Lamellipodial Protrusion Research Articles

Rac function is critical for cell migration but not required for spreading and focal adhesion formation

SummaryCell migration is commonly accompanied by protrusion of membrane ruffles and lamellipodia. In two-dimensional migration, protrusion of these thin sheets of cytoplasm is considered relevant to both exploration of new space and initiation of nascent adhesion to the substratum. Lamellipodium formation can be potently stimulated by Rho GTPases of the Rac subfamily, but also by RhoG or Cdc42. Here we describe viable fibroblast cell lines genetically deficient for Rac1 that lack detectable levels of Rac2 and Rac3. Rac-deficient cells were devoid of apparent lamellipodia, but these structures were restored by expression of either Rac subfamily member, but not by Cdc42 or RhoG. Cells deficient in Rac showed strong reduction in wound closure and random cell migration and a notable loss of sensitivity to a chemotactic gradient. Despite these defects, Rac-deficient cells were able to spread, formed filopodia and established focal adhesions. Spreading in these cells was achieved by the extension of filopodia followed by the advancement of cytoplasmic veils between them. The number and size of focal adhesions as well as their intensity were largely unaffected by genetic removal of Rac1. However, Rac deficiency increased the mobility of different components in focal adhesions, potentially explaining how Rac – although not essential – can contribute to focal adhesion assembly. Together, our data demonstrate that Rac signaling is essential for lamellipodium protrusion and for efficient cell migration, but not for spreading or filopodium formation. Our findings also suggest that Rac GTPases are crucial to the establishment or maintenance of polarity in chemotactic migration.

On the existence and strength of stable membrane protrusions

We present a mathematical model for the protrusion of lamellipodia in motile cells. The model lamellipodium consists of a viscoelastic actin gel in the bulk and a dynamic boundary layer of newly polymerized filaments at the leading edge called the semiflexible region (SR). The density of filaments in the SR can increase due to nucleation of new filaments and decrease due to capping and severing of existing filaments. Following on from previous publications, we present important approximations that make the model feasible and accessible to fast computational analysis. It reveals that there are three qualitatively different parameter regimes: a stable, stationarily protruding lamellipodium; a stable lamellipodium showing oscillatory motion of the leading edge; and zero filament density and no stable lamellipodium. Hence, the model defines criteria for the existence of lamellipodia and the ability of cells to move effectively, and we discuss which parameter changes can induce transitions between the different states. Furthermore, stable lamellipodia have to be able to exert and withstand substantial forces. We can fit the experimentally measured dynamic force–velocity relation that describes how cells can adapt to increasing external forces when encountering an obstacle in their environment during motion. Moreover, we predict a different stationary force–velocity relation that should apply if cells experience a constant force, e.g. exerted by the surrounding tissue.

Sarcomere-Like Units Contract Cell Edges

Extracellular matrix rigidity directs cell behavior and key processes in development and tumorigenesis. During spreading and migration, cells produce periodic lamellipodial protrusion retractions cycles that require a rigid substrate and the development of adhesions1. Moreover, there is evidence that rigidity is detected locally in the region near the cell edge2. The motor protein myosin II powers cell retractions, which are simultaneous with rearward flow of the actin filament network1. However, the pattern and mechanism of forces exerted by cells to test matrix rigidity have not been characterized. using high-resolution force sensing pillars, we show here that the primary forces powering periodic edge retraction come from local contraction units, which exert inward forces near the cell edge, correlated with outward forces 2-3 micrometers rearwards from the edge. Pillar displacement correlates with the local concentrations of active myosin II and the actin crosslinking protein alpha-actinin, but not with local levels of other common actin binding proteins. The contractile units therefore display similarity to striated muscle and stress fiber units, which are ∼1-3 micrometers long, rely on myosin II for contractility, and contain alpha-actinin as a major component of the complex that anchors actin filaments. Thus, our results indicate that local contractile units, with sarcomere-like organization, are responsible for periodic cell edge retractions and rigidity sensing.

Free-radical polymer science structural cancer model: a review.

