Regulation of Rho GTPase activity at the leading edge of migrating cells by p190RhoGAP.

Cell migration, a key feature of embryonic development, immunity, angiogenesis, and tumor metastasis, is based on the coordinated regulation of actin dynamics and integrin-mediated adhesion. Rho GTPases play a major role in this phenomenon by regulating the onset and maintenance of actin-based protruding structures at cell leading edges (i.e. lamellipodia and filopodia) and contractile structures (i.e., stress fibers) at their trailing edge. While spatio-temporal analysis demonstrated the tight regulation of Rho GTPases at the migration front during cell locomotion, little is known about how the main regulators of Rho GTPase activity, such as GAPs, GEFs and GDIs, play a role in this process. In this review, we focus on a major negative regulator of RhoA, p190RhoGAP-A and its close isoform p190RhoGAP-B, which are necessary for efficient cell migration. Recent studies, including our, demonstrated that p190RhoGAP-A localization and activity undergo a complex regulatory mechanism, accounting for the tight regulation of RhoA, but also other members of the Rho GTPase family, at the cell periphery.

Cancer-associated mutations in the protrusion-targeting region of p190RhoGAP impact tumor cell migration.

Spatiotemporal regulation of RhoGTPases such as RhoA is required at the cell leading edge to achieve cell migration. p190RhoGAP (p190A) is the main negative regulator of RhoA and localizes to membrane protrusions, where its GTPase-activating protein (GAP) activity is required for directional migration. In this study, we investigated the molecular processes responsible for p190A targeting to actin protrusions. By analyzing the subcellular localization of truncated versions of p190A in hepatocellular carcinoma cells, we identified a novel functional p190A domain: the protrusion localization sequence (PLS) necessary and sufficient for p190A targeting to leading edges. Interestingly, the PLS is also required for the negative regulation of p190A RhoGAP activity. Further, we show that the F-actin binding protein cortactin binds the PLS and is required for p190A targeting to protrusions. Lastly, we demonstrate that cancer-associated mutations in PLS affect p190A localization and function, as well as tumor cell migration. Altogether, our data unveil a new mechanism of regulation of p190A in migrating tumor cells.

The NG2 Proteoglycan Protects Oligodendrocyte Precursor Cells against Oxidative Stress via Interaction with OMI/HtrA2.

The NG2 proteoglycan is characteristically expressed by oligodendrocyte progenitor cells (OPC) and also by aggressive brain tumours highly resistant to chemo- and radiation therapy. Oligodendrocyte-lineage cells are particularly sensitive to stress resulting in cell death in white matter after hypoxic or ischemic insults of premature infants and destruction of OPC in some types of Multiple Sclerosis lesions. Here we show that the NG2 proteoglycan binds OMI/HtrA2, a mitochondrial serine protease which is released from damaged mitochondria into the cytosol in response to stress. In the cytosol, OMI/HtrA2 initiates apoptosis by proteolytic degradation of anti-apoptotic factors. OPC in which NG2 has been downregulated by siRNA, or OPC from the NG2-knockout mouse show an increased sensitivity to oxidative stress evidenced by increased cell death. The proapoptotic protease activity of OMI/HtrA2 in the cytosol can be reduced by the interaction with NG2. Human glioma expressing high levels of NG2 are less sensitive to oxidative stress than those with lower NG2 expression and reducing NG2 expression by siRNA increases cell death in response to oxidative stress. Binding of NG2 to OMI/HtrA2 may thus help protect cells against oxidative stress-induced cell death. This interaction is likely to contribute to the high chemo- and radioresistance of glioma.

Oligodendroglial p130Cas is a target of Fyn kinase involved in process formation, cell migration and survival.

Oligodendrocytes are the myelinating glial cells of the central nervous system. In the course of brain development, oligodendrocyte precursor cells migrate, scan the environment and differentiate into mature oligodendrocytes with multiple cellular processes which recognize and ensheath neuronal axons. During differentiation, oligodendrocytes undergo dramatic morphological changes requiring cytoskeletal rearrangements which need to be tightly regulated. The non-receptor tyrosine kinase Fyn plays a central role in oligodendrocyte differentiation and myelination. In order to improve our understanding of the role of oligodendroglial Fyn kinase, we have identified Fyn targets in these cells. Purification and mass-spectrometric analysis of tyrosine-phosphorylated proteins in response to overexpressed active Fyn in the oligodendrocyte precursor cell line Oli-neu, yielded the adaptor molecule p130Cas. We analyzed the function of this Fyn target in oligodendroglial cells and observed that reduction of p130Cas levels by siRNA affects process outgrowth, the thickness of cellular processes and migration behavior of Oli-neu cells. Furthermore, long term p130Cas reduction results in decreased cell numbers as a result of increased apoptosis in cultured primary oligodendrocytes. Our data contribute to understanding the molecular events taking place during oligodendrocyte migration and morphological differentiation and have implications for myelin formation.

NG2 regulates directional migration of oligodendrocyte precursor cells via Rho GTPases and polarity complex proteins.

