary antibodies were incubated for 2 h at RT in blocking solution. Images were captured using a Leica DM6000 microscope coupled with a Coolsnap HQ2 camera, using a Leica 63X APO 1.4 lens, powered by metamorph 7.1 software. For mitotic cells 0.3 m Z-sections were taken, and deconvoluted using Huygens 3.0 software. Maximum projections and total fluorescence measurements were performed with Image J, metaphase plate measurements and false coloring were performed using Photoshop CS4 Extended software.

Live cell imaging

Thymidine synchronized cells were filmed with a Micromax YHS 1300 camera coupled to a Zeiss Axiovert 200M inverted microscope fitted with a Zeiss slider PlasDIC 32X LD A plan NA 0.4 lens, Zeiss mercury lamp, and controlled by Metamorph 7.1 software (Molecular Devices, Sunnyvale, CA, USA). Images, both phase-contrast and GFP/CherryFP fluorescence, were taken every 10-15 min. The resulting images were processed, analyzed and false colored using Photoshop CS4 Extended software. Box Graphs and statistical analysis was performed using GraphPad Prism 5 (La Jolla, CA). Mitotic entry, length, exit, and cell death were determined as described in Figure S7. Briefly, the following parameters were used to identify mitotic entry: first appearance of a mitotic spindle with condensed chromosomes, loss of a nucleolus and rounding up of the cell from the culture plate. Exit was scored based on the first appearance of anaphase, thus visualization of chromosome separation, pinching of the membrane and appearance of a mid-body.

Figure 1 :Figure 2 :

 12 Figure 1: hGwl siRNA knockdown induces multiple mitotic defects (A) HeLa cells were synchronized by either Thymidine (Thy) or Nocodazole (Noc) shake-off, and

Figure 3 :

 3 Figure 3: hGwl knockdown phenotypes can be rescued by the inactivation of PP2A

Figure 4 :

 4 Figure 4: Hypothetical model showing the different steps required for mitotic entry

ACKNOWLEDGMENTS

We thank Dr. J. Ellenberg and Dr. V Doye for the generous gift of the Hela EB3/H2B cell line, to Montpellier RIO Imaging for microscopy facilities and B. Gabrielli and K Rogowski for helpful comments on the manuscript. This work was supported by the Ligue Nationale Contre le Cancer (Equipe Labellisée, 2004-2008). A.B. and E.B. are "Fondation pour la Recherche Medicale" and "Ligue Nationale Contre le Cancer" fellows respectively. ABBREVIATIONS Gwl: Greatwall, MAST-L: Microtubule Associated Serine/Threonine Kinase Like, SAC: Spindle Assembly Checkpoint, FACS: Flow Cytometry Analysis, PP2A: Protein Phosphatase 2A, OA: Okadaic Acid, CSF: M-phase frog egg extracts, SC: Scramble.

activation of the cyclin B-Cdc2 amplification loop. Once activated cyclin B-Cdc2 will promote the complete phosphorylation of Gwl and the total inhibition of PP2A. Cyclin B-Cdc2 also promotes the phosphorylation of all the other mitotic substrates that now cannot be dephosphorylated by PP2A and therefore cells enter mitosis. Once in mitosis, PP2A activity must be inhibited to ensure that cyclin B-Cdc2 substrates remain phosphorylated long enough to performe mitosis correctly. This inhibition is assured by Gwl-dependent phosphorylation, which itself is maintained by cyclin B-Cdc2 activity. Finally, at metaphase-anaphase transition the ubiquitin-dependent degradation of cyclin B results in a loss of Cdc2 activity triggering Gwl dephosphorylation and inactivation, which then allows re-activation of PP2A.

Once activated, PP2A will promote the irreversible dephosphorylation of mitotic substrates and cell will exit mitosis until next G2 phase in when cyclin B is synthesised again. Black arrows denote pathways active during mitotic entry. Dotted arrow denotes pathways that are inhibited at mitotic entry, during mitosis or at mitotic exit.


Sylvain Contassot-Vivier
email: sylvain.contassotvivier@loria.fr

Thomas Jost
email: thomas.jost@loria.fr

Stéphane Vialle
email: stephane.vialle@supelec.fr

Impact of asynchronism on GPU accelerated parallel iterative computations

Keywords: Parallelism, GPGPU, Asynchronism, Scientific computing

We study the impact of asynchronism on parallel iterative algorithms in the particular context of local clusters of workstations including GPUs. The application test is a classical PDE problem of advection-diffusion-reaction in 3D. We propose an asynchronous version of a previously developed PDE solver using GPUs for the inner computations. The algorithm is tested with two kinds of clusters, a homogeneous one and a heterogeneous one.

