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Although the role of the b-cell lymphoma (Bcl)-2 family of apoptosis inhibitors is well documented in tumor cells and tissue morphogenesis, their role during the early development of vertebrates is unknown. Here, we characterize Nrz, a new Bcl-2-related inhibitor of apoptosis in zebrafish. Nrz is a mitochondrial protein, antagonizing the death-accelerator Bax. The nrz gene is mainly expressed during gastrulation and somitogenesis. The knockdown of nrz with antisense morpholinos leads to alterations of the somites, correlated with an increase in apoptosis. In addition, earlier during development, in the zebrafish gastrula, nrz knockdown results in an increase of snail-1 expression at the margin and frequent gastrulation arrest at the shield stage, independently of apoptosis. Together these data suggest that Nrz, in addition to its effect on apoptosis, contributes to cell movements during gastrulation by negatively regulating the expression of Snail-1, a transcription factor that controls cell adhesion.Cell Death and Differentiation (2006) 13, 1128–1137. doi:10.1038/sj.cdd.4401797; published online 11 November 2005 [ABSTRACT FROM AUTHOR]

Entry into mitosis has been classically attributed to the activation of a cyclin B/Cdk1 amplification loop via a partial pool of this kinase becoming active at the end of G2 phase. However, how this initial pool is activated is still unknown. Here we discovered a new role of the recently identified PP2A-B55 inhibitor FAM122A in triggering mitotic entry. Accordingly, depletion of the orthologue of FAM122A in C. elegans prevents entry into mitosis in germline stem cells. Moreover, data from Xenopus egg extracts strongly suggest that FAM122A-dependent inhibition of PP2A-B55 could be the initial event promoting mitotic entry. Inhibition of this phosphatase allows subsequent phosphorylation of early mitotic substrates by cyclin A/Cdk, resulting in full cyclin B/Cdk1 and Greatwall (Gwl) kinase activation. Subsequent to Greatwall activation, Arpp19/ENSA become phosphorylated and now compete with FAM122A, promoting its dissociation from PP2A-B55 and taking over its phosphatase inhibition role until the end of mitosis., (© 2024. The Author(s).)

During eukaryotic cell division a microtubule-based structure, the mitotic spindle, aligns and segregates chromosomes between daughter cells. Understanding how this cellular structure is assembled and coordinated in space and in time requires measuring microtubule dynamics and visualizing spindle assembly with high temporal and spatial resolution. Visualization is often achieved by the introduction and the detection of molecular probes and fluorescence microscopy. Microtubules and mitotic spindles are highly conserved across eukaryotes; however, several technical limitations have restricted these investigations to only a few species. The ability to monitor microtubule and chromosome choreography in a wide range of species is fundamental to reveal conserved mechanisms or unravel unconventional strategies that certain forms of life have developed to ensure faithful partitioning of chromosomes during cell division. Here, we describe a technique based on injection of purified proteins that enables the visualization of microtubules and chromosomes with a high contrast in several divergent marine embryos. We also provide analysis methods and tools to extract microtubule dynamics and monitor spindle assembly. These techniques can be adapted to a wide variety of species in order to measure microtubule dynamics and spindle assembly kinetics when genetic tools are not available or in parallel to the development of such techniques in non-model organisms., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cells require major physical changes to induce a proper repartition of the DNA. Nuclear envelope breakdown, DNA condensation and spindle formation are promoted at mitotic entry by massive protein phosphorylation and reversed at mitotic exit by the timely and ordered dephosphorylation of mitotic substrates. This phosphorylation results from the balance between the activity of kinases and phosphatases. The role of kinases in the control of mitosis has been largely studied, however, the impact of phosphatases has long been underestimated. Recent data have now established that the regulation of phosphatases is crucial to confer timely and ordered cellular events required for cell division. One major phosphatase involved in this process is the phosphatase holoenzyme PP2A-B55. This review will be focused in the latest structural, biochemical and enzymatic insights provided for PP2A-B55 phosphatase as well as its regulators and mechanisms of action., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lacroix, Lorca and Castro.)

Clathrin-mediated endocytosis (CME) is a central trafficking pathway in eukaryotic cells regulated by phosphoinositides. The plasma membrane phosphatidylinositol-4,5-bisphosphate (PI(4,5)P 2 ) plays an instrumental role in driving CME initiation. The F-BAR domain-only protein 1 and 2 complex (FCHo1/2) is among the early proteins that reach the plasma membrane, but the exact mechanisms triggering its recruitment remain elusive. Here, we show the molecular dynamics of FCHo2 self-assembly on membranes by combining minimal reconstituted in vitro and cellular systems. Our results indicate that PI(4,5)P 2 domains assist FCHo2 docking at specific membrane regions, where it self-assembles into ring-like-shaped protein patches. We show that the binding of FCHo2 on cellular membranes promotes PI(4,5)P 2 clustering at the boundary of cargo receptors and that this accumulation enhances clathrin assembly. Thus, our results provide a mechanistic framework that could explain the recruitment of early PI(4,5)P 2 -interacting proteins at endocytic sites., Competing Interests: FE, IC, DS, CA, RR, VB, AC, TL, JV, SV, AC, LP No competing interests declared, (© 2022, El Alaoui et al.)