The transmembrane proteoglycan NG2 is expressed by oligodendrocyte precursor cells (OPC), which migrate to axons during developmental myelination and remyelinate in the adult after migration to injured sites. Highly invasive glial tumors also express NG2. Despite the fact that NG2 has been implicated in control of OPC migration, its mode of action remains unknown. Here, we show in vitro and in vivo that NG2 controls migration of OPC through the regulation of cell polarity. In stab wounds in adult mice we show that NG2 controls orientation of OPC toward the wound. NG2 stimulates RhoA activity at the cell periphery via the MUPP1/Syx1 signaling pathway, which favors the bipolar shape of migrating OPC and thus directional migration. Upon phosphorylation of Thr-2256, downstream signaling of NG2 switches from RhoA to Rac stimulation. This triggers process outgrowth through regulators of front-rear polarity and we show using a phospho-mimetic form of NG2 that indeed NG2 recruits proteins of the CRB and the PAR polarity complexes to stimulate Rac activity via the GEF Tiam1. Our findings demonstrate that NG2 is a core organizer of Rho GTPase activity and localization in the cell, which controls OPC polarity and directional migration. This work also reveals CRB and PAR polarity complexes as new effectors of NG2 signaling in the establishment of front-rear polarity.

Cell shape and TGF-beta signaling define the choice of lineage during in vitro differentiation of mouse primary hepatic precursors.

Cellular differentiation relies on both physical and chemical environmental cues. The bipotential mouse embryonic liver (BMEL) cells are early progenitors of liver epithelial cells with an apparently infinite proliferative potential. These cells, which remain undifferentiated in a monolayer culture, differentiate upon release from geometrical constraints imposed by growth on a stiff plastic plate. In a complex three dimensional environment of a Matrigel extracellular matrix, BMEL cells form two types of polarized organoids of distinct morphologies: cyst-like structures suggesting cholangiocyte-type organization or complex organoids, reminiscent of liver parenchyma and associated with acquisition of hepatocyte-specific phenotypic markers. The choice of the in vitro differentiation lineage is governed by Transforming Growth Factor-beta (TGF-beta) signaling. Our results suggest that morphological cues initiate the differentiation of early hepatic precursors and confirm the inhibitory role of TGF-beta on hepatocytic lineage differentiation.

What makes cells move: requirements and obstacles for spontaneous cell motility.

Movement of individual cells and of cellular cohorts, chains or sheets requires physical forces that are established through interactions of cells with their environment. In vivo, migration occurs extensively during embryonic development and in adults during wound healing and tumorigenesis. In order to identify the molecular events involved in cell movement, in vitro systems have been developed. These have contributed to the definition of a number of molecular pathways put into play in the course of migratory behaviours, such as mesenchymal and amoeboid movement. More recently, our knowledge of migratory modes has been enriched by analyses of cells exploring and moving through three-dimensional (3D) matrices. While the cells' morphologies differ in 2D and 3D environments, the basic mechanisms that put a cellular body into motion are remarkably similar. Thus, in both 2D and 3D, the polarity of the migrating cell is initially defined by a specific subcellular localization of signalling molecules and components of molecular machines required for motion. While the polarization can be initiated either in response to extracellular signalling or be a chance occurrence, it is reinforced and sustained by positive feedback loops of signalling molecules. Second, adhesion to a substratum is necessary to generate forces that will propel the cell engaged in either mesenchymal or ameboid migration. For collective cell movement, intercellular coordination constitutes an additional requirement: a cell cohort remains stationary if individual cells pull in opposite directions. Finally, the availability of space to move into is a general requirement to set cells into motion. Lack of free space is probably the main obstacle for migration of most healthy cells in an adult multicellular organism. Thus, the requirements for cell movement are both intrinsic to the cell, involving coordinated signalling and interactions with molecular machines, and extrinsic, imposed by the physicochemical nature of the environment. In particular, the geometry and stiffness of the support act on a range of signalling pathways that induce specific cell migratory responses. These issues are discussed in the present review in the context of published work and our own data on collective migration of hepatocyte cohorts.

Calpain activation by hepatitis C virus proteins inhibits the extrinsic apoptotic signaling pathway.

UNLABELLED: An unresolved question regarding the physiopathology of hepatitis C virus (HCV) infection is the remarkable efficiency with which host defenses are neutralized to establish chronic infection. Modulation of an apoptotic response is one strategy used by viruses to escape immune surveillance. We previously showed that HCV proteins down-regulate expression of BH3-only Bcl2 interacting domain (Bid) in hepatocytes of HCV transgenic mice. As a consequence, cells acquire resistance to Fas-mediated apoptosis, which in turn leads to increased persistence of experimental viral infections in vivo. This mechanism might participate in the establishment of chronic infections and the resulting pathologies, including hepatocellular carcinoma. We now report that Bid is also down-regulated in patients in the context of noncirrhotic HCV-linked tumorigenesis and in the HCV RNA replicon system. We show that the nonstructural HCV viral protein NS5A is sufficient to activate a calpain cysteine protease, leading to degradation of Bid. Moreover, pharmacological inhibitors of calpains restore both the physiological levels of Bid and the sensitivity of cells toward a death receptor-mediated apoptotic signal. Finally, human HCV-related tumors and hepatocytes from HCV transgenic mice that display low Bid expression contain activated calpains. CONCLUSION: Calpains activated by HCV proteins degrade Bid and thus dampen apoptotic signaling. These results suggest that inhibiting calpains could lead to an improved efficiency of immune-mediated elimination of HCV-infected cells.