Quantitative imaging and semiotic phenotyping of mitochondrial network morphology in live human cells.

The importance of mitochondria in tissue homeostasis, stress responses and human diseases, combined to their ability to transition between various structural and functional states, makes them excellent organelles for monitoring cell health. There is therefore a need for technologies to accurately analyze and quantify changes in mitochondrial organization in a variety of cells and cellular contexts. Here we present an innovative computerized method that enables accurate, multiscale, fast and cost-effective analysis of mitochondrial shape and network architecture from confocal fluorescence images by providing more than thirty features. In order to facilitate interpretation of the quantitative results, we introduced two innovations: the use of Kiviat-graphs (herein named MitoSpider plots) to present highly multidimensional data and visualization of the various mito-cellular configurations in the form of morphospace diagrams (called MitoSigils). We tested our fully automated image analysis tool on rich datasets gathered from live normal human skin cells cultured under basal conditions or exposed to specific stress including UVB irradiation and pesticide exposure. We demonstrated the ability of our proprietary software (named MitoTouch) to sensitively discriminate between control and stressed dermal fibroblasts, and between normal fibroblasts and other cell types (including cancer tissue-derived fibroblasts and primary keratinocytes), showing that our automated analysis captures subtle differences in morphology. Based on this novel algorithm, we report the identification of a protective natural ingredient that mitigates the deleterious impact of hydrogen peroxide (H2O2) on mitochondrial organization. Hence we conceived a novel wet-plus-dry pipeline combining cell cultures, quantitative imaging and semiotic analysis for exhaustive analysis of mitochondrial morphology in living adherent cells. Our tool has potential for broader applications in other research areas such as cell biology and medicine, high-throughput drug screening as well as predictive and environmental toxicology.

Quantitative assessment of mitochondrial morphology relevant for studies on cellular health and environmental toxicity.

Mitochondria are essential organelles that play crucial roles in cellular energy metabolism, calcium signaling and apoptosis. Their importance in tissue homeostasis and stress responses, combined to their ability to transition between various structural and functional states, make them excellent organelles for monitoring cellular health. Quantitative assessment of mitochondrial morphology can therefore provide valuable insights into environmentally-induced cell damage. High-content screening (HCS) provides a powerful tool for analyzing organelles and cellular substructures. We developed a fully automated and miniaturized HCS wet-plus-dry pipeline (MITOMATICS) exploiting mitochondrial morphology as a marker for monitoring cellular health or damage. MITOMATICS uses an in-house, proprietary software (MitoRadar) to enable fast, exhaustive and cost-effective analysis of mitochondrial morphology and its inherent diversity in live cells. We applied our pipeline and big data analytics software to assess the mitotoxicity of selected chemicals, using the mitochondrial uncoupler CCCP as an internal control. Six different pesticides (inhibiting complexes I, II and III of the mitochondrial respiratory chain) were tested as individual compounds and five other pesticides present locally in Occitanie (Southern France) were assessed in combination to determine acute mitotoxicity. Our results show that the assayed pesticides exhibit specific signatures when used as single compounds or chemical mixtures and that they function synergistically to impact mitochondrial architecture. Study of environment-induced mitochondrial damage has the potential to open new fields in mechanistic toxicology, currently underexplored by regulatory toxicology and exposome research. Such exploration could inform health policy guidelines and foster pharmacological intervention, water, air and soil pollution control and food safety.