Polymer free-radical lipid alkene chain-growth biological models particularly for hypoxic cellular mitochondrial metabolic waste can be used to better understand abnormal cancer cell morphology and invasive metastasis. Without oxygen as the final electron acceptor for mitochondrial energy synthesis, protons cannot combine to form water and instead mitochondria produce free radicals and acid during hypoxia. Nonuniform bond-length shrinkage of membranes related to erratic free-radical covalent crosslinking can explain cancer-cell pleomorphism with epithelial-mesenchymal transition for irregular membrane borders that “ruffle” and warp over stiff underlying actin fibers. Further, mitochondrial hypoxic conditions produce acid that can cause molecular degradation. Subsequent low pH-activated enzymes then provide paths for invasive cell movement through tissue and eventually blood-born metastasis. Although free-radical crosslinking creates irregularly shaped membranes with structural actin-polymerized fiber extensions as filopodia and lamellipodia, due to rapid cell division the overall cell modulus (approximately stiffness) is lower than normal cells. When combined with low pH-activated enzymes and lower modulus cells, smaller cancer stem cells subsequently have a large advantage to follow molecular destructive pathways and leave the central tumor. In addition, forward structural spike-like lamellipodia protrusions can leverage to force lower-modulus cancer cells through narrow openings. By squeezing and deforming even smaller to allow for easier movement through difficult passageways, cancer cells can travel into adjacent tissues or possibly metastasize through the blood to new tissue.

Cortactin Expression Is Tightly Connected to B-Cell Chronic Lymphocytic Leukemia Aggressiveness

AbstractAbstract 4561 Background.Cortactin is an ubiquitous actin-binding protein, encoded by EMS1 gene and localized in chromosome 11q13 region. This protein is expressed in nearly all mammalian tissues and mostly localizes to dynamic actin structures. As a consequence, cortactin is involved in regulating several actin-dependent processes, including lamellipodium protrusion, trafficking of the key invadopodia proteases and intracellular transducer downstream to kinase-mediated cell signalling upon phosphorylation by Src and Syk tyrosine kinase families. Cortactin is over-expressed in several tumors, such as esophageal squamous cell carcinoma, head and neck squamous cell carcinoma and gastric carcinoma, and so tied to tumor aggressiveness by the promotion of cell invasion and metastasis. We previously showed that cortactin is over-expressed also in neoplastic B cells of patients with B-Cell Chronic Lymphocytic Leukemia (B-CLL). In the present study we investigated the regulation of cortactin activity by the Src kinase Lyn in neoplastic B-CLL cells and the biological implications of cortactin overexpression in this leukemia. Methods.Twenty patients and ten normal controls were enrolled. Informed consent was obtained from all patients according to the Declaration of Helsinki. CD19+ lymphocytes were purified from peripheral blood by negative selection using the RosetteSep cells isolation kit (StemCell Technologies). Cortactin localization was performed by Confocal Microscope analysis at basal condition, in presence of the chemotactic stimulus CXCL12 and Src kinase inhibitor PP2. Lyn kinase and cortactin phosphorylation levels were evaluated by western blotting analysis. Migration assay was performed using 3 μm pore filters (Transwell Permeable Supports) in the presence of CXCL12, Src kinase inhibitor PP2 and after cortactin silencing. Results.We found that cortactin localization is regulated by Lyn kinase through its phosphorylation. In CLL cells, cortactin was over-phosphorylated and it did not co-localize with actin, as compared to normal cells. PP2-inhibition of Lyn decreased cortactin phosphorylation and triggered its co-localization with actin. Cortactin over-expression proved to be associated to the increased B-CLL spreading through Lyn activity. In fact, not only cortactin over-expression correlated with leukemic cell increased response to CXCL12 (r = 0.9), but also the inhibition of cortactin activity, through PP2 inhibitor or cortactin silencing, drastically reduced neoplastic cell migration after chemotactic triggering. Conclusions.These results suggest that cortactin is involved in aggressiveness and spreading of B-CLL cells and that Lyn-cortactin axis could represent an alternative target for the development of new therapeutic strategies. Disclosures:No relevant conflicts of interest to declare.

Progressive Bundling of Fibrillin Microfibrils into Oxytalan Fibers in the Chick Presumptive Dermis

Dorsoventral fibers in the presumptive dermis of the chick limb bud reported first by Hurle's group in 1989 are now revealed as bundles of fibrillin microfibrils (Isokawa et al., 2004). The bundles, which could be called oxytalan fibers at the light microscopic level, are aligned perpendicularly to the overlying ectoderm and form a unique fiber array, originating directly from the basal lamina. This well-oriented organization is beneficial in examining the process of in vivo bundling of microfibrils into oxytalan fibers. In this study, sections through the presumptive limb dermis were preferentially prepared from chick embryos at Days 4-6 (ED4-6). Immunohistochemically, fibrillin-positive dots representing cross-sectioned surfaces of individual fibers, increased in size from ED4 to 6, but their number per unit area remained constant. Ultrastructurally, a single oxytalan fiber at ED4 consisted of ∼15 microfibrils; the latter number increased fourfold from ED4 to 5 and threefold from ED5 to 6. Oxytalan fibers were all closely associated with mesenchymal cell; notably, the fibers at ED5 and 6 were held in a shallow ditch on the cell body or by lamellipodial cytoplasmic protrusion. In the sites of cell-fiber adhesion, microfibrils in the periphery of an oxytalan fiber appeared to adhere directly or by means of short flocculent strands to a nearby cell membrane; the latter showed a thickening of plasmalemma and its undercoat, indicating the presence of adhesive membrane specification. These findings suggest that the bundling of microfibrils is a progressive and closely cell-associated process.