Introduction

Scientific computing generally involves a huge amount of computations to obtain accurate results on representative data sets in reasonable time. This is why it is important to take as much advantage as possible of any new device which can be used in the parallel systems and bring a sensible gain in performances. In that context, one of our previous works was focused on the use of clusters of GPUs for solving PDEs [START_REF] Jost | On the interest of clusters of gpus[END_REF]. The underlying scheme is a twostage iterative algorithm in which the inner linear computations are performed on the GPUs [START_REF] Jost | An efficient multialgorithms sparse linear solver for GPUs[END_REF]. Important gains were obtained both in performance and energy consumption. In the meantime, we also showed in our works related to asynchronism in parallel iterative algorithms [START_REF] Bahi | Parallel Iterative Algorithms: from sequential to grid computing[END_REF][START_REF] Bahi | Evaluation of the asynchronous iterative algorithms in the context of distant heterogeneous clusters[END_REF] that this algorithmic scheme could be very interesting in some specific combinations of parallel system and algorithm. Moreover, we also identified the context in which this algorithmic scheme is advantageous compared to the synchronous one. As asynchronism allows an efficient and implicit overlapping of communications by computations, it is especially well suited to contexts where there is a sensible ratio of communication time according to the computation time. This is for example the case in large local clusters or grids where communications through the system are expensive compared to local accesses.

Our motivation for conducting the study presented in this paper comes from the fact that a local cluster of GPUs represents a similar context of costly communications according to computations. Indeed, the cost of data transfers between the GPU memory and the CPU memory inside each machine is added to the classical cost of local communications between the machines. So, we propose in this work to study the interest of using asynchronism in our PDE solver in that specific context.

The test application used for our experiments is the classical advection-diffusion-reaction problem in a 3D environment and two chemical species (see [START_REF] Bahi | Synchronous and asynchronous solution of a 3D transport model in a grid computing environment[END_REF] for further details). Two series of experiments have been performed, one with a heterogeneous cluster with two couples of CPU-GPU, and another one with a homogeneous cluster.

The following section presents the algorithmic scheme of our iterative PDE solver together with the implementation sketch of the asynchronous version. Then, the experiments are presented and the results are discussed in Section 3.

Asynchronous PDE Solver

It is quite obvious that over the last few years, the classical algorithmic schemes used to exploit parallel systems have shown their limit. As the most recent systems are more and more complex and often include multiple levels of parallelism with very heterogeneous communication links between those levels, one of the major drawbacks of the previous schemes has become their synchronous nature. Indeed, synchronizations may sensibly degrade performances in large or hierarchical systems, even for local systems.

For a few years now, asynchronous algorithmic schemes have emerged, and although they cannot be used for all problems, they are efficiently usable for a majority of them. In scientific computing, it can be expressed only in iterative algorithms. Although those methods are generally slower than direct ones, they are often the only known way to solve some problems and they are also less memory consuming.

The asynchronous feature consists in suppressing any idle time induced by the waiting for the dependency data to be exchanged between the computing units of the par-allel system. Hence, each unit performs the successive iterations on its local data with the dependency data versions it owns at the current time. The main advantage of this scheme is to allow an efficient and implicit overlapping of communications by computations. On the other hand, the major drawbacks of asynchronous iterations are: a more complex behavior which requires a specific convergence study, and a larger number of iterations to reach convergence. However, the convergence conditions in asynchronous iterations are verified for numerous problems and, in many computing contexts, the time overhead induced by the additional iterations is largely compensated by the gain in the communications [START_REF] Bahi | Evaluation of the asynchronous iterative algorithms in the context of distant heterogeneous clusters[END_REF][START_REF] Bahi | Asynchronism for iterative algorithms in a global computing environment[END_REF]. In fact, as partly mentioned in the introduction, as soon as the frequency of communications relatively to computations is high enough and the communication costs are larger than local accesses, an asynchronous version may provide better performances than a synchronous version.

Multisplitting-Newton algorithm

There are several methods to solve PDE problems, each of them including different degrees of synchronism/asynchronism. The method used in this study is the multisplitting-Newton which allows for a rather important level of asynchronism. In that context, we use a finite difference method to solve the PDE system. Hence, the system is linearized, a regular discretization of the spatial domain is used and the Jacobian matrix of the system is computed at the beginning. The Euler equations are used to approximate the derivatives. The algorithmic scheme of the method is as follows:

• Rewriting of the problem under a fixed point problem formulation:

x = T (x), x ∈ R where T (x) = x -F ′ (x) -1 F(x) and F ′ is the Jacobian • We get F ′ × ∆X = -F with F ′ a sparse matrix (in most cases)
• F ′ and F are distributed over the computing units

• Each unit computes a different part of ∆X using the quasi-Newton algorithm over its sub-domain as can be seen in Fig. 1 • The local elements of X are directly updated with the local part of ∆X

• The non-local elements of X come from the other units using messages exchanges

• F is updated by using the entire vector X

Inner linear solver

The method described above is a two-stage algorithm in which a linear solver is needed in the inner stage. In fact, most of the time of the algorithm is spent in that linear solver. This is why we chose to use the most powerful elements of the parallel system on that part. Thus, the linear computations have been placed on the GPUs. Due to their regularity, those treatments are very well suited to the SIMD architecture of the GPU. Hence, on each computing unit, the linear computations required to solve the partial system are performed on the local GPU while all the algorithmic control, non-linear computations and data exchanges between the units are done on the CPU.

