Cell-to-cell heterogeneity of EWSR1-FLI1 activity determines proliferation/migration choices in Ewing sarcoma cells.

Ewing sarcoma is characterized by the expression of the chimeric EWSR1-FLI1 transcription factor. Proteomic analyses indicate that the decrease of EWSR1-FLI1 expression leads to major changes in effectors of the dynamics of the actin cytoskeleton and the adhesion processes with a shift from cell-to-cell to cell-matrix adhesion. These changes are associated with a dramatic increase of in vivo cell migration and invasion potential. Importantly, EWSR1-FLI1 expression, evaluated by single-cell RT-ddPCR/immunofluorescence analyses, and activity, assessed by expression of EWSR1-FLI1 downstream targets, are heterogeneous in cell lines and in tumours and can fluctuate along time in a fully reversible process between EWSR1-FLI1high states, characterized by highly active cell proliferation, and EWSR1-FLI1low states where cells have a strong propensity to migrate, invade and metastasize. This new model of phenotypic plasticity proposes that the dynamic fluctuation of the expression level of a dominant oncogene is an intrinsic characteristic of its oncogenic potential.

p63/MT1-MMP axis is required for in situ to invasive transition in basal-like breast cancer.

The transition of ductal carcinoma in situ (DCIS) to invasive breast carcinoma requires tumor cells to cross the basement membrane (BM). However, mechanisms underlying BM transmigration are poorly understood. Here, we report that expression of membrane-type 1 (MT1)-matrix metalloproteinase (MMP), a key component of the BM invasion program, increases during breast cancer progression at the in situ to invasive breast carcinoma transition. In the intraductal xenograft model, MT1-MMP is required for BM transmigration of MCF10DCIS.com breast adenocarcinoma cells and is overexpressed in cell clusters overlying focal BM disruptions and at the invasive tumor front. Mirrored upregulation of p63 and MT1-MMP is observed at the edge of MCF10DCIS.com xenograft tumors and p63 is required for induction of MT1-MMP-dependent invasive program in response to microenvironmental signals. Immunohistochemistry and analysis of public database reveal that p63 and MT1-MMP are upregulated in human basal-like breast tumors suggesting that p63/MT1-MMP axis contributes to progression of basal-like breast cancers with elevated p63 and MT1-MMP levels.

Collective migration of an epithelial monolayer in response to a model wound.

Using an original microfabrication-based technique, we experimentally study situations in which a virgin surface is presented to a confluent epithelium with no damage made to the cells. Although inspired by wound-healing experiments, the situation is markedly different from classical scratch wounding because it focuses on the influence of the free surface and uncouples it from the other possible contributions such as cell damage and/or permeabilization. Dealing with Madin-Darby canine kidney cells on various surfaces, we found that a sudden release of the available surface is sufficient to trigger collective motility. This migration is independent of the proliferation of the cells that mainly takes place on the fraction of the surface initially covered. We find that this motility is characterized by a duality between collective and individual behaviors. On the one hand, the velocity fields within the monolayer are very long range and involve many cells in a coordinated way. On the other hand, we have identified very active "leader cells" that precede a small cohort and destabilize the border by a fingering instability. The sides of the fingers reveal a pluricellular actin "belt" that may be at the origin of a mechanical signaling between the leader and the followers. Experiments performed with autocrine cells constitutively expressing hepatocyte growth factor (HGF) or in the presence of exogenous HGF show a higher average velocity of the border and no leader.

Regulation of phagocytosis by Rho GTPases.

Phagocytosis is the mechanism of internalization used by specialized cells such as macrophages, dendritic cells, and neutrophils to internalize, degrade, and eventually present peptides derived from particulate antigens. The phagocytic process comprises several sequential and complex events initiated by the recognition ofligands on the surface of the particles by specific receptors on the surface of the phagocytic cells. Receptor clustering at the attachment site generates a phagocytic signal that in turn leads to local polymerization of actin filaments and to particle internalization. Depending on the particles and receptors involved, it appears that the structures and mechanisms associated with particle ingestion are diverse. However, work during the past few years has highlighted the importance of small GTP-binding proteins of the Rho family in various types of phagocytosis. As reviewed here, Rho family GTPases, their activators, and their downstream effectors control the local reorganization of the actin cytoskeleton beneath bound particles.

