OSGN-1 is a conserved flavin-containing monooxygenase required to stabilize the intercellular bridge in late cytokinesis.

Cytokinesis is the last step of cell division and is regulated by the small GTPase RhoA. RhoA activity is required for all steps of cytokinesis, including prior to abscission when daughter cells are ultimately physically separated. Like germ cells in all animals, the Caenorhabditis elegans embryonic germline founder cell initiates cytokinesis but does not complete abscission, leaving a stable intercellular bridge between the two daughter cells. Here, we identify and characterize C. elegans OSGN-1 as a cytokinetic regulator that promotes RhoA activity during late cytokinesis. Sequence analyses and biochemical reconstitutions reveal that OSGN-1 is a flavin-containing monooxygenase (MO). Genetic analyses indicate that the MO activity of OSGN-1 is required to maintain active RhoA at the end of cytokinesis in the germline founder cell and to stabilize the intercellular bridge. Deletion of OSGIN1 in human cells results in an increase in binucleation as a result of cytokinetic furrow regression, and this phenotype can be rescued by expressing a catalytically active form of C. elegans OSGN-1, indicating that OSGN-1 and OSGIN1 are functional orthologs. We propose that OSGN-1 and OSGIN1 are conserved MO enzymes required to maintain RhoA activity at the intercellular bridge during late cytokinesis and thus favor its stability, enabling proper abscission in human cells and bridge stabilization in C. elegans germ cells.

Increases in cyclin A/Cdk activity and in PP2A-B55 inhibition by FAM122A are key mitosis-inducing events.

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.

Phosphoproteomic analysis identifies supervillin as an ERK3 substrate regulating cytokinesis and cell ploidy.

Extracellular signal-regulated kinase 3 (ERK3) is a poorly characterized member of the mitogen-activated protein (MAP) kinase family. Functional analysis of the ERK3 signaling pathway has been hampered by a lack of knowledge about the substrates and downstream effectors of the kinase. Here, we used large-scale quantitative phosphoproteomics and targeted gene silencing to identify direct ERK3 substrates and gain insight into its cellular functions. Detailed validation of one candidate substrate identified the gelsolin/villin family member supervillin (SVIL) as a bona fide ERK3 substrate. We show that ERK3 phosphorylates SVIL on Ser245 to regulate myosin II activation and cytokinesis completion in dividing cells. Depletion of SVIL or ERK3 leads to increased cytokinesis failure and multinucleation, a phenotype rescued by wild type SVIL but not by the non-phosphorylatable S245A mutant. Our results unveil a new function of the atypical MAP kinase ERK3 in cell division and the regulation of cell ploidy.

Uncoupling cell division and cytokinesis during germline development in metazoans.

The canonical eukaryotic cell cycle ends with cytokinesis, which physically divides the mother cell in two and allows the cycle to resume in the newly individualized daughter cells. However, during germline development in nearly all metazoans, dividing germ cells undergo incomplete cytokinesis and germ cells stay connected by intercellular bridges which allow the exchange of cytoplasm and organelles between cells. The near ubiquity of incomplete cytokinesis in animal germ lines suggests that this is an ancient feature that is fundamental for the development and function of this tissue. While cytokinesis has been studied for several decades, the mechanisms that enable regulated incomplete cytokinesis in germ cells are only beginning to emerge. Here we review the current knowledge on the regulation of germ cell intercellular bridge formation, focusing on findings made using mouse, Drosophila melanogaster and Caenorhabditis elegans as experimental systems.

The pentacyclic triterpenoid phytosterol lupeol promotes antioxidant response in the nematode C. elegans.

Plants of the Mimosa genus are studied and used for their bioactive properties. Among bioactive phytochemicals are quercetin and myricetin, which have been demonstrated to act as antioxidants in many contexts (Taheri et al. 2020; Xu et al. 2019), including in C. elegans (Buchter et al. 2013; Grünz et al. 2012; Sugawara and Sakamoto 2020). Other phytochemicals from these plants, such as the triterpenoid phytosterol lupeol, have been shown to have antioxidant properties but have not been as extensively characterized in model organisms (Liu et al. 2021; Shai et al. 2009). Here we employed the nematode C. elegans to assess whether lupeol elicits antioxidant response in vivo . Using reporter assays for oxidative stress, we find that treatment of animals with lupeol rescues some of the effects resulting from treatment with the prooxidant paraquat. Our results demonstrate that lupeol displays antioxidant properties in vivo in C. elegans .

PP2A-B55: substrates and regulators in the control of cellular functions.

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.

The primordial germ line is refractory to perturbations of actomyosin regulator function in C. elegans L1 larvae.

