ARHGAP17 regulates the spatiotemporal activity of Cdc42 at invadopodia.

Invadopodia formation is regulated by Rho GTPases. However, the molecular mechanisms that control Rho GTPase signaling at invadopodia remain poorly understood. Here, we have identified ARHGAP17, a Cdc42-specific RhoGAP, as a key regulator of invadopodia in breast cancer cells and characterized a novel ARHGAP17-mediated signaling pathway that controls the spatiotemporal activity of Cdc42 during invadopodia turnover.

Quantification of ruffle area and dynamics in live or fixed lung adenocarcinoma cells.

Ruffles are actin-rich membrane protrusions implicated in actin reorganization and initiation of cell motility. Here, we describe methods for measuring and analyzing ruffle dynamics in live cells and average ruffle area per cell in fixed samples. The specific steps described are for the analysis of A549 lung adenocarcinoma cells, but the protocol can be applied to other cell types.

FARP1, ARHGEF39, and TIAM2 are essential receptor tyrosine kinase effectors for Rac1-dependent cell motility in human lung adenocarcinoma.

Despite the undisputable role of the small GTPase Rac1 in the regulation of actin cytoskeleton reorganization, the Rac guanine-nucleotide exchange factors (Rac-GEFs) involved in Rac1-mediated motility and invasion in human lung adenocarcinoma cells remain largely unknown. Here, we identify FARP1, ARHGEF39, and TIAM2 as essential Rac-GEFs responsible for Rac1-mediated lung cancer cell migration upon EGFR and c-Met activation. Noteworthily, these Rac-GEFs operate in a non-redundant manner by controlling distinctive aspects of ruffle dynamics formation.

SGEF forms a complex with Scribble and Dlg1 and regulates epithelial junctions and contractility.

The canonical Scribble polarity complex is implicated in regulation of epithelial junctions and apical polarity. Here, we show that SGEF, a RhoG-specific GEF, forms a ternary complex with Scribble and Dlg1, two members of the Scribble complex. SGEF targets to apical junctions in a Scribble-dependent fashion and functions in the regulation of actomyosin-based contractility and barrier function at tight junctions as well as E-cadherin-mediated formation of adherens junctions.

The small GTPase RhoG regulates microtubule-mediated focal adhesion disassembly.

Focal adhesions (FA) are a complex network of proteins that allow the cell to form physical contacts with the extracellular matrix (ECM). FA assemble and disassemble in a dynamic process, orchestrated by a variety of cellular components. However, the underlying mechanisms that regulate adhesion turnover remain poorly understood.

Regulation of circular dorsal ruffles, macropinocytosis, and cell migration by RhoG and its exchange factor, Trio.

Circular dorsal ruffles (CDRs) are actin-rich structures that form on the dorsal surface of many mammalian cells in response to growth factor stimulation. CDRs represent a unique type of structure that forms transiently and only once upon stimulation. The formation of CDRs involves a drastic rearrangement of the cytoskeleton, which is regulated by the Rho family of GTPases.

A RhoG-mediated signaling pathway that modulates invadopodia dynamics in breast cancer cells.

One of the hallmarks of cancer is the ability of tumor cells to invade surrounding tissues and metastasize. During metastasis, cancer cells degrade the extracellular matrix, which acts as a physical barrier, by developing specialized actin-rich membrane protrusion structures called invadopodia. The formation of invadopodia is regulated by Rho GTPases, a family of proteins that regulates the actin cytoskeleton.

mDia2 and CXCL12/CXCR4 chemokine signaling intersect to drive tumor cell amoeboid morphological transitions.

Morphological plasticity in response to environmental cues in migrating cancer cells requires F-actin cytoskeletal rearrangements. Conserved formin family proteins play critical roles in cell shape, tumor cell motility, invasion and metastasis, in part, through assembly of non-branched actin filaments. Diaphanous-related formin-2 (mDia2/Diaph3/Drf3/Dia) regulates mesenchymal-to-amoeboid morphological conversions and non-apoptotic blebbing in tumor cells by interacting with its inhibitor diaphanous-interacting protein (DIP), and disrupting cortical F-actin assembly and bundling.

The actin crosslinking protein palladin modulates force generation and mechanosensitivity of tumor associated fibroblasts.

Cells organize actin filaments into higher-order structures by regulating the composition, distribution and concentration of actin crosslinkers. Palladin is an actin crosslinker found in the lamellar actin network and stress fibers, which are critical for mechanosensing of the environment. Palladin also serves as a molecular scaffold for α-actinin, another key actin crosslinker.

Palladin expression is a conserved characteristic of the desmoplastic tumor microenvironment and contributes to altered gene expression.

The stroma surrounding solid tumors contributes in complex ways to tumor progression. Cancer-associated fibroblasts (CAFs) are the predominant cell type in the tumor stroma. Previous studies have shown that the actin-binding protein palladin is highly expressed in the stroma of pancreas tumors, but the interpretation of these results is complicated by the fact that palladin exists as multiple isoforms.

I'm coming to GEF you: Regulation of RhoGEFs during cell migration.

Cell migration is a highly regulated multistep process that requires the coordinated regulation of cell adhesion, protrusion, and contraction. These processes require numerous protein-protein interactions and the activation of specific signaling pathways. The Rho family of GTPases plays a key role in virtually every aspect of the cell migration cycle.