Actin dynamics during phagocytosis.

Bacteria, apoptotic cells and other particulate material are taken up through phagocytosis, a conserved cellular function driven by actin polymerization. As reviewed here, small GTPases of the Rho family, their activators and effectors control the local reorganization of the actin cytoskeleton underneath bound particles. Remarkably, the molecular actors and regulatory mechanisms involved during phagocytosis through the FcR or the CR3 receptors are very similar to those underlying the cytoskeletal rearrangements that take place at the leading edge of motile cell and at adhesion sites, respectively.

A WASp-VASP complex regulates actin polymerization at the plasma membrane.

Proteins of the Wiskott-Aldrich syndrome and Ena/VASP families both play essential functions in the regulation of actin dynamics at the cell leading edge. However, possibilities of functional interplay between members of these two families have not been addressed. Here we show that, in hemopoietic cells, recruitment of the C-terminal VCA (Verprolin homology, Cofilin homology, Acidic) domain of WASp at the plasma membrane by a ligand technique using rapamycin as an intermediate is not sufficient to elicit efficient Arp2/3 complex-mediated actin polymerization. Other domains of WASp, in particular the proline-rich domain, are required for the formation of actin-rich structures. An in vitro analysis demonstrates that the proline-rich domain of WASp binds VASP with an affinity of approximately 10(6) M(-1). In addition, WASp and VASP both accumulate in actin-rich phagocytic cups. Finally, in a reconstituted motility medium, VASP enhances actin-based propulsion of WASp-coated beads in a fashion reminiscent of its effect on Listeria movement. We propose that VASP and WASp cooperation is essential in stimulating actin assembly and membrane protrusion at the leading edge.

Function of Rho family proteins in actin dynamics during phagocytosis and engulfment.

Phagocytosis is the uptake of large particles by cells by a mechanism that is based on local rearrangement of the actin microfilament cytoskeleton. In higher organisms, phagocytic cells are essential for host defence against invading pathogens, and phagocytosis contributes to inflammation and the immune response. In addition, engulfment, defined as the phagocytic clearance of cell corpses generated by programmed cell death or apoptosis, has an essential role in tissue homeostasis. Although morphologically distinct phagocytic events can be observed depending on the type of surface receptor engaged, work over the past two years has revealed the essential underlying role of Rho family proteins and their downstream effectors in controlling actin dynamics during phagocytosis.

Membrane recruitment of Rac1 triggers phagocytosis.

Rac1 is a &Rgr;-family GTP-binding protein that controls lamellipodia formation and membrane ruffling in fibroblasts. Recently, Rac1 and Cdc42, another member of the &Rgr;-family, have been shown to regulate Fc receptor-mediated phagocytosis in macrophages by controlling different steps of membrane and actin dynamics leading to particle engulfment. Here, we investigated the function of Rac1 using a membrane recruitment system that mimics phagocytosis. Recruitment of an activated Rac1 protein to the cytoplasmic domain of an engineered membrane receptor by using rapamycin as a bridge induces ingestion of latex beads bound to the receptor. Rac1-mediated bead uptake depends on actin polymerisation since actin filaments accumulate at the bead/membrane binding sites and internalisation is inhibited by cytochalasin D. Internalisation is also abolished upon substitution of Phe37 to Leu in the Rac1 effector region. Our results indicate that by promoting actin polymerisation at particle attachment sites, Rac1 by acting through specific downstream effectors induces plasma membrane remodeling that allows particle internalisation in a membrane-enclosed phagosome.

The role of ARF and Rab GTPases in membrane transport.

Two key events of intracellular transport and membrane trafficking in eukaryotic cells, the formation of transport vesicles and their specific delivery to target membranes, are controlled by small GTPases of the ADP-ribosylation factor (ARF) and Rab families, respectively. The past 18 months have seen the identification of proteins that regulate ARF and Rab GDP/GTP cycle, as well as the characterization of their effectors, shedding light on the molecular mechanisms of ARF and Rab function.

Inducible recruitment of Cdc42 or WASP to a cell-surface receptor triggers actin polymerization and filopodium formation.