PP2A is a major serine/threonine phosphatase class involved in the regulation of cell signaling through the removal of protein phosphorylation. This class of phosphatases is comprised of different heterotrimeric complexes displaying distinct substrate specificities. The present review will focus on one specific heterocomplex, the phosphatase PP2A-B55. Herein, we will report the direct substrates of this phosphatase identified to date, and its impact on different cell signaling cascades. We will additionally describe its negative regulation by its inhibitors Arpp19 and ENSA and their upstream kinase Greatwall. Finally, we will describe the essential molecular features defining PP2A-B55 substrate specificity that confer the correct temporal pattern of substrate dephosphorylation. The main objective of this review is to provide the reader with a unique source compiling all the knowledge of this particular holoenzyme that has evolved as a key enzyme for cell homeostasis and cancer development., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Arpp19 is a potent PP2A-B55 inhibitor that regulates this phosphatase to ensure the stable phosphorylation of mitotic/meiotic substrates. At G2-M, Arpp19 is phosphorylated by the Greatwall kinase on S67. This phosphorylated Arpp19 form displays a high affinity to PP2A-B55 and a slow dephosphorylation rate, acting as a competitor of PP2A-B55 substrates. The molecular determinants conferring slow dephosphorylation kinetics to S67 are unknown. PKA also phosphorylates Arpp19. This phosphorylation performed on S109 is essential to maintain prophase I-arrest in Xenopus oocytes although the underlying signalling mechanism is elusive. Here, we characterize the molecular determinants conferring high affinity and slow dephosphorylation to S67 and controlling PP2A-B55 inhibitory activity of Arpp19. Moreover, we show that phospho-S109 restricts S67 phosphorylation by increasing its catalysis by PP2A-B55. Finally, we discover a double feed-back loop between these two phospho-sites essential to coordinate the temporal pattern of Arpp19-dependent PP2A-B55 inhibition and Cyclin B/Cdk1 activation during cell division.

Protein phosphorylation is a post-translational modification essential for the control of the activity of most enzymes in the cell. This protein modification results from a fine-tuned balance between kinases and phosphatases. PP2A is one of the major serine/threonine phosphatases that is involved in the control of a myriad of different signaling cascades. This enzyme, often misregulated in cancer, is considered a tumor suppressor. In this review, we will focus on PP2A-B55, a particular holoenzyme of the family of the PP2A phosphatases whose specific role in cancer development and progression has only recently been highlighted. The discovery of the Greatwall (Gwl)/Arpp19-ENSA cascade, a new pathway specifically controlling PP2A-B55 activity, has been shown to be frequently altered in cancer. Herein, we will review the current knowledge about the mechanisms controlling the formation and the regulation of the activity of this phosphatase and its misregulation in cancer.

Reprogramming of cultured cells using Xenopus egg extract involves controlling four major steps: plasma membrane permeabilization, egg factors import into the nucleus, membrane resealing, and cell proliferation. Using propidium iodide to assess plasma membrane permeability, we established that 90% of the cultured fin cells were permeabilized by digitonin without any cell losses. We showed that egg extract at metaphase II stage was essential to maintain nuclear import function in the permeabilized cells, as assessed with a fusion GFP protein carrying the nuclear import signal NLS. Moreover, the Xenopus-egg-specific Lamin B3 was detected in 87% of the cell nuclei, suggesting that other egg extract reprogramming factors of similar size could successfully enter the nucleus. Lamin B3 labelling was maintained in most cells recovered 24 h after membrane resealing with calcium, and cells successfully resumed cell cycle in culture. In contrast, permeabilized cells that were not treated with egg extract failed to proliferate in culture and died, implying that egg extract provided factor essential to the survival of those cells. To conclude, fish fin cells were successfully primed for treatment with reprogramming factors, and egg extract was shown to play a major role in their survival and recovery after permeabilization.

