Pathogenic TRIO variants associated with neurodevelopmental disorders perturb the molecular regulation of TRIO and axon pathfinding in vivo.

The RhoGEF TRIO is known to play a major role in neuronal development by controlling actin cytoskeleton remodeling, primarily through the activation of the RAC1 GTPase. Numerous de novo mutations in the TRIO gene have been identified in individuals with neurodevelopmental disorders (NDDs). We have previously established the first phenotype/genotype correlation in TRIO-associated diseases, with striking correlation between the clinical features of the individuals and the opposite modulation of RAC1 activity by TRIO variants targeting different domains. The mutations hyperactivating RAC1 are of particular interest, as they are recurrently found in patients and are associated with a severe form of NDD and macrocephaly, indicating their importance in the etiology of the disease. Yet, it remains unknown how these pathogenic TRIO variants disrupt TRIO activity at a molecular level and how they affect neurodevelopmental processes such as axon outgrowth or guidance. Here we report an additional cohort of individuals carrying a pathogenic TRIO variant that reinforces our initial phenotype/genotype correlation. More importantly, by performing conformation predictions coupled to biochemical validation, we propose a model whereby TRIO is inhibited by an intramolecular fold and NDD-associated variants relieve this inhibition, leading to RAC1 hyperactivation. Moreover, we show that in cultured primary neurons and in the zebrafish developmental model, these gain-of-function variants differentially affect axon outgrowth and branching in vitro and in vivo, as compared to loss-of-function TRIO variants. In summary, by combining clinical, molecular, cellular and in vivo data, we provide compelling new evidence for the pathogenicity of novel genetic variants targeting the TRIO gene in NDDs. We report a novel mechanism whereby the fine-tuned regulation of TRIO activity is critical for proper neuronal development and is disrupted by pathogenic mutations.

Upregulated flotillins and sphingosine kinase 2 derail AXL vesicular traffic to promote epithelial-mesenchymal transition.

Altered endocytosis and vesicular trafficking are major players during tumorigenesis. Flotillin overexpression, a feature observed in many invasive tumors and identified as a marker of poor prognosis, induces a deregulated endocytic and trafficking pathway called upregulated flotillin-induced trafficking (UFIT). Here, we found that in non-tumoral mammary epithelial cells, induction of the UFIT pathway promotes epithelial-to-mesenchymal transition (EMT) and accelerates the endocytosis of several transmembrane receptors, including AXL, in flotillin-positive late endosomes. AXL overexpression, frequently observed in cancer cells, is linked to EMT and metastasis formation. In flotillin-overexpressing non-tumoral mammary epithelial cells and in invasive breast carcinoma cells, we found that the UFIT pathway-mediated AXL endocytosis allows its stabilization and depends on sphingosine kinase 2, a lipid kinase recruited in flotillin-rich plasma membrane domains and endosomes. Thus, the deregulation of vesicular trafficking following flotillin upregulation, and through sphingosine kinase 2, emerges as a new mechanism of AXL overexpression and EMT-inducing signaling pathway activation.

Flotillin membrane domains in cancer.

Flotillins 1 and 2 are two ubiquitous, highly conserved homologous proteins that assemble to form heterotetramers at the cytoplasmic face of the plasma membrane in cholesterol- and sphingolipid-enriched domains. Flotillin heterotetramers can assemble into large oligomers to form molecular scaffolds that regulate the clustering of at the plasma membrane and activity of several receptors. Moreover, flotillins are upregulated in many invasive carcinomas and also in sarcoma, and this is associated with poor prognosis and metastasis formation. When upregulated, flotillins promote plasma membrane invagination and induce an endocytic pathway that allows the targeting of cargo proteins in the late endosomal compartment in which flotillins accumulate. These late endosomes are not degradative, and participate in the recycling and secretion of protein cargos. The cargos of this Upregulated Flotillin-Induced Trafficking (UFIT) pathway include molecules involved in signaling, adhesion, and extracellular matrix remodeling, thus favoring the acquisition of an invasive cellular behavior leading to metastasis formation. Thus, flotillin presence from the plasma membrane to the late endosomal compartment influences the activity, and even modifies the trafficking and fate of key protein cargos, favoring the development of diseases, for instance tumors. This review summarizes the current knowledge on flotillins and their role in cancer development focusing on their function in cellular membrane remodeling and vesicular trafficking regulation.

P-cadherin-induced decorin secretion is required for collagen fiber alignment and directional collective cell migration.