N-cadherin/p120 catenin association at cell-cell contacts occurs in cholesterol-rich membrane domains and is required for RhoA activation and myogenesis.

p120 catenin is a major regulator of cadherin stability at cell-cell contacts and a modulator of Rho GTPase activities. In C2C12 myoblasts, N-cadherin is stabilized at cell contacts through its association with cholesterol-rich membrane domains or lipid rafts (LR) and acts as an adhesion-activated receptor that activates RhoA, an event required for myogenesis induction. Here, we report that association of p120 catenin with N-cadherin at cell contacts occurs specifically in LR. We demonstrate that interaction of p120 catenin with N-cadherin is required for N-cadherin association with LR and for its stabilization at cell contacts. LR disruption inhibits myogenesis induction and N-cadherin-dependent RhoA activation as does the perturbation of the N-cadherin-p120 catenin complex after p120 catenin knockdown. Finally, we observe an N-cadherin-dependent accumulation of RhoA at phosphatidylinositol 4,5-bisphosphate-enriched cell contacts which is lost after LR disruption. Thus, a functional N-cadherin-catenin complex occurs in cholesterol-rich membrane microdomains which allows the recruitment of RhoA and the regulation of its activity during myogenesis induction.

M-cadherin activates Rac1 GTPase through the Rho-GEF trio during myoblast fusion.

Cadherins are transmembrane glycoproteins that mediate Ca(2+)-dependent homophilic cell-cell adhesion and play crucial role during skeletal myogenesis. M-cadherin is required for myoblast fusion into myotubes, but its mechanisms of action remain unknown. The goal of this study was to cast some light on the nature of the M-cadherin-mediated signals involved in myoblast fusion into myotubes. We found that the Rac1 GTPase activity is increased at the time of myoblast fusion and it is required for this process. Moreover, we showed that M-cadherin-dependent adhesion activates Rac1 and demonstrated the formation of a multiproteic complex containing M-cadherin, the Rho-GEF Trio, and Rac1 at the onset of myoblast fusion. Interestingly, Trio knockdown efficiently blocked both the increase in Rac1-GTP levels, observed after M-cadherin-dependent contact formation, and myoblast fusion. We conclude that M-cadherin-dependent adhesion can activate Rac1 via the Rho-GEF Trio at the time of myoblast fusion.

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.

N-cadherin-dependent cell-cell contact regulates Rho GTPases and beta-catenin localization in mouse C2C12 myoblasts.

N-cadherin, a member of the Ca(2+)-dependent cell-cell adhesion molecule family, plays an essential role in skeletal muscle cell differentiation. We show that inhibition of N-cadherin-dependent adhesion impairs the upregulation of the two cyclin-dependent kinase inhibitors p21 and p27, the expression of the muscle-specific genes myogenin and troponin T, and C2C12 myoblast fusion. To determine the nature of N-cadherin-mediated signals involved in myogenesis, we investigated whether N-cadherin-dependent adhesion regulates the activity of Rac1, Cdc42Hs, and RhoA. N-cadherin-dependent adhesion decreases Rac1 and Cdc42Hs activity, and as a consequence, c-jun NH2-terminal kinase (JNK) MAPK activity but not that of the p38 MAPK pathway. On the other hand, N-cadherin-mediated adhesion increases RhoA activity and activates three skeletal muscle-specific promoters. Furthermore, RhoA activity is required for beta-catenin accumulation at cell-cell contact sites. We propose that cell-cell contacts formed via N-cadherin trigger signaling events that promote the commitment to myogenesis through the positive regulation of RhoA and negative regulation of Rac1, Cdc42Hs, and JNK activities.

RhoA GTPase regulates M-cadherin activity and myoblast fusion.

The Rho family of GTP-binding proteins plays critical roles during myogenesis induction. To elucidate their role later during myogenesis, we have analyzed RhoA function during myoblast fusion into myotubes. We find that RhoA activity is rapidly and transiently increased when cells are shifted into differentiation medium and then is decreased until myoblast fusion. RhoA activity must be down-regulated to allow fusion, because expression of a constitutively active form of RhoA (RhoAV14) inhibits this process. RhoAV14 perturbs the expression and localization of M-cadherin, a member of the Ca2+-dependent cell-cell adhesion molecule family that has an essential role in skeletal muscle cell differentiation. This mutant does not affect N-cadherin and other proteins involved in myoblast fusion, beta1-integrin and ADAM12. Active RhoA induces the entry of M-cadherin into a degradative pathway and thus decreases its stability in correlation with the monoubiquitination of M-cadherin. Moreover, p120 catenin association with M-cadherin is decreased in RhoAV14-expressing cells, which is partially reverted by the inhibition of the RhoA effector Rho-associated kinase ROCK. ROCK inhibition also restores M-cadherin accumulation at the cell-cell contact sites. We propose that the sustained activation of the RhoA pathway inhibits myoblast fusion through the regulation of p120 activity, which controls cadherin internalization and degradation.