¢ 0 0 0 0 F Loc ∆X F Loc
The linear solver has been implemented both on CPU and GPU, using the biconjugate gradient algorithm (see [START_REF] Jost | An efficient multialgorithms sparse linear solver for GPUs[END_REF] for further details). This linear solver was chosen because it performs well on non-symmetric matrices (on both convergence time and numerical accuracy), it has a low memory footprint, and it is relatively easy to implement.

GPU implementation

Several aspects are critical in a GPU: the regularity of the computations and the memory which is of limited amount and the way the data are accessed. In order to reduce the memory consumption of our sparse matrix, we have used a compact representation, depicted in Fig. 2, similar to the DIA (diagonal) format in BLAS, but with several additional advantages. The first one is the regularity of the structure which allows us to do coalesced memory accesses most of the time. The second one is that it provides an efficient access to the transpose of the matrix, which is required in the biconjugate gradient method. In order to be as efficient as possible, the shared memory has been used as a cache memory whenever it was possible in order to avoid the slower accesses to the global memory of the GPU. The different kernels used in the solver are divided to reuse as much data as possible at each call, hence minimizing transfers between the global memory and the registers. To get full details on those kernels, the reader should refer to [START_REF] Jost | An efficient multialgorithms sparse linear solver for GPUs[END_REF].

Asynchronous aspects

Since the size of the simulation domain can be huge, the domain is distributed among several nodes of a cluster. Each node solves a part of the resulting linear system and sends the relevant data to the other units that need them. In the asynchronous version, this is that part which is performed asynchronously. One synchronization is still required between each time step of the simulation, as illustrated in Fig. 3. At the practical level, the main differences with the synchronous version lie in the suppression of some barriers and in the way the communications between the units are managed. Concerning the first aspect, all the barriers between the inner iterations inside each time step of the simulation are suppressed. The only remaining synchronization is the one between each time step as pointed out above.

Simulation

The communications management is a bit more complex than in the synchronous version as it must enable sending and receiving operations at any time during the algorithm. Although the use of non-blocking communications seems appropriate, it is not sufficient, especially concerning receptions. This is why a multi-threaded programming is required. The principle is to use separated threads to perform the communications, while the computations are continuously done in the main thread without any interruption, until convergence detection. In our version, we used nonblocking sends in the main thread and an additional thread to manage the receptions. It must be noted that in order to be as reactive as possible, some communications may be initiated by the receiving thread (for example to send back the local state of the unit).

Subsequently to the multi-threading, the use of mutex is necessary to protect the accesses to data and some variables in order to avoid concurrency and potentially incoherent modifications.

Another difficulty brought by the asynchronism comes from the detection of the convergence. Some specific mechanisms must replace the simple global reduction of local states of the units to ensure the validity of the detection [START_REF] Bahi | An efficient and robust decentralized algorithm for detecting the global convergence in asynchronous iterative algorithms[END_REF]. The most general scheme may be too expensive in some simple contexts such as local clusters. So, when some information about the system are available (for example bounded communication delay), it is often more pertinent to use a simplified mechanism whose efficiency is better and whose validity is still ensured in that context. Although both general and simplified schemes have been developed for this study, the performances presented in the following section are related to the simplified scheme which gave the best results.

Experimental results

The platform used to conduct our experiments is a set of two clusters hosted by SUPELEC in Metz. The first one is composed of 15 machines with Intel Core2 Duo CPUs running at 2.66GHz, 4GB of RAM and one Nvidia GeForce 8800GT GPU with 512MB per machine. The operating system is Linux Fedora. The second cluster is composed of 17 machines with Intel Nehalem CPUs (4 cores + hyperthreading) running at 2.67GHz, 6GB RAM and one Nvidia GeForce GTX 285 with 1GB per machine. The OS is the same as the previous cluster. Concerning the interconnection network, both clusters use a Gigabit Ethernet network. Moreover, they are connected to each other and can be used as a single heterogeneous cluster via the OAR management system.

In that hardware context, two series of experiments seemed particularly interesting to us. The first one consists in running our application for several problem sizes on one of the homogeneous clusters. We chose the most recent one, with the Nehalem CPUs and GTX 285 GPUs. The second series of experiments is similar to the first one except that instead of using only one cluster, we used the two clusters to obtain a heterogeneous system with 32 nodes.