BACKGROUND: Cdc42, a GTP-binding protein of the Rho family, controls actin cytoskeletal organization and helps to generate actin-based protruding structures, such as filopodia. In vitro, Cdc42 regulates actin polymerization by facilitating the creation of free barbed ends - the more rapidly growing ends of actin filaments - and subsequent elongation at these ends. The Wiskott- Aldrich syndrome protein, WASP, which has a pleckstrin-homology domain and a Cdc42/Rac-binding motif, has been implicated in cell signaling and cytoskeleton reorganization. We have investigated the consequences of local recruitment of activated Cdc42 or WASP to the plasma membrane. RESULTS: We used an activated Cdc42 protein that could be recruited to an engineered membrane receptor by adding rapamycin as a bridge, and added antibody-coupled beads to aggregate these receptors. Inducible recruitment of Cdc42 to clusters of receptors stimulated actin polymerization, resulting in the formation of membrane protrusions. Cdc42-induced protrusions were enriched in the vasodilator-stimulated phosphoprotein VASP and the focal-adhesion-associated proteins zyxin and ezrin. The Cdc42 effector WASP could also induce the formation of protrusions, albeit of different morphology. CONCLUSIONS: This is the first demonstration that the local recruitment of activated Cdc42 or its downstream effector, WASP, to a membrane receptor in whole cells is sufficient to trigger actin polymerization that results in the formation of membrane protrusions. Our data suggest that Cdc42-induced actin-based protrusions result from the local and serial recruitment of cytoskeletal proteins including zyxin, VASP, and ezrin.

EFA6, a sec7 domain-containing exchange factor for ARF6, coordinates membrane recycling and actin cytoskeleton organization.

We have identified a human cDNA encoding a novel protein, exchange factor for ARF6 (EFA6), which contains Sec7 and pleckstrin homology domains. EFA6 promotes efficient guanine nucleotide exchange on ARF6 and is distinct from the ARNO family of ARF1 exchange factors. The protein localizes to a dense matrix on the cytoplasmic face of plasma membrane invaginations, induced on its expression. We show that EFA6 regulates endosomal membrane recycling and promotes the redistribution of transferrin receptors to the cell surface. Furthermore, expression of EFA6 induces actin-based membrane ruffles that are inhibited by co-expression of dominant-inhibitory mutant forms of ARF6 or Rac1. Our results demonstrate that by catalyzing nucleotide exchange on ARF6 at the plasma membrane and by regulating Rac1 activation, EFA6 coordinates endocytosis with cytoskeletal rearrangements.

Fc receptor-mediated phagocytosis requires CDC42 and Rac1.

At the surface of phagocytes, antibody-opsonized particles are recognized by surface receptors for the Fc portion of immunoglobulins (FcRs) that mediate their capture by an actin-driven process called phagocytosis which is poorly defined. We have analyzed the function of the Rho proteins Rac1 and CDC42 in the high affinity receptor for IgE (FcepsilonRI)-mediated phagocytosis using transfected rat basophil leukemia (RBL-2H3) mast cells expressing dominant inhibitory forms of CDC42 and Rac1. Binding of opsonized particles to untransfected RBL-2H3 cells led to the accumulation of F-actin at the site of contact with the particles and further, to particle internalization. This process was inhibited by Clostridium difficile toxin B, a general inhibitor of Rho GTP-binding proteins. Dominant inhibition of Rac1 or CDC42 function severely inhibited particle internalization but not F-actin accumulation. Inhibition of CDC42 function resulted in the appearance of pedestal-like structures with particles at their tips, while particles bound at the surface of the Rac1 mutant cell line were enclosed within thin membrane protrusions that did not fuse. These phenotypic differences indicate that Rac1 and CDC42 have distinct functions and may act cooperatively in the assembly of the phagocytic cup. Inhibition of phagocytosis in the mutant cell lines was accompanied by the persistence of tyrosine-phosphorylated proteins around bound particles. Phagocytic cup closure and particle internalization were also blocked when phosphotyrosine dephosphorylation was inhibited by treatment of RBL-2H3 cells with phenylarsine oxide, an inhibitor of protein phosphotyrosine phosphatases. Altogether, our data show that Rac1 and CDC42 are required to coordinate actin filament organization and membrane extension to form phagocytic cups and to allow particle internalization during FcR-mediated phagocytosis. Our data also suggest that Rac1 and CDC42 are involved in phosphotyrosine dephosphorylation required for particle internalization.