Cytokinesis, the separation of daughter cells at the end of mitosis, relies on the coordinated activity of several regulators of actomyosin assembly and contractility (Green et al. 2012). These include the small GTPase RhoA (RHO-1) and its guanine-nucleotide exchange factor Ect2 (ECT-2), the scaffold protein Anillin (ANI-1), the non-muscle myosin II (NMY-2), the formin CYK-1 and the centralspindlin complex components ZEN-4 and CYK-4. These regulators were also shown to be required for maintenance of C. elegans germline syncytial organization by stabilizing intercellular bridges in embryos and adults (Amini et al. 2014; Goupil et al. 2017; Green et al. 2011; Priti et al. 2018; Zhou et al. 2013). We recently demonstrated that many of these regulators are enriched at intercellular bridges in the small rachis (proto-rachis) of L1-stage larvae (Bauer et al. 2021). We sought to assess whether these contractility regulators are functionally required for stability of intercellular bridges and maintenance of the primordial germ line syncytial architecture in L1-stage C. elegans animals. Here we report that temperature-sensitive alleles, RNAi-mediated depletion and latrunculin A treatment are largely ineffective to perturb actomyosin function in the L1-stage primordial germ line.

The initial expansion of the C. elegans syncytial germ line is coupled to incomplete primordial germ cell cytokinesis.

The C. elegans germline is organized as a syncytium in which each germ cell possesses an intercellular bridge that is maintained by a stable actomyosin ring and connected to a common pool of cytoplasm, termed the rachis. How germ cells undergo cytokinesis while maintaining this syncytial architecture is not completely understood. Here, we use live imaging to characterize primordial germ cell (PGC) division in C. elegans first-stage larvae. We show that each PGC possesses a stable intercellular bridge that connects it to a common pool of cytoplasm, which we term the proto-rachis. We further show that the first PGC cytokinesis is incomplete and that the stabilized cytokinetic ring progressively moves towards the proto-rachis and eventually integrates with it. Our results support a model in which the initial expansion of the C. elegans syncytial germline occurs by incomplete cytokinesis, where one daughter germ cell inherits the actomyosin ring that was newly formed by stabilization of the cytokinetic ring, while the other inherits the pre-existing stable actomyosin ring. We propose that such a mechanism of iterative cytokinesis incompletion underpins C. elegans germline expansion and maintenance.

The study of the determinants controlling Arpp19 phosphatase-inhibitory activity reveals an Arpp19/PP2A-B55 feedback loop.

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.

CentTracker: a trainable, machine-learning-based tool for large-scale analyses of Caenorhabditis elegans germline stem cell mitosis.

Investigating the complex interactions between stem cells and their native environment requires an efficient means to image them in situ. Caenorhabditis elegans germline stem cells (GSCs) are distinctly accessible for intravital imaging; however, long-term image acquisition and analysis of dividing GSCs can be technically challenging. Here we present a systematic investigation into the technical factors impacting GSC physiology during live imaging and provide an optimized method for monitoring GSC mitosis under minimally disruptive conditions. We describe CentTracker, an automated and generalizable image analysis tool that uses machine learning to pair mitotic centrosomes and that can extract a variety of mitotic parameters rapidly from large-scale data sets. We employ CentTracker to assess a range of mitotic features in a large GSC data set. We observe spatial clustering of mitoses within the germline tissue but no evidence that subpopulations with distinct mitotic profiles exist within the stem cell pool. We further find biases in GSC spindle orientation relative to the germline's distal-proximal axis and thus the niche. The technical and analytical tools provided herein pave the way for large-scale screening studies of multiple mitotic processes in GSCs dividing in situ, in an intact tissue, in a living animal, under seemingly physiological conditions.

PP2A-B55 Holoenzyme Regulation and Cancer.

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.

Cyclin A-cdk1-Dependent Phosphorylation of Bora Is the Triggering Factor Promoting Mitotic Entry.

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.

Spindle assembly checkpoint strength is linked to cell fate in the Caenorhabditis elegans embryo.

The spindle assembly checkpoint (SAC) is a conserved mitotic regulator that preserves genome stability by monitoring kinetochore-microtubule attachments and blocking anaphase onset until chromosome biorientation is achieved. Despite its central role in maintaining mitotic fidelity, the ability of the SAC to delay mitotic exit in the presence of kinetochore-microtubule attachment defects (SAC "strength") appears to vary widely. How different cellular aspects drive this variation remains largely unknown. Here we show that SAC strength is correlated with cell fate during development of Caenorhabditis elegans embryos, with germline-fated cells experiencing longer mitotic delays upon spindle perturbation than somatic cells. These differences are entirely dependent on an intact checkpoint and only partially attributable to differences in cell size. In two-cell embryos, cell size accounts for half of the difference in SAC strength between the larger somatic AB and the smaller germline P1 blastomeres. The remaining difference requires asymmetric cytoplasmic partitioning downstream of PAR polarity proteins, suggesting that checkpoint-regulating factors are distributed asymmetrically during early germ cell divisions. Our results indicate that SAC activity is linked to cell fate and reveal a hitherto unknown interaction between asymmetric cell division and the SAC.

