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.

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.

BIN1 regulates actin-membrane interactions during IRSp53-dependent filopodia formation.

Amphiphysin 2 (BIN1) is a membrane and actin remodeling protein mutated in congenital and adult centronuclear myopathies. Here, we report an unexpected function of this N-BAR domain protein BIN1 in filopodia formation. We demonstrated that BIN1 expression is necessary and sufficient to induce filopodia formation. BIN1 is present at the base of forming filopodia and all along filopodia, where it colocalizes with F-actin. We identify that BIN1-mediated filopodia formation requires IRSp53, which allows its localization at negatively-curved membrane topologies. Our results show that BIN1 bundles actin in vitro. Finally, we identify that BIN1 regulates the membrane-to-cortex architecture and functions as a molecular platform to recruit actin-binding proteins, dynamin and ezrin, to promote filopodia formation.

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.

A major sea-level drop briefly precedes the Toarcian oceanic anoxic event: implication for Early Jurassic climate and carbon cycle.

Sea-level change is an important parameter controlling the expansion of oxygen-depleted conditions in neritic settings during oceanic anoxic events (OAEs). Despite this fundamental role, it remains on a short timescale (<1 Myr) one of the least constrained parameters for numerous OAEs. Here we present sedimentological and geochemical evidence from Morocco and East Greenland showing that a forced regression shortly precedes (ca.102 kyr) the major transgression associated with the Toarcian OAE. The forced regression can be correlated over distances greater than 3000 km in numerous Tethyan and Boreal basins, indicating that the relative sea-level change was driven by eustastic fluctuations. The major amplitude (>50 m) and short duration of the forced regression suggests that it was most likely related to the transient waxing and waning of polar ice sheet. We suggest that this short-lived glaciation might have a genetic link with the inception of the Toarcian OAE. Indeed, during the deglaciation and the accompanying sea-level rise, the thawing permafrost may have released important quantities of methane into the atmosphere that would have contributed to the Toarcian OAE rapid warming and its characteristic negative carbon isotope excursion. This study offers a hypothesis on how some hyperthermal events might be rooted in short-lived "cold-snap" episodes.

Plasma membrane organization is essential for balancing competing pseudopod- and uropod-promoting signals during neutrophil polarization and migration.

Exposure of neutrophils to chemoattractant induces cell polarization and migration. These behaviors require the asymmetric activation of distinct signaling pathways and cytoskeletal elements in the protruding pseudopod at the front of cells and the retracting uropod at the rear. An important outstanding question is, how does the organization of the plasma membrane participate in establishing asymmetry during polarization and migration? To answer this question, we investigated the function of cholesterol, a lipid known to influence membrane organization. Using controlled cholesterol depletion, we found that a cholesterol-dependent membrane organization enabled cell polarization and migration by promoting uropod function and suppressing ectopic pseudopod formation. At a mechanistic level, we showed that cholesterol was directly required for suppressing inappropriate activation of the pseudopod-promoting Gi/PI3-kinase signaling pathway. Furthermore, cholesterol was required for dampening Gi-dependent negative feedback on the RhoA signaling pathway, thus enabling RhoA activation and uropod function. Our findings suggest a model in which a cholesterol-dependent membrane organization plays an essential role in the establishment of cellular asymmetry by balancing the activation and segregating the localization of competing pseudopod- and uropod-inducing signaling pathways during neutrophil polarization and migration.

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.

Lipid rafts are critical membrane domains in blood platelet activation processes.

Among the various hematopoi;etic cells, platelets are critical for maintaining the integrity of the vascular system. They must be rapidly activated by sequential and coordinated mechanisms in order to efficiently prevent haemorrhage upon vascular injury. Several signal transduction pathways lead to platelet activation in vitro and in vivo, among them, several are initiated via receptors or co-receptors containing immuno-receptor tyrosine-based activation motifs (ITAM) which trigger downstream signalling like the immune receptors in lymphocytes. However, in contrast to immune cells for which the role of lipid rafts in signalling has largely been described, the involvement of laterally segregated membrane microdomains in platelet activation has been investigated only recently. The results obtained until now strongly suggest that early steps of platelet activation via the collagen receptor GpVI or via FcgammaRIIa occur preferentially in these microdomains where specific proteins efficiently organize key downstream signalling pathways. In addition, lipid rafts also contribute to platelet activation via heterotrimeric G-protein-coupled receptors. They are sites where the phosphoinositide (PI) metabolism is highly active, leading to a local generation of lipid second messengers such as phosphatidylinositol 3,4,5-trisphosphate. Here, evidence is accumulating that cholesterol-enriched membrane microdomains are part of a general process that contributes to the efficiency and the coordination of platelet activation mechanisms. Here we will discuss the biochemical and functional characterizations of human platelet rafts and their potential impact in platelet physiopathology.

SH2-containing inositol 5-phosphatases 1 and 2 in blood platelets: their interactions and roles in the control of phosphatidylinositol 3,4,5-trisphosphate levels.