ENSA and ARPP19 differentially control cell cycle progression and development.

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.

Rac1 and RhoA GTPases have antagonistic functions during N-cadherin-dependent cell-cell contact formation in C2C12 myoblasts.

BACKGROUND INFORMATION: N-cadherin, a member of the Ca(2+)-dependent cell-cell adhesion molecule family, plays an essential role in the induction of the skeletal muscle differentiation programme. However, the molecular mechanisms which govern the formation of N-cadherin-dependent cell-cell contacts in myoblasts remain unexplored. RESULTS: In the present study, we show that N-cadherin-dependent cell contact formation in myoblasts is defined by two stages. In the first phase, N-cadherin is highly mobile in the lamellipodia extensions between the contacting cells. The second stage corresponds to the formation of mature N-cadherin-dependent cell contacts, characterized by the immobilization of a pool of N-cadherin which appears to be clustered in the interdigitated membrane structures that are also membrane attachment sites for F-actin filaments. We also demonstrated that the formation of N-cadherin-dependent cell-cell contacts requires a co-ordinated and sequential activity of Rac1 and RhoA. Rac1 is involved in the first stage and facilitates N-cadherin-dependent cell-cell contact formation, but it is not absolutely required. Conversely, RhoA is necessary for N-cadherin-dependent cell contact formation, since, via ROCK (Rho-associated kinase) signalling and myosin 2 activation, it allows the stabilization of N-cadherin at the cell-cell contact sites. CONCLUSIONS: We have shown that Rac1 and RhoA have opposite effects on N-cadherin-dependent cell-cell contact formation in C2C12 myoblasts and act sequentially to allow its formation.

Biogenesis of N-cadherin-dependent cell-cell contacts in living fibroblasts is a microtubule-dependent kinesin-driven mechanism.

Cadherin-mediated cell-cell adhesion is a dynamic process that is regulated during embryonic development, cell migration, and differentiation. Different cadherins are expressed in specific tissues consistent with their roles in cell type recognition. In this study, we examine the formation of N-cadherin-dependent cell-cell contacts in fibroblasts and myoblasts. In contrast to E-cadherin, both endogenous and ectopically expressed N-cadherin shuttles between an intracellular and a plasma membrane pool. Initial formation of N-cadherin-dependent cell-cell contacts results from the recruitment of the intracellular pool of N-cadherin to the plasma membrane. N-cadherin also localizes to the Golgi apparatus and both secretory and endocytotic vesicles. We demonstrate that the intracellular pool of N-cadherin is tightly associated with the microtubule (MT) network and that junction formation requires MTs. In addition, localization of N-cadherin to the cortex is dependent on an intact F-actin cytoskeleton. We show that N-cadherin transport requires the MT network as well as the activity of the MT-associated motor kinesin. In conclusion, we propose that N-cadherin distribution is a regulated process promoted by cell-cell contact formation, which controls the biogenesis and turnover of the junctions through the MT network.

Loss of Centromere Cohesion in Aneuploid Human Oocytes Correlates with Decreased Kinetochore Localization of the Sac Proteins Bub1 and Bubr1.

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.

Ensa controls S-phase length by modulating Treslin levels.

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.

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.

Variation in cadherins and catenins expression is linked to both proliferation and transformation of Rhabdomyosarcoma.