Actomyosin contractility regulators stabilize the cytoplasmic bridge between the two primordial germ cells during Caenorhabditis elegans embryogenesis.

Stable cytoplasmic bridges arise from failed cytokinesis, the last step of cell division, and are a key feature of syncytial architectures in the germline of most metazoans. Whereas the Caenorhabditiselegans germline is syncytial, its formation remains poorly understood. We found that the germline precursor blastomere, P4 , fails cytokinesis, leaving a stable cytoplasmic bridge between the two daughter cells, Z2 and Z3 Depletion of several regulators of actomyosin contractility resulted in a regression of the membrane partition between Z2 and Z3, indicating that they are required to stabilize the cytoplasmic bridge. Epistatic analysis revealed a pathway in which Rho regulators promote accumulation of the noncannonical anillin ANI-2 at the stable cytoplasmic bridge, which in turns promotes the accumulation of the nonmuscle myosin II NMY-2 and the midbody component CYK-7 at the bridge, in part by limiting the accumulation of canonical anillin ANI-1. Our results uncover key steps in C. elegans germline formation and define a set of conserved regulators that are enriched at the primordial germ cell cytoplasmic bridge to ensure its stability during embryonic development.

DAF-18/PTEN signals through AAK-1/AMPK to inhibit MPK-1/MAPK in feedback control of germline stem cell proliferation.

Under replete growth conditions, abundant nutrient uptake leads to the systemic activation of insulin/IGF-1 signalling (IIS) and the promotion of stem cell growth/proliferation. Activated IIS can stimulate the ERK/MAPK pathway, the activation of which also supports optimal stem cell proliferation in various systems. Stem cell proliferation rates can further be locally refined to meet the resident tissue's need for differentiated progeny. We have recently shown that the accumulation of mature oocytes in the C. elegans germ line, through DAF-18/PTEN, inhibits adult germline stem cell (GSC) proliferation, despite high systemic IIS activation. We show here that this feedback occurs through a novel cryptic signalling pathway that requires PAR-4/LKB1, AAK-1/AMPK and PAR-5/14-3-3 to inhibit the activity of MPK-1/MAPK, antagonize IIS, and inhibit both GSC proliferation and the production of additional oocytes. Interestingly, our results imply that DAF-18/PTEN, through PAR-4/LKB1, can activate AAK-1/AMPK in the absence of apparent energy stress. As all components are conserved, similar signalling cascades may regulate stem cell activities in other organisms and be widely implicated in cancer.

The master Greatwall kinase, a critical regulator of mitosis and meiosis.

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.

Bigger Isn't Always Better: Cell Size and the Spindle Assembly Checkpoint.

Variation in the activity of the spindle assembly checkpoint has been observed in different cell types, yet the reason for this variability remains poorly understood. Reporting in Developmental Cell, Galli and Morgan (2016) show that checkpoint activity increases during development as cell size, and the cytoplasm-to-kinetochore ratio, decreases.

DAF-18/PTEN locally antagonizes insulin signalling to couple germline stem cell proliferation to oocyte needs in C. elegans.

During development, stem cell populations rapidly proliferate to populate the expanding tissues and organs. During this phase, nutrient status, by systemically affecting insulin/IGF-1 signalling, largely dictates stem cell proliferation rates. In adults, however, differentiated stem cell progeny requirements are generally reduced and vary according to the spatiotemporal needs of each tissue. We demonstrate here that differential regulation of germline stem cell proliferation rates in Caenorhabditis elegans adults is accomplished through localized neutralization of insulin/IGF-1 signalling, requiring DAF-18/PTEN, but not DAF-16/FOXO. Indeed, the specific accumulation of oocytes, the terminally differentiated stem cell progeny, triggers a feedback signal that locally antagonizes insulin/IGF-1 signalling outputs in the germ line, regardless of their systemic levels, to block germline stem cell proliferation. Thus, during adulthood, stem cells can differentially respond within tissues to otherwise equal insulin/IGF-1 signalling inputs, according to the needs for production of their immediate terminally differentiated progeny.