Directional collective cell migration (DCCM) is crucial for morphogenesis and cancer metastasis. P-cadherin (also known as CDH3), which is a cell-cell adhesion protein expressed in carcinoma and aggressive sarcoma cells and associated with poor prognosis, is a major DCCM regulator. However, it is unclear how P-cadherin-mediated mechanical coupling between migrating cells influences force transmission to the extracellular matrix (ECM). Here, we found that decorin, a small proteoglycan that binds to and organizes collagen fibers, is specifically expressed and secreted upon P-cadherin, but not E- and R-cadherin (also known as CDH1 and CDH4, respectively) expression. Through cell biological and biophysical approaches, we demonstrated that decorin is required for P-cadherin-mediated DCCM and collagen fiber orientation in the migration direction in 2D and 3D matrices. Moreover, P-cadherin, through decorin-mediated collagen fiber reorientation, promotes the activation of ß1 integrin and of the ß-Pix (ARHGEF7)/CDC42 axis, which increases traction forces, allowing DCCM. Our results identify a novel P-cadherin-mediated mechanism to promote DCCM through ECM remodeling and ECM-guided cell migration.

MT1-MMP targeting to endolysosomes is mediated by upregulation of flotillins.

Tumor cell invasion and metastasis formation are the major cause of death in cancer patients. These processes rely on extracellular matrix (ECM) degradation mediated by organelles termed invadopodia, to which the transmembrane matrix metalloproteinase MT1-MMP (also known as MMP14) is delivered from its reservoir, the RAB7-containing endolysosomes. How MT1-MMP is targeted to endolysosomes remains to be elucidated. Flotillin-1 and -2 are upregulated in many invasive cancers. Here, we show that flotillin upregulation triggers a general mechanism, common to carcinoma and sarcoma, which promotes RAB5-dependent MT1-MMP endocytosis and its delivery to RAB7-positive endolysosomal reservoirs. Conversely, flotillin knockdown in invasive cancer cells greatly reduces MT1-MMP accumulation in endolysosomes, its subsequent exocytosis at invadopodia, ECM degradation and cell invasion. Our results demonstrate that flotillin upregulation is necessary and sufficient to promote epithelial and mesenchymal cancer cell invasion and ECM degradation by controlling MT1-MMP endocytosis and delivery to the endolysosomal recycling compartment.

Flotillins control zebrafish epiboly through their role in cadherin-mediated cell-cell adhesion.

Zebrafish gastrulation and particularly epiboly that involves coordinated movements of several cell layers is a dynamic process for which regulators remain to be identified. We show here that Flotillin 1 and 2, ubiquitous and highly conserved proteins, are required for epiboly. Flotillins knockdown compromised embryo survival, strongly delayed epiboly and impaired deep cell radial intercalation and directed collective migration without affecting enveloping layer cell movement. At the molecular level, we identified that Flotillins are required for the formation of E-cadherin-mediated cell-cell junctions. These results provide the first in vivo evidence that Flotillins regulate E-cadherin-mediated cell-cell junctions to allow epiboly progression.

The RhoE/ROCK/ARHGAP25 signaling pathway controls cell invasion by inhibition of Rac activity.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of skeletal muscle origin in children and adolescents. Among RMS subtypes, alveolar rhabdomyosarcoma (ARMS), which is characterized by the presence of the PAX3-FOXO1A or PAX7-FOXO1A chimeric oncogenic transcription factor, is associated with poor prognosis and a strong risk of metastasis compared with the embryonal subtype (ERMS). To identify molecular pathways involved in ARMS aggressiveness, we first characterized the migratory behavior of cell lines derived from ARMS and ERMS biopsies using a three-dimensional spheroid cell invasion assay. ARMS cells were more invasive than ERMS cells and adopted an ellipsoidal morphology to efficiently invade the extracellular matrix. Moreover, the invasive potential of ARMS cells depended on ROCK activity, which is regulated by the GTPase RhoE. Specifically, RhoE expression was low in ARMS biopsies, and its overexpression in ARMS cells reduced their invasion potential. Conversely, ARHGAP25, a GTPase-activating protein for Rac, was up-regulated in ARMS biopsies. Moreover, we found that ARHGAP25 inhibits Rac activity downstream of ROCKII and is required for ARMS cell invasion. Our results indicate that the RhoE/ROCK/ARHGAP25 signaling pathway promotes ARMS invasive potential and identify these proteins as potential therapeutic targets for ARMS treatment.

P-cadherin promotes collective cell migration via a Cdc42-mediated increase in mechanical forces.

Collective cell migration (CCM) is essential for organism development, wound healing, and metastatic transition, the primary cause of cancer-related death, and it involves cell-cell adhesion molecules of the cadherin family. Increased P-cadherin expression levels are correlated with tumor aggressiveness in carcinoma and aggressive sarcoma; however, how P-cadherin promotes tumor malignancy remains unknown. Here, using integrated cell biology and biophysical approaches, we determined that P-cadherin specifically induces polarization and CCM through an increase in the strength and anisotropy of mechanical forces. We show that this mechanical regulation is mediated by the P-cadherin/ß-PIX/Cdc42 axis; P-cadherin specifically activates Cdc42 through ß-PIX, which is specifically recruited at cell-cell contacts upon CCM. This mechanism of cell polarization and migration is absent in cells expressing E- or R-cadherin. Thus, we identify a specific role of P-cadherin through ß-PIX-mediated Cdc42 activation in the regulation of cell polarity and force anisotropy that drives CCM.

