A hydraulic instability drives the cell death decision in the nematode germline.

Oocytes are large cells that develop into an embryo upon fertilization1. As interconnected germ cells mature into oocytes, some of them grow-typically at the expense of others that undergo cell death2-4. We present evidence that in the nematode Caenorhabditis elegans, this cell-fate decision is mechanical and related to tissue hydraulics. An analysis of germ cell volumes and material fluxes identifies a hydraulic instability that amplifies volume differences and causes some germ cells to grow and others to shrink, a phenomenon that is related to the two-balloon instability5. Shrinking germ cells are extruded and they die, as we demonstrate by artificially reducing germ cell volumes via thermoviscous pumping6. Our work reveals a hydraulic symmetry-breaking transition central to the decision between life and death in the nematode germline.

Syncytium biogenesis: It's all about maintaining good connections.

At the end of mitosis, cells typically complete their division with cytokinesis. In certain tissues however, incomplete cytokinesis can give rise to cells that remain connected by intercellular bridges, thus forming a syncytium. Examples include the germline of many species, from fruitfly to humans, yet the mechanisms regulating syncytial formation and maintenance is unclear, and the biological relevance of syncytial organization remains largely speculative. To better understand these processes, we recently used the germline of Caenorhabditis elegans as a model for syncytium development. Analysis of the germline syncytial architecture throughout development revealed that it arises progressively during larval growth and that it relies on the activity of 2 actomyosin scaffold proteins of the Anillin family. Our work also showed that the gonad can sustain elastic deformation when under mechanical stress and that this property may be conferred by the malleability of syncytial openings. We suggest that elasticity and resistance to mechanical stress constitutes a general property of syncytial tissues.

C. elegans Anillin proteins regulate intercellular bridge stability and germline syncytial organization.

Cytokinesis generally produces two separate daughter cells, but in some tissues daughter nuclei remain connected to a shared cytoplasm, or syncytium, through incomplete cytokinesis. How syncytia form remains poorly understood. We studied syncytial formation in the Caenorhabditis elegans germline, in which germ cells connect to a shared cytoplasm core (the rachis) via intercellular bridges. We found that syncytial architecture initiates early in larval development, and germ cells become progressively interconnected until adulthood. The short Anillin family scaffold protein ANI-2 is enriched at intercellular bridges from the onset of germ cell specification, and ANI-2 loss resulted in destabilization of intercellular bridges and germ cell multinucleation defects. These defects were partially rescued by depleting the canonical Anillin ANI-1 or blocking cytoplasmic streaming. ANI-2 is also required for elastic deformation of the gonad during ovulation. We propose that ANI-2 promotes germ cell syncytial organization and allows for compensation of the mechanical stress associated with oogenesis by conferring stability and elasticity to germ cell intercellular bridges.

PAR-4/LKB1 regulates DNA replication during asynchronous division of the early C. elegans embryo.

Regulation of cell cycle duration is critical during development, yet the underlying molecular mechanisms are still poorly understood. The two-cell stage Caenorhabditis elegans embryo divides asynchronously and thus provides a powerful context in which to study regulation of cell cycle timing during development. Using genetic analysis and high-resolution imaging, we found that deoxyribonucleic acid (DNA) replication is asymmetrically regulated in the two-cell stage embryo and that the PAR-4 and PAR-1 polarity proteins dampen DNA replication dynamics specifically in the posterior blastomere, independently of regulators previously implicated in the control of cell cycle timing. Our results demonstrate that accurate control of DNA replication is crucial during C. elegans early embryonic development and further provide a novel mechanism by which PAR proteins control cell cycle progression during asynchronous cell division.

MEX-3 proteins: recent insights on novel post-transcriptional regulators.

RNA-binding proteins of the evolutionarily-conserved MEX-3 family are mediators of post-transcriptional regulation in different organisms. Recent studies highlight their involvement in diverse physiological settings, including the maintenance of a balance between stem cell self-renewal and differentiation. Here, we draw attention to their putative role in tissue homeostasis and disease, particularly cancer.

CDX2 regulation by the RNA-binding protein MEX3A: impact on intestinal differentiation and stemness.