The results are respectively presented in Table 1 and Table 3. The problem size indicated in the left column corresponds to the number of elements for each dimension of the spatial domain (3D). Thus, for a size of 50, there are 50 3 elements, and as we have two chemical species, the global linear system is a square matrix with 2 × 50 3 lines and columns. Fortunately, the local nature of dependencies in the advection-diffusion-reaction problem implies that only 9 diagonals in that matrix are non-zero. The results obtained in that context are interesting but not as good as could be expected. The decrease of the gain when the problem size increases is quite natural as the ratio of communications according to the computations decreases and the impact of synchronizations becomes less preponderant over the overall performances. However, the rather limited maximal gain is a bit deceiving. In fact, it can be explained, at least partially, by the very high performance network used in the cluster, the rather small amount of data exchanged between the nodes and the homogeneity of the nodes and loads. In such a context, it is clear that the synchronous communications are not so expensive compared to the extra iterations required by the asynchronous version. Also, it can be deduced that although the GPU ↔ CPU data transfers play a role in the overall performances, their impact on our PDE solver is less important than one could have thought at first glance.

Two additional experiments have been done with the same cluster but with less processors in order to observe the behavior of our PDE solver when the number of processors varies. The results are provided in Table 2 Those results confirm the general trend of gain decrease when the problem size increases. It can also be observed that for smaller clusters, the limit of gain brought by asynchronism is reached sooner. And finally, there are some fluctuations in the gains which denote a complex behavior of this kind of algorithm according to the context of use. This would require a deeper study to identify the frontier of gain between synchronism and asynchronism in function of the number of processors and the problem size.

Concerning the second context of use, the heterogeneous cluster, the results presented in Table 3 In fact, the heterogeneity of the machines should imply different computation speeds and the synchronizations should induce a global slow down imposed by the slowest machine. Nevertheless, the results tend to show that the difference in the powers of the machines is not large enough to induce a sufficiently sensible unbalancing between them. Moreover, it clearly appears that the overhead of the asynchronism in terms of iterations is rapidly more important than the gain in the communications, leading to a loss in performances.

Also, another point that may explain the degraded performances of the asynchronous version in the heteroge-neous cluster is that the GPU cards used in the older cluster do not support double precision real numbers. Thus, the program is compiled to use only single precision numbers, which divides the data size by a factor two and then also the communications volumes, reducing even more the impact of the communications on the overall execution times.

Conclusion

Two versions of a PDE solver algorithm have been implemented and tested on two clusters of GPUs. The conclusion that can be drawn concerning the interest of asynchronism in such a context of parallel system for that kind of application is mitigated. Some gains can be observed but they are rather limited. Moreover, the asynchronous version is not always better than the synchronous one, denoting a combination application-system not completely suited to that kind of algorithm.

However, as far as we know, that study is among the very firsts of its kind and it shows that this subject requires further works. For example, an interesting topic would be to precisely identify the areas in which one of the operating modes (sync or async) is better suited than the other to a given context of number of processors and problem size. In addition, using load-balancing in that context should also improve performances of both versions.

Figure 1 :

 1 Figure 1: Local computations associated to the subdomain of one unit

Figure 2 :

 2 Figure 2: Compact and regular sparse matrix representation

Figure 3 :

 3 Figure 3: Asynchronous iterations inside each time step of the computation

Table 1 :

 1 Execution times (in seconds) with the homogeneous cluster (17 machines).

	Pb size	Sync	Async	Speed up Gain (%)
	50	16.52	14.85	1.11	10.10
	100	144.52	106.09	1.36	26.59
	150	392.79	347.40	1.13	11.55
	200	901.18	866.31	1.04	3.87
	250	1732.60 1674.30	1.03	3.36

Table 2 :

 2 . Execution times (in seconds) with 14 and 9 homogeneous machines.

		14 Machines of the newer cluster
	Pb size	Sync	Async	Speed up Gain (%)
	50	20.95	17.83	1.17	14.89
	100	182.85	132.35	1.38	27.62
	150	486.69	442.16	1.10	9.15
	200	1101.29 1029.61	1.07	6.51
		9 Machines of the newer cluster
	Pb size	Sync	Async	Speed up Gain (%)
	50	39.68	25.81	1.54	34.95
	100	249.63	200.25	1.25	19.78
	150	714.85	635.78	1.12	11.06
	200	1599.01 1617.28	0.99	-1.14

Table 3 :

 3 are very surprising. Execution times (in seconds) with the heterogeneous cluster (15 + 17 machines).

	Pb size	Sync	Async Speed up Gain (%)
	100	53.21	52.01	1.02	2.25
	150	155.13 164.05	0.94	-5.75
	200	322.11 395.11	0.81	-22.66