Src homology domain 2-containing inositol 5-phosphatases 1 and 2 (SHIP1 and SHIP2) are capable of dephosphorylating the second messenger PtdIns(3,4,5) P3 (phosphatidylinositol 3,4,5-trisphosphate) and interacting with several signalling proteins. SHIP1 is essentially expressed in haematopoietic cells, whereas SHIP2, a closely related enzyme, is ubiquitous. In the present study, we show that SHIP1 and SHIP2 are expressed as functional PtdIns(3,4,5) P3 5-phosphatases in human blood platelets and are capable of interacting when these two lipid phosphatases are co-expressed, either naturally (platelets and A20 B lymphoma cells) or artificially (COS-7 cells). Using COS-7 cells transfected with deletion mutants of SHIP2, we demonstrate that the Src homology domain 2 of SHIP2 is the minimal and sufficient protein motif responsible for the interaction between the two phosphatases. These results prompted us to investigate the relative importance of SHIP1 and SHIP2 in the control of PtdIns(3,4,5) P3 levels in platelets using homozygous or heterozygous SHIP1- or SHIP2-deficient mice. Our results strongly suggest that SHIP1, rather than SHIP2, plays a major role in controlling PtdIns(3,4,5) P3 levels in response to thrombin or collagen activation of mouse blood platelets.

A critical role of lipid rafts in the organization of a key FcgammaRIIa-mediated signaling pathway in human platelets.

The involvement of platelet FcgammaRIIa in heparin-associated thrombocytopenia (HIT) is now well established. However, the precise sequence of molecular events initiated by FcgammaRIIa cross-linking in platelets remains partly characterized. We investigated here the role of lipid rafts in the spatio-temporal organization of the FcgammaRIIa-dependent signaling events. Upon cross-linking, FcgammaRIIa relocated in rafts where the kinase Lyn and the adapter LAT were among the major phosphotyrosyl proteins. Upon stimulation by HIT sera, the second messenger phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) accumulated in rafts in a P(2)Y(12) adenosine diphosphate (ADP) receptor-dependent manner. PtdIns(3,4,5)P(3) was then essential to specifically recruit phospholipase Cgamma2 (PLCgamma2) to these membrane microdomains. Controlled disruption of rafts by methyl beta-cyclodextrin reversibly abolished PtdIns(3,4,5)P(3) production, PLC activation and platelet responses induced by FcgammaRIIa cross-linking without affecting the tyrosine phosphorylation events. This work demonstrates that platelet rafts are essential for the integration of a key signaling complex leading to the rapid production of PtdIns(3,4,5)P(3) and in turn PLCgamma2 activation during HIT.

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.

Integrin-dependent interaction of lipid rafts with the actin cytoskeleton in activated human platelets.

Dynamic connections between actin filaments and the plasma membrane are crucial for the regulation of blood platelet functions. Protein complexes associated with alphaIIbbeta3 integrin-based cytoskeleton structures are known to play a role in these processes. However, mechanisms involving lateral organizations of the plasma membrane remain to be investigated. Here, we demonstrate that a large fraction of platelet lipid rafts specifically associates with the actin cytoskeleton upon activation. This association was inhibited by antagonists of fibrinogen-alphaIIbbeta3 binding and did not occur in type I Glanzman's thrombasthenic platelets. The raft-cytoskeleton interaction is a reversible process correlating with the intensity and stability of platelet aggregation. Although only a minor fraction of alphaIIbbeta3 was recovered in rafts upon activation, this integrin specifically upregulated the level of PtdIns(4,5)P2 in membrane microdomains and induced the recruitment of several actin-modulating proteins known to directly or indirectly interact with this lipid. Controlled disruption of rafts did not affect alphaIIbbeta3-mediated platelet aggregation in response to high concentrations of thrombin but significantly inhibited fibrin clot retraction. We propose that rafts participate in the organization of membrane-cytoskeleton interactions where alphaIIbbeta3-mediated tension forces apply during the late phase of platelet activation.

The tyrosine phosphatase 1B regulates linker for activation of T-cell phosphorylation and platelet aggregation upon FcgammaRIIa cross-linking.

Human platelets express the receptor for immunoglobulin G, FcgammaRIIa, that triggers cell aggregation upon interaction with immune complexes. Here, we report that the rapid tyrosine phosphorylation of the Linker for Activation of T-cell (LAT) in human platelets stimulated by FcgammaRIIa cross-linking was followed by its complete dephosphorylation in an alphaIIb/beta3 integrin-dependent manner. Concomitant to LAT dephosphorylation, the protein tyrosine phosphatase 1B (PTP1B) was activated through a mechanism involving its proteolysis by calpains downstream of integrins. Both PTP1B and LAT were associated with the actin cytoskeleton complex formed during platelet aggregation. Moreover, phospho-LAT appeared as a good substrate of activated PTP1B in vitro and these two proteins interacted upon platelet activation by FcgammaRIIa cross-linking. The permeant substrate-trapping PTP1B (TAT-PTP1B D181A) partly inhibited LAT dephosphorylation in human platelets, strongly suggesting that this tyrosine phosphatase was involved in this regulatory pathway. Using a pharmacological inhibitor, we provide evidence that PTP1B activation and LAT dephosphorylation processes were required for irreversible platelet aggregation. Altogether, our results demonstrate that PTP1B plays an important role in the integrin-mediated dephosphorylation of LAT in human platelets and is involved in the control of irreversible aggregation upon FcgammaRIIa stimulation.