The homeobox transcription factor CDX2 plays a crucial role in intestinal cell fate specification, both during normal development and in tumorigenic processes involving intestinal reprogramming. The CDX2 regulatory network is intricate, but it has not yet been fully uncovered. Through genome-wide screening of a 3D culture system, the RNA-binding protein MEX3A was identified as putatively involved in CDX2 regulation; therefore, its biological relevance was addressed by setting up cell-based assays together with expression studies in murine intestine. We demonstrate here that MEX3A has a repressive function by controlling CDX2 levels in gastric and colorectal cellular models. This is dependent on the interaction with a specific binding determinant present in CDX2 mRNA 3'untranslated region. We have further determined that MEX3A impairs intestinal differentiation and cellular polarization, affects cell cycle progression and promotes increased expression of intestinal stem cell markers, namely LGR5, BMI1 and MSI1. Finally, we show that MEX3A is expressed in mouse intestine, supporting an in vivo context for interaction with CDX2 and modulation of stem cell properties. Therefore, we describe a novel CDX2 post-transcriptional regulatory mechanism, through the RNA-binding protein MEX3A, with a major impact in intestinal differentiation, polarity and stemness, likely contributing to intestinal homeostasis and carcinogenesis.

Enterocytic differentiation is modulated by lipid rafts-dependent assembly of adherens junctions.

Integrity of the epithelial barrier is determined by apical junctional complexes which also participate in the signalling pathways inducing intestinal cell differentiation. Lipid rafts (LR) have been proposed to play a role in the organization and the function of these intercellular complexes. This study investigated potential mechanisms by which LR could participate in the establishment of adherens junctions (AJ) and the initiation of enterocytic differentiation. We showed that the differentiation of epithelial cells in rat colons correlates with the emergence of LR. Using HT-29 cells we demonstrated that during the differentiation process, LR are required for the recruitment and the association of p120ctn to E-cadherin. Silencing of flotillin-1, a LR component, alters the recruitment of AJ proteins in LR and delays the expression of differentiation markers. Furthermore, the ability of p120ctn/E-cadherin complexes to support cell differentiation is altered in HT-29 Rac1N17 cells. These results show a contributory role of LR in the enterocytic differentiation process, which serve as signalling platforms for Rac1-mediated organization of AJ. A better understanding of the mechanism involved in the establishment of junctional complex and their role in enterocytic differentiation provides new insights into the regulation of intestinal homeostasis.

PAR-4/LKB1 mobilizes nonmuscle myosin through anillin to regulate C. elegans embryonic polarization and cytokinesis.

BACKGROUND: The serine/threonine kinase LKB1 regulates cell growth and polarity in metazoans, and loss of LKB1 function is implicated in the development of some epithelial cancers. Despite its fundamental role, the mechanism by which LKB1 regulates polarity establishment and/or maintenance is unclear. In the present study, we use the nematode C. elegans to investigate the role of the LKB1 ortholog PAR-4 in actomyosin contractility, a cellular process essential for polarity establishment and cell division in the early embryo. RESULTS: Using high-resolution time-lapse imaging of GFP-tagged nonmuscle myosin II (NMY-2), we found that par-4 mutations reduce actomyosin contractility during polarity establishment, leading to the mispositioning of anterior PAR proteins and to defects in contractile ring ingression during cytokinesis. Fluorescence recovery after photobleaching analysis revealed that the mobility of a cortical population of NMY-2 was reduced in par-4 mutants. Interestingly, the contractility defects of par-4 mutants depend on the reciprocal activity of ANI-1 and ANI-2, two C. elegans homologs of the actin cytoskeletal scaffold protein anillin. CONCLUSION: Because loss of PAR-4 promoted inappropriate accumulation of ANI-2 at the cell cortex, we propose that PAR-4 controls C. elegans embryonic polarity by regulating the activity of anillin family scaffold proteins, thus enabling turnover of cortical myosin and efficient actomyosin contractility. This work provides the first description of a cellular mechanism by which PAR-4/LKB1 mediates cell polarization.

[Mechanisms of asymmetric cell division: from model organisms to tumorigenesis]. / Mécanismes de division cellulaire asymétrique : des organismes modèles au développement tumoral.