Cadherins are a family of transmembrane glycoproteins that mediate Ca(2+)-dependent homophilic cell-cell adhesion and play a crucial role in cell differentiation. E-cadherin-mediated cell-cell adhesion is lost during the development of most epithelial cancers. This study examines cadherin-dependent adhesion in cell lines derived from rhabdomyosarcoma (RMS), a highly malignant soft-tissue tumor committed to the myogenic lineage, but arrested prior to terminal differentiation. We analysed the expression of cadherins and associated catenins at the mRNA and protein levels as well as their localization in nine RMS-derived cell lines relative to normal myoblasts. We show a decrease in the expression of cadherins and catenins in all RMS-derived cell lines compared to control cells. This decrease in the expression of N- and M-cadherin was confirmed in RMS biopsies. In contrast, R-cadherin is found expressed in RMS, whereas it is normally absent in normal myoblasts. We show that a decrease of R-cadherin expression using RNA interference inhibits cell proliferation of the RD cell line. In addition to their diminished expression, cadherins and catenins do not localize to intercellular contacts in embryonal RMS (ERMS), whereas specific persistent localization is seen in alveolar RMS (ARMS)-derived cell lines. Thus, RMS exhibit defects in the expression of molecules of the cadherin family. Defects in the localization of these adhesion molecules at the sites of cell-cell contact are specifically observed in the ERMS subtype. In addition, our data suggest that R-cadherin is a specific diagnostic marker for RMS and is also an important factor of RMS cell proliferation.

Participation of small GTPases Rac1 and Cdc42Hs in myoblast transformation.

We have previously shown that expression of active Rac1 and Cdc4Hs inhibits skeletal muscle cell differentiation. We show here, by bromodeoxyuridine incorporation and cyclin D1 expression, that the expression of active Rac1 and Cdc42Hs but not RhoA impairs cell cycle exit of L6 myoblasts cultured in differentiation medium. Furthermore, expression of activated forms of Rac1 and Cdc42Hs elicits the loss of cell contact inhibition and anchorage-dependent growth as measured by focus forming activity and growth in soft agar. RhoA was once again not found to have this effect. We found a constitutive Rac1 and Cdc42Hs activation in three human rhabdomyosarcoma-derived cell lines, one of the most common causes of solid tumours arising from muscle precursors during childhood. Finally, dominant negative forms of Rac1 and Cdc42Hs inhibit cell proliferation of the RD rhabdomyosarcoma cell line. These data suggest an important role for the small GTPases Rac1 and Cdc42Hs in the generation of skeletal muscle tumours.

Rab35 regulates cadherin-mediated adherens junction formation and myoblast fusion.

Cadherins are homophilic cell-cell adhesion molecules implicated in many fundamental processes, such as morphogenesis, cell growth, and differentiation. They accumulate at cell-cell contact sites and assemble into large macromolecular complexes named adherens junctions (AJs). Cadherin targeting and function are regulated by various cellular processes, many players of which remain to be uncovered. Here we identify the small GTPase Rab35 as a new regulator of cadherin trafficking and stabilization at cell-cell contacts in C2C12 myoblasts and HeLa cells. We find that Rab35 accumulates at cell-cell contacts in a cadherin-dependent manner. Knockdown of Rab35 or expression of a dominant-negative form of Rab35 impaired N- and M-cadherin recruitment to cell-cell contacts, their stabilization at the plasma membrane, and association with p120 catenin and led to their accumulation in transferrin-, clathrin-, and AP-2-positive intracellular vesicles. We also find that Rab35 function is required for PIP5KIγ accumulation at cell-cell contacts and phosphatidyl inositol 4,5-bisphosphate production, which is involved in cadherin stabilization at contact sites. Finally, we show that Rab35 regulates myoblast fusion, a major cellular process under the control of cadherin-dependent signaling. Taken together, these results reveal that Rab35 regulates cadherin-dependent AJ formation and myoblast fusion.