Structure and function of palladin's actin binding domain.

Here, we report the NMR structure of the actin-binding domain contained in the cell adhesion protein palladin. Previously, we demonstrated that one of the immunoglobulin domains of palladin (Ig3) is both necessary and sufficient for direct filamentous actin binding in vitro. In this study, we identify two basic patches on opposite faces of Ig3 that are critical for actin binding and cross-linking.

The actin associated protein palladin is important for the early smooth muscle cell differentiation.

Palladin, an actin associated protein, plays a significant role in regulating cell adhesion and cell motility. Palladin is important for development, as knockdown in mice is embryonic lethal, yet its role in the development of the vasculature is unknown. We have shown that palladin is essential for the expression of smooth muscle cells (SMC) marker genes and force development in response to agonist stimulation in palladin deficient SMCs.

Isoform-specific upregulation of palladin in human and murine pancreas tumors.

Pancreatic ductal adenocarcinoma (PDA) is a lethal disease with a characteristic pattern of early metastasis, which is driving a search for biomarkers that can be used to detect the cancer at an early stage. Recently, the actin-associated protein palladin was identified as a candidate biomarker when it was shown that palladin is mutated in a rare inherited form of PDA, and overexpressed in many sporadic pancreas tumors and premalignant precursors. In this study, we analyzed the expression of palladin isoforms in murine and human PDA and explored palladin's potential use in diagnosing PDA.

Cytoplasmic Ig-domain proteins: cytoskeletal regulators with a role in human disease.

Immunoglobulin domains are found in a wide variety of functionally diverse transmembrane proteins, and also in a smaller number of cytoplasmic proteins. Members of this latter group are usually associated with the actin cytoskeleton, and most of them bind directly to either actin or myosin, or both. Recently, studies of inherited human disorders have identified disease-causing mutations in five cytoplasmic Ig-domain proteins: myosin-binding protein C, titin, myotilin, palladin, and myopalladin.

Roles of the small GTPases RhoA and Rac1 in cell behavior.

The Rho-family GTPases are proving to have a variety of biological functions apart from their well known effects on the cytoskeleton. Recent work indicates their involvement in signaling between the adhesion receptors integrin and syndecan, effects on the recruitment of beta-catenin to the nucleus, and potential roles in the nucleus as well as the cytoplasm.

Characterization of cortactin as an in vivo protein kinase D substrate: interdependence of sites and potentiation by Src.

Protein Kinase D (PKD) has been implicated in the regulation of actin turnover at the leading edge, invasion and migration. In particular, a complex between cortactin, paxillin and PKD in the invadopodia of invasive breast cancer cells has been described earlier, but so far this complex remained ill defined. Here we have investigated the possible role of PKD as a cortactin kinase.

The role of palladin in actin organization and cell motility.

Palladin is a widely expressed protein found in stress fibers, focal adhesions, growth cones, Z-discs, and other actin-based subcellular structures. It belongs to a small gene family that includes the Z-disc proteins myopalladin and myotilin, all of which share similar Ig-like domains. Recent advances have shown that palladin shares with myotilin the ability to bind directly to F-actin, and to crosslink actin filaments into bundles, in vitro.

Palladin binds to Eps8 and enhances the formation of dorsal ruffles and podosomes in vascular smooth muscle cells.

Palladin is a widely expressed phosphoprotein that plays an important role in organizing the actin cytoskeleton. Palladin is concentrated in multiple actin-based structures involved in cell motility and adhesion, including stress fibers, focal adhesions, cell-cell junctions, growth cones and Z-discs. Here, we show that palladin also localizes to the dorsal, circular ruffles that form transiently in response to growth factor stimulation.

Low density lipoprotein receptor-related protein is a calreticulin coreceptor that signals focal adhesion disassembly.

Thrombospondin (TSP) signals focal adhesion disassembly (the intermediate adhesive state) through interactions with cell surface calreticulin (CRT). TSP or a peptide (hep I) of the active site induces focal adhesion disassembly through binding to CRT, which activates phosphoinositide 3-kinase (PI3K) and extracellular signal-related kinase (ERK) through Galphai2 proteins. Because CRT is not a transmembrane protein, it is likely that CRT signals as part of a coreceptor complex.

The anti-adhesive activity of thrombospondin is mediated by the N-terminal domain of cell surface calreticulin.

Thrombospondin (TSP) induces reorganization of the actin cytoskeleton and restructuring of focal adhesions through binding of amino acids (aa) 17-35 (hep I peptide) of thrombospondin to a cell surface form of calreticulin (CRT). In this report we provide further evidence for the involvement of calreticulin in thrombospondin signaling and characterize thrombospondin-calreticulin interactions. Wild type but not crt(-/-) cells respond to hep I/TSP.

Nck-2 interacts with focal adhesion kinase and modulates cell motility.

Nck-2 is a ubiquitously expressed adaptor protein comprising primarily three N-terminal SH3 domains and one C-terminal SH2 domain. We report here that Nck-2 interacts with focal adhesion kinase (FAK), a cytoplasmic protein tyrosine kinase critically involved in the cellular control of motility. Using a mutational strategy, we have found that the formation of the Nck-2-FAK complex is mediated by interactions involving multiple SH2 and SH3 domains of Nck-2.