Greatwall (GWL) is an essential kinase that indirectly controls PP2A-B55, the phosphatase counterbalancing cyclin B/CDK1 activity during mitosis. In Xenopus laevis egg extracts, GWL-mediated phosphorylation of overexpressed ARPP19 and ENSA turns them into potent PP2A-B55 inhibitors. It has been shown that the GWL/ENSA/PP2A-B55 axis contributes to the control of DNA replication, but little is known about the role of ARPP19 in cell division. By using conditional knockout mouse models, we investigated the specific roles of ARPP19 and ENSA in cell division. We found that Arpp19 , but not Ensa , is essential for mouse embryogenesis. Moreover, Arpp19 ablation dramatically decreased mouse embryonic fibroblast (MEF) viability by perturbing the temporal pattern of protein dephosphorylation during mitotic progression, possibly by a drop of PP2A-B55 activity inhibition. We show that these alterations are not prevented by ENSA, which is still expressed in Arpp19 Δ/Δ MEFs, suggesting that ARPP19 is essential for mitotic division. Strikingly, we demonstrate that unlike ARPP19, ENSA is not required for early embryonic development. Arpp19 knockout did not perturb the S phase, unlike Ensa gene ablation. We conclude that, during mouse embryogenesis, the Arpp19 and Ensa paralog genes display specific functions by differentially controlling cell cycle progression., (© 2019 Hached et al.)

Mitosis is controlled by a subtle balance between kinase and phosphatase activities that involve the master mitotic kinase cyclin-B-Cdk1 and its antagonizing protein phosphatase 2A-B55 (PP2A-B55). Importantly, the Greatwall (Gwl; known as Mastl in mammals, Rim15 in budding yeast and Ppk18 in fission yeast) kinase pathway regulates PP2A-B55 activity by phosphorylating two proteins, cAMP-regulated phosphoprotein 19 (Arpp19) and α-endosulfine (ENSA). This phosphorylation turns these proteins into potent inhibitors of PP2A-B55, thereby promoting a correct timing and progression of mitosis. In this Cell Science at a Glance article and the accompanying poster, we discuss how Gwl is regulated in space and time, and how the Gwl-Arpp19-ENSA-PP2A-B55 pathway plays an essential role in the control of M and S phases from yeast to human. We also summarize how Gwl modulates oncogenic properties of cells and how nutrient deprivation influences Gwl activity., (© 2018. Published by The Company of Biologists Ltd.)

Mitosis is induced by the activation of the cyclin B/cdk1 feedback loop that creates a bistable state. The triggering factor promoting active cyclin B/cdk1 switch has been assigned to cyclin B/cdk1 accumulation during G2. However, this complex is rapidly inactivated by Wee1/Myt1-dependent phosphorylation of cdk1 making unlikely a triggering role of this kinase in mitotic commitment. Here we show that cyclin A/cdk1 kinase is the factor triggering mitosis. Cyclin A/cdk1 phosphorylates Bora to promote Aurora A-dependent Plk1 phosphorylation and activation and mitotic entry. We demonstrate that Bora phosphorylation by cyclin A/cdk1 is both necessary and sufficient for mitotic commitment. Finally, we identify a site in Bora whose phosphorylation by cyclin A/cdk1 is required for mitotic entry. We constructed a mathematical model confirming the essential role of this kinase in mitotic commitment. Overall, our results uncover the molecular mechanism by which cyclin A/cdk1 triggers mitotic entry., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The Greatwall/Ensa/PP2A-B55 pathway is essential for controlling mitotic substrate phosphorylation and mitotic entry. Here, we investigate the effect of the knockdown of the Gwl substrate, Ensa, in human cells. Unexpectedly, Ensa knockdown promotes a dramatic extension of S phase associated with a lowered density of replication forks. Notably, Ensa depletion results in a decrease of Treslin levels, a pivotal protein for the firing of replication origins. Accordingly, the extended S phase in Ensa-depleted cells is completely rescued by the overexpression of Treslin. Our data herein reveal a new mechanism by which normal cells regulate S-phase duration by controlling the ubiquitin-proteasome degradation of Treslin in a Gwl/Ensa-dependent pathway.The Greatwall/Ensa/PP2A-B55 pathway controls mitotic substrate phosphorylation and mitotic entry. Here the authors show that cells regulate S phase duration by controlling the ubiquitin-proteasome degradation of Treslin in a Gwl/Ensa-dependent pathway.