ADP-ribosylation factor 6 regulates mammalian myoblast fusion through phospholipase D1 and phosphatidylinositol 4,5-bisphosphate signaling pathways.

Myoblast fusion is an essential step during myoblast differentiation that remains poorly understood. M-cadherin-dependent pathways that signal through Rac1 GTPase activation via the Rho-guanine nucleotide exchange factor (GEF) Trio are important for myoblast fusion. The ADP-ribosylation factor (ARF)6 GTPase has been shown to bind to Trio and to regulate Rac1 activity. Moreover, Loner/GEP(100)/BRAG2, a GEF of ARF6, has been involved in mammalian and Drosophila myoblast fusion, but the specific role of ARF6 has been not fully analyzed. Here, we show that ARF6 activity is increased at the time of myoblast fusion and is required for its implementation in mouse C2C12 myoblasts. Specifically, at the onset of myoblast fusion, ARF6 is associated with the multiproteic complex that contains M-cadherin, Trio, and Rac1 and accumulates at sites of myoblast fusion. ARF6 silencing inhibits the association of Trio and Rac1 with M-cadherin. Moreover, we demonstrate that ARF6 regulates myoblast fusion through phospholipase D (PLD) activation and phosphatidylinositol 4,5-bis-phosphate production. Together, these data indicate that ARF6 is a critical regulator of C2C12 myoblast fusion and participates in the regulation of PLD activities that trigger both phospholipids production and actin cytoskeleton reorganization at fusion sites.

A cell active chemical GEF inhibitor selectively targets the Trio/RhoG/Rac1 signaling pathway.

RhoGEFs (guanine nucleotide exchange factors of the Rho GTPase family) are upstream regulators of cell adhesion and migration pathways, thus representing attractive yet relatively unexplored targets for the development of anti-invasive drugs. We screened for chemical inhibitors of TrioN, the N-terminal GEF domain of the multidomain Trio protein, and identified ITX3 as a nontoxic inhibitor. In transfected mammalian cells, ITX3 blocked TrioN-mediated dorsal membrane ruffling and Rac1 activation while having no effect on GEF337-, Tiam1-, or Vav2-mediated RhoA or Rac1 activation. ITX3 specifically inhibited endogenous TrioN activity, as evidenced by its ability to inhibit neurite outgrowth in nerve growth factor (NGF)-stimulated PC12 cells or C2C12 differentiation into myotubes. This study introduces a selective cell active inhibitor of the Trio/RhoG/Rac1 pathway and validates RhoGEFs as druggable targets.

R-Cadherin expression inhibits myogenesis and induces myoblast transformation via Rac1 GTPase.

Cadherins are transmembrane glycoproteins that mediate Ca(2+)-dependent homophilic cell-cell adhesion and play a crucial role in proliferation, differentiation, and cell transformation. The goal of this study was to understand why R-cadherin is found in rhabdomyosarcomas (RMS), tumors of skeletal muscle origin, whereas it is absent in normal myoblasts. We show that R-cadherin expression in C2C12 myoblasts causes inhibition of myogenesis induction and impairment of cell cycle exit when cells are cultured in differentiation medium. Furthermore, R-cadherin expression elicits myoblast transformation, as shown by anchorage-independent growth in soft agar in vivo tumor formation assays and increased cell motility. In contrast, inhibition of R-cadherin expression using RNA interference hinders growth of RD cell line in soft agar and its tumorigenicity in mice. The analysis of the nature of R-cadherin-mediated signals shows that R-cadherin-dependent adhesion increases Rac1 activity. Dominant-negative forms of Rac1 inhibit R-cadherin-mediated signaling and transformation. In addition, expression of R-cadherin results in perturbed function of endogenous N-cadherin and M-cadherin. Together, these data suggest that R-cadherin expression inhibits myogenesis and induces myoblast transformation through Rac1 activation. Therefore, the properties of R-cadherin make it an attractive target for therapeutic intervention in RMS.