Asymmetric cell division is the process by which a single cell gives rise to two different daughter cells. This process is important to generate cell diversity during the development of multicellular organisms, as well as for stem cell self-renewal in adults. Current knowledge on so-called cancer stem cells suggests that a loss of asymmetry during their division could lead to overproliferation and favour tumorigenesis, highlighting the importance of deciphering the mechanisms governing asymmetric cell division. Two mechanisms can lead to an asymmetric cell division: asymmetry can either be governed by proximity to a given cellular environment (or niche), in which case the mechanism is referred to as extrinsic, or the mother cell polarizes itself without external intervention, in which case the mechanism is referred to as intrinsic. In the last 20 years, our understanding of intrinsic mechanisms leading to asymmetric cell division has progressed, largely after studies carried out in model organisms such as the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster. These models allowed the identification of molecular complexes used by nearly all the cells that divide asymmetrically, including human cells. Here we review the main intrinsic mechanisms of asymmetric cell division as described in model organisms and discuss their relevance towards mammalian tumorigenesis.

Understanding the role of asymmetric cell division in cancer using C. elegans.

Asymmetric cell division is an important process to generate cell diversity and maintain tissue homeostasis. Recent evidence suggests that this process may also be crucial to prevent tumor formation. In the past 30 years, the embryo of the nematode Caenorhabditis elegans has proven to be a very powerful model to study the molecular and cellular basis of asymmetric cell division. Understanding this process in Caenorhabditis elegans may thus lead to a better understanding of stem cell function and tumorigenesis in humans.

Cyclin-dependent kinase 2/cyclin E complex is involved in p120 catenin (p120ctn)-dependent cell growth control: a new role for p120ctn in cancer.

Depending on its cellular localization, p120 catenin (p120ctn) can participate in various processes, such as cadherin-dependent cell-cell adhesion, actin cytoskeleton remodeling, and intracellular trafficking. Recent studies also indicate that p120ctn could regulate cell proliferation and contact inhibition. This report describes a new function of p120ctn in the regulation of cell cycle progression. Overexpression of the p120ctn isoform 3A in human colon adenocarcinoma cells (HT-29) results in cytoplasmic accumulation of the protein, as observed in many tumors. This cytoplasmic increase is correlated with a reduction in proliferation and inhibition of DNA synthesis. Under these conditions, experiments on synchronized cells revealed a prolonged S phase associated with cyclin E stabilization. Both confocal microscopy and biochemical analysis showed that cyclin E and cyclin-dependent kinase 2 colocalized with p120ctn in centrosomes during mitosis. These proteins are associated in a functional complex evidenced by coimmunoprecipitation experiments and the emergence of Thr199-phosphorylated nucleophosmin/B23. Such post-translational modification of this centrosomal target has been shown to trigger the initiation of centrosome duplication. Therefore, p120ctn-mediated accumulation of cyclin E in centrosomes may participate in abnormal amplification of centrosomes and the inhibition of DNA replication, thus leading to aberrant mitosis and polyploidy. Because these modifications are often observed in cancer, p120ctn may represent a new therapeutic target for future therapy.

Laminin-5-integrin interaction signals through PI 3-kinase and Rac1b to promote assembly of adherens junctions in HT-29 cells.

Human intestinal cell differentiation is mediated by signaling pathways that remain largely undefined. We and others have shown that cell migration and differentiation along the crypt-villus axis is associated with temporal and spatial modulations of the repertoire, as well as with the function of integrins and E-cadherins and their substrates. Cross-talk between integrin and cadherin signaling was previously described and seems to coordinate this differentiation process. Here, we report that engagement of alpha6 and, to a lesser extent, alpha3 integrin subunits after HT-29 cell adhesion on laminin 5 increases the expression of E-cadherin, which then organizes into nascent adherens junctions. We further identify that phosphoinositide 3-kinase (PI 3-kinase) activation plays a key role in this cross-talk. Indeed, integrin-dependent adhesion on laminin 5 stimulates PI 3-kinase activity. Immunofluorescence and immunoprecipitation experiments revealed that activated PI 3-kinase is recruited at cell-cell contacts. Using LY294002, an inhibitor of PI 3-kinase activity, we found that this activation is essential for E-cadherin connection with the cytoskeleton and for biogenesis of adherens junctions. Finally, we demonstrated that PI 3-kinase could signal through Rac1b activation to control adherens junction assembly. Our results provide a mechanistic insight into integrin-cadherin cross-talk and identify a novel role for PI 3-kinase in the establishment of adherens junctions.