In human eggs, aneuploidy increases with age and can result in infertility and genetic diseases. Studies in mouse oocytes suggest that reduced centromere cohesion and spindle assembly checkpoint (SAC) activity could be at the origin of chromosome missegregation. Little is known about these two features in humans. Here, we show that in human eggs, inter-kinetochore distances of bivalent chromosomes strongly increase with age. This results in the formation of univalent chromosomes during metaphase I (MI) and of single chromatids in metaphase II (MII). We also investigated SAC activity by checking the localization of BUB1 and BUBR1. We found that they localize at the kinetochore with a similar temporal timing than in mitotic cells and in a MPS1-dependent manner, suggesting that the SAC signalling pathway is active in human oocytes. Moreover, our data also suggest that this checkpoint is inactivated when centromere cohesion is lost in MI and consequently cannot inhibit premature sister chromatid separation. Finally, we show that the kinetochore localization of BUB1 and BUBR1 decreases with the age of the oocyte donors. This could contribute to oocyte aneuploidy.

Centrioles are essential for the assembly of both centrosomes and cilia. Centriole biogenesis occurs once and only once per cell cycle and is temporally coordinated with cell-cycle progression, ensuring the formation of the right number of centrioles at the right time. The formation of new daughter centrioles is guided by a pre-existing, mother centriole. The proximity between mother and daughter centrioles was proposed to restrict new centriole formation until they separate beyond a critical distance. Paradoxically, mother and daughter centrioles overcome this distance in early mitosis, at a time when triggers for centriole biogenesis Polo-like kinase 4 (PLK4) and its substrate STIL are abundant. Here we show that in mitosis, the mitotic kinase CDK1-CyclinB binds STIL and prevents formation of the PLK4-STIL complex and STIL phosphorylation by PLK4, thus inhibiting untimely onset of centriole biogenesis. After CDK1-CyclinB inactivation upon mitotic exit, PLK4 can bind and phosphorylate STIL in G1, allowing pro-centriole assembly in the subsequent S phase. Our work shows that complementary mechanisms, such as mother-daughter centriole proximity and CDK1-CyclinB interaction with centriolar components, ensure that centriole biogenesis occurs once and only once per cell cycle, raising parallels to the cell-cycle regulation of DNA replication and centromere formation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Entry into mitosis is induced by the activation of cyclin-B-Cdk1 and Greatwall (Gwl; also known as MASTL in mammals) kinases. Cyclin-B-Cdk1 phosphorylates mitotic substrates, whereas Gwl activation promotes the phosphorylation of the small proteins Arpp19 and ENSA. Phosphorylated Arpp19 and/or ENSA bind to and inhibit PP2A comprising the B55 subunit (PP2A-B55; B55 is also known as PPP2R2A), the phosphatase responsible for cyclin-B-Cdk1 substrate dephosphorylation, allowing the stable phosphorylation of mitotic proteins. Upon mitotic exit, cyclin-B-Cdk1 and Gwl kinases are inactivated, and mitotic substrates are dephosphorylated. Here, we have identified protein phosphatase-1 (PP1) as the phosphatase involved in the dephosphorylation of the activating site (Ser875) of Gwl. Depletion of PP1 from meioticXenopusegg extracts maintains phosphorylation of Ser875, as well as the full activity of this kinase, resulting in a block of meiotic and mitotic exit. By contrast, preventing the reactivation of PP2A-B55 through the addition of a hyperactive Gwl mutant (GwlK72M) mainly affected Gwl dephosphorylation on Thr194, resulting in partial inactivation of Gwl and in the incomplete exit from mitosis or meiosis. We also show that when PP2A-B55 is fully reactivated by depleting Arpp19, this protein phosphatase is able to dephosphorylate both activating sites, even in the absence of PP1., (© 2016. Published by The Company of Biologists Ltd.)

Entry into mitosis requires the coordinated activation of various protein kinases and phosphatases that together activate sequential signaling pathways allowing entry, progression and exit of mitosis. The limiting step is thought to be the activation of the mitotic Cdk1-cyclin B kinase. However, this model has recently evolved with new data showing that in addition to the Cdk1-cyclin B complex, Greatwall (Gwl) kinase is also required to enter into and maintain mitosis. This new concept proposes that entry into mitosis is now based on the combined activation of both kinases Cdk1-cyclin B and Gwl, the former promoting massive phosphorylation of mitotic substrates and the latter inhibiting PP2A-B55 phosphatase responsible for dephosphorylation of these substrates. Activated Gwl phosphorylates both Arpp19 and ENSA, which associate and inhibit PP2A-B55. This pathway seems relatively well conserved from yeast to humans, although some differences appear based on models or techniques used. While Gwl is activated by phosphorylation, its inactivation requires dephosphorylation of critical residues. Several phosphatases such as PP1, PP2A-B55 and FCP1 are required to control the dephosphorylation and inactivation of Gwl and a properly regulated mitotic exit. Gwl has also been reported to be involved in cancer processes and DNA damage recovery. These new findings support the idea that the Gwl-Arpp19/ENSA-PP2A-B55 pathway is essential to achieve an efficient division of cells and to maintain genomic stability.