Flotillins in intercellular adhesion - from cellular physiology to human diseases.

Flotillin 1 and 2 are ubiquitous and highly conserved proteins. They were initially discovered in 1997 as being associated with specific caveolin-independent cholesterol- and glycosphingolipid-enriched membrane microdomains and as being expressed during axon regeneration. Flotillins have a role in a large number of physiopathological processes, mainly through their function in membrane receptor clustering and in the regulation of clathrin-independent endocytosis. In this Commentary, we summarize the research performed so far on the role of flotillins in cell-cell adhesion. Recent studies have demonstrated that flotillins directly regulate the formation of cadherin complexes. Indeed, flotillin microdomains are required for the dynamic association and stabilization of cadherins at cell-cell junctions and also for cadherin signaling. Moreover, because flotillins regulate endocytosis and also the actin cytoskeleton, they could have an indirect role in the assembly and stabilization of cadherin complexes. Because it has also recently been shown that flotillins are overexpressed during neurodegenerative diseases and in human cancers, where their upregulation is associated with metastasis formation and poor prognosis, understanding to what extent flotillin upregulation participates in the development of such pathologies is thus of particular interest, as well as how, at the molecular level, it might affect cell adhesion processes.

Flotillin microdomains stabilize cadherins at cell-cell junctions.

Cadherins are essential in many fundamental processes and assemble at regions of cell-cell contact in large macromolecular complexes named adherens junctions. We have identified flotillin 1 and 2 as new partners of the cadherin complexes. We show that flotillins are localised at cell-cell junctions (CCJs) in a cadherin-dependent manner. Flotillins and cadherins are constitutively associated at the plasma membrane and their colocalisation at CCJ increases with CCJ maturation. Using three-dimensional structured illumination super-resolution microscopy, we found that cadherin and flotillin complexes are associated with F-actin bundles at CCJs. The knockdown of flotillins dramatically affected N- and E-cadherin recruitment at CCJs in mesenchymal and epithelial cell types and perturbed CCJ integrity and functionality. Moreover, we determined that flotillins are required for cadherin association with GM1-containing plasma membrane microdomains. This allows p120 catenin binding to the cadherin complex and its stabilization at CCJs. Altogether, these data demonstrate that flotillin microdomains are required for cadherin stabilization at CCJs and for the formation of functional CCJs.

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.

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.

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.

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.

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.

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.

The GTPase RhoA increases utrophin expression and stability, as well as its localization at the plasma membrane.

The Rho family of small GTPases are signalling molecules involved in cytoskeleton remodelling and gene transcription. Their activities are important for many cellular processes, including myogenesis. In particular, RhoA positively regulates skeletal-muscle differentiation. We report in the present study that the active form of RhoA increases the expression of utrophin, the autosomal homologue of dystrophin in the mouse C2C12 and rat L8 myoblastic cell lines. Even though this RhoA-dependent utrophin increase is higher in proliferating myoblasts, it is maintained during myogenic differentiation. This occurs via two mechanisms: (i) transcriptional activation of the utrophin promoter A and (ii) post-translational stabilization of utrophin. In addition, RhoA increases plasma-membrane localization of utrophin. Thus RhoA activation up-regulates utrophin levels and enhances its localization at the plasma membrane.

N-cadherin association with lipid rafts regulates its dynamic assembly at cell-cell junctions in C2C12 myoblasts.

Cadherins are homophilic cell-cell adhesion molecules implicated in cell growth, differentiation, and organization into tissues during embryonic development. They accumulate at cell-cell contact sites and act as adhesion-activated signaling receptors. Here, we show that the dynamic assembly of N-cadherin at cell-cell contacts involves lipid rafts. In C2C12 myoblasts, immunofluorescence and biochemical experiments demonstrate that N-cadherin present at cell-cell contacts is colocalized with lipid rafts. Disruption of lipid rafts leads to the inhibition of cell-cell adhesion and disorganization of N-cadherin-dependent cell-cell contacts without modifying the association of N-cadherin with catenins and its availability at the plasma membrane. Fluorescent recovery after photobleaching experiments demonstrate that at the dorsal plasma membrane, lipid rafts are not directly involved in the diffusional mobility of N-cadherin. In contrast, at cell-cell junctions N-cadherin association with lipid rafts allows its stabilization enabling the formation of a functional adhesive complex. We show that lipid rafts, as homophilic interaction and F-actin association, stabilize cadherin-dependent adhesive complexes. Homophilic interactions and F-actin association of N-cadherin are both required for its association to lipid rafts. We thus identify lipid rafts as new regulators of cadherin-mediated cell adhesion.

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.