Evidence for a cell cycle checkpoint that senses branched actin in the lamellipodium

Recent evidence indicates that branched actin might control cell progression through G1 in addition to lamellipodium protrusion.

Transient receptor potential canonical channel 1 impacts on mechanosignaling during cell migration

Cell migration is crucial for many important physiological and pathophysiological processes ranging from embryogenesis to tumor metastasis. It requires the coordination of mechanical forces generated in different regions of the migrating cell. It has been proposed that stretch-activated, Ca(2+)-permeable channels are involved in mechanosignaling during cell migration. To date, the molecular identity of these channels is only poorly defined. Here, we investigated the contribution of TRPC1 channels to mechanosignaling during cell migration. We used primary cultures of synovial fibroblasts from TRPC1(-/-) mice and the wild-type littermates or Madin-Darby canine kidney (MDCK-F) cells with increased or decreased TRPC1 expression. TRPC1(-/-) fibroblasts have the same migratory phenotype as siTRPC1 MDCK-F cells, with a largely increased projected cell area and impaired directionality. Measurements of the intracellular Ca(2+) concentration ([Ca(2+)](i)) were combined with time-lapse video microscopic cell migration experiments. Cells were seeded on elastic silicone membranes. Uniaxial stretch elicits a graded elevation of the [Ca(2+)](i) in TRPC1-expressing cells. In contrast, TRPC1(-/-) fibroblasts or siTRPC1 MDCK-F cells do not react to 0.4%, and the response to 4% stretch is attenuated. Similarly, siTRPC1 MDCK-F cells do not alter their direction of migration upon mechanical stimulation, which contrasts the behavior of TRPC1-overexpressing cells which turn into the direction of stretch. Impaired mechanosignaling in siTRPC1 MDCK-F cells leads to accelerated lamellipodial protrusions. Finally, artificially decreasing membrane tension with the detergent deoxycholate impairs the migration of TRPC1-overexpressing cells, but not of siTRPC1 cells. Taken together, our findings indicate that TRPC1 channels are linked to mechanosignaling during cell migration.

Function of Ahnak protein in aortic smooth muscle cell migration through Rac activation

Ahnak protein acts as a scaffold protein networking phospholipase C-γ and protein kinase C-α, which subsequently stimulate an extracellular signal-regulated kinase (Erk) pathway. In mouse aortic smooth muscle cells (ASMCs), the activation of the signalling cascade ultimately promotes the cell migration through an unknown mechanism. We aimed to dissect the Ahnak-mediated cell signalling network involved in the migration of ASMCs. Migration of ASMCs from wild-type mice was significantly increased by platelet-derived growth factor (PDGF) stimulation in transwell chamber and wound-healing assays, whereas migration of ASMCs from Ahnak knockout mice was reduced. Consistently, stimulation of wild-type ASMCs with PDGF resulted in Rac activation-mediated lamellipodial protrusion in migrating cells. In contrast, Ahnak knockout ASMCs displayed lower activation of Rac in response to PDGF and slow lamellipodial protrusion rate and cell migration. Ahnak signalling complex was analysed by immunoprecipitation with antibody to p21-activated protein kinase (PAK). Ahnak protein was shown to function as the signalling scaffold interacting with the multiple protein complex of Erk, PAK, and p21-activated kinase-interacting exchange factor β. The proposed role of Ahnak in cell migration was examined using a restenosis model in which the carotid arteries of mice were subjected to post-ligation injury. We show neointimal formation and SMC migration after ligation injury in Ahnak knockout mice were significantly retarded compared with wild-type mice. Ahnak protein plays an important scaffolding function connecting Erk and Rac activation in PDGF-dependent migration of ASMC.