Tyrosine phosphorylation of the Wiskott-Aldrich syndrome protein by Lyn and Btk is regulated by CDC42.

The Wiskott-Aldrich syndrome (WAS) is a rare immunodeficiency disease affecting mainly platelets and lymphocytes. Here, we show that the WAS gene product, WASp, is tyrosine phosphorylated upon aggregation of the high affinity IgE receptor (Fc epsilonRI) at the surface of RBL-2H3 rat tumor mast cells. Lyn and the Bruton's tyrosine kinase (Btk), two protein tyrosine kinases involved in Fc epsilonRI-signaling phosphorylate WASp and interact with WASp in vivo. Interestingly, expression of a GTPase defective mutant form of CDC42, that interacts with WASp, is accompanied by a substantial increase in WASp tyrosine phosphorylation. This study suggests that activated CDC42 recruits WASp to the plasma membrane where it becomes phosphorylated by Lyn and Btk. We conclude that WASp represents a connection between protein tyrosine kinase signaling pathways and CDC42 function in cytoskeleton and cell growth regulation in hematopoietic cells.

ARNO3, a Sec7-domain guanine nucleotide exchange factor for ADP ribosylation factor 1, is involved in the control of Golgi structure and function.

Budding of transport vesicles in the Golgi apparatus requires the recruitment of coat proteins and is regulated by ADP ribosylation factor (ARF) 1. ARF1 activation is promoted by guanine nucleotide exchange factors (GEFs), which catalyze the transition to GTP-bound ARF1. We recently have identified a human protein, ARNO (ARF nucleotide-binding-site opener), as an ARF1-GEF that shares a conserved domain with the yeast Sec7 protein. We now describe a human Sec7 domain-containing GEF referred to as ARNO3. ARNO and ARNO3, as well as a third GEF called cytohesin-1, form a family of highly related proteins with identical structural organization that consists of a central Sec7 domain and a carboxy-terminal pleckstrin homology domain. We show that all three proteins act as ARF1 GEF in vitro, whereas they have no effect on ARF6, an ARF protein implicated in the early endocytic pathway. Substrate specificity of ARNO-like GEFs for ARF1 depends solely on the Sec7 domain. Overexpression of ARNO3 in mammalian cells results in (i) fragmentation of the Golgi apparatus, (ii) redistribution of Golgi resident proteins as well as the coat component beta-COP, and (iii) inhibition of SEAP transport (secreted form of alkaline phosphatase). In contrast, the distribution of endocytic markers is not affected. This study indicates that Sec7 domain-containing GEFs control intracellular membrane compartment structure and function through the regulation of specific ARF proteins in mammalian cells.

Differential properties of D4/LyGDI versus RhoGDI: phosphorylation and rho GTPase selectivity.

RhoA/B/C and CDC42/Rac, which form two subgroups of the rho guanosine triphosphatase (GTPase) family, regulate various aspects of actin cytoskeleton organisation. In cytosol, guanosine diphosphate (GDP) dissociation inhibitor (GDI) interacts with and maintains rho GTPases in their inactive GDP-bound form. RhoGDI is a ubiquitously expressed GDI, whereas D4/LyGDI is hematopoietic cell-specific and 10-fold less potent than RhoGDI in binding to and regulating rho GTPases. We have combined microanalytical liquid chromatography with the use of specific antibodies in order to separate D4/LyGDI and RhoDGI-complexes from the cytosol of U937 cells and to demonstrate that the two GDIs associate with different rho protein partners. RhoGDI can form a complex with CDC42Hs, RhoA, Rac1 and Rac2, while none of these GTPases was found to interact with D4/LyGDI. In addition, we found that stimulation of U937 cells with phorbol ester leads to phosphorylation of D4/LyGDI. Our results suggest that LyGDI forms complexes with specific rho GTPases expressed in hematopoietic cells where it may regulate specific pathways.