The Palmitoylation State of PMP22 Modulates Epithelial Cell Morphology and Migration

PMP22 (peripheral myelin protein 22), also known as GAS 3 (growth-arrest-specific protein 3), is a disease-linked tetraspan glycoprotein of peripheral nerve myelin and constituent of intercellular junctions in epithelia. To date, our knowledge of the post-translational modification of PMP22 is limited. Using the CSS-Palm 2.0 software we predicted that C85 (cysteine 85), a highly conserved amino acid located between the second and third transmembrane domains, is a potential site for palmitoylation. To test this, we mutated C85S (C85 to serine) and established stable cells lines expressing the WT (wild-type) or the C85S-PMP22. In Schwann and MDCK (Madin–Darby canine kidney) cells mutating C85 blocked the palmitoylation of PMP22, which we monitored using 17-ODYA (17-octadecynoic acid). While palmitoylation was not necessary for processing the newly synthesized PMP22 through the secretory pathway, overexpression of C85S-PMP22 led to pronounced cell spreading and uneven monolayer thinning. To further investigate the functional significance of palmitoylated PMP22, we evaluated MDCK cell migration in a wound-healing assay. While WT-PMP22 expressing cells were resistant to migration, C85S cells displayed lamellipodial protrusions and migrated at a similar rate to vector control. These findings indicate that palmitoylation of PMP22 at C85 is critical for the role of the protein in modulating epithelial cell shape and motility.

Cells Lacking β-Actin are Genetically Reprogrammed and Maintain Conditional Migratory Capacity*

Vertebrate nonmuscle cells express two actin isoforms: cytoplasmic β- and γ-actin. Because of the presence and localized translation of β-actin at the leading edge, this isoform is generally accepted to specifically generate protrusive forces for cell migration. Recent evidence also implicates β-actin in gene regulation. Cell migration without β-actin has remained unstudied until recently and it is unclear whether other actin isoforms can compensate for this cytoplasmic function and/or for its nuclear role. Primary mouse embryonic fibroblasts lacking β-actin display compensatory expression of other actin isoforms. Consistent with this preservation of polymerization capacity, β-actin knockout cells have unchanged lamellipodial protrusion rates despite a severe migration defect. To solve this paradox we applied quantitative proteomics revealing a broad genetic reprogramming of β-actin knockout cells. This also explains why reintroducing β-actin in knockout cells does not restore the affected cell migration. Pathway analysis suggested increased Rho-ROCK signaling, consistent with observed phenotypic changes. We therefore developed and tested a model explaining the phenotypes in β-actin knockout cells based on increased Rho-ROCK signaling and increased TGFβ production resulting in increased adhesion and contractility in the knockout cells. Inhibiting ROCK or myosin restores migration of β-actin knockout cells indicating that other actins compensate for β-actin in this process. Consequently, isoactins act redundantly in providing propulsive forces for cell migration, but β-actin has a unique nuclear function, regulating expression on transcriptional and post-translational levels, thereby preventing myogenic differentiation.

Functions of Nonmuscle Myosin II in Assembly of the Cellular Contractile System

The contractile system of nonmuscle cells consists of interconnected actomyosin networks and bundles anchored to focal adhesions. The initiation of the contractile system assembly is poorly understood structurally and mechanistically, whereas system’s maturation heavily depends on nonmuscle myosin II (NMII). Using platinum replica electron microscopy in combination with fluorescence microscopy, we characterized the structural mechanisms of the contractile system assembly and roles of NMII at early stages of this process. We show that inhibition of NMII by a specific inhibitor, blebbistatin, in addition to known effects, such as disassembly of stress fibers and mature focal adhesions, also causes transformation of lamellipodia into unattached ruffles, loss of immature focal complexes, loss of cytoskeleton-associated NMII filaments and peripheral accumulation of activated, but unpolymerized NMII. After blebbistatin washout, assembly of the contractile system begins with quick and coordinated recovery of lamellipodia and focal complexes that occurs before reappearance of NMII bipolar filaments. The initial formation of focal complexes and subsequent assembly of NMII filaments preferentially occurred in association with filopodial bundles and concave actin bundles formed by filopodial roots at the lamellipodial base. Over time, accumulating NMII filaments help to transform the precursor structures, focal complexes and associated thin bundles, into stress fibers and mature focal adhesions. However, semi-sarcomeric organization of stress fibers develops at much slower rate. Together, our data suggest that activation of NMII motor activity by light chain phosphorylation occurs at the cell edge and is uncoupled from NMII assembly into bipolar filaments. We propose that activated, but unpolymerized NMII initiates focal complexes, thus providing traction for lamellipodial protrusion. Subsequently, the mechanical resistance of focal complexes activates a load-dependent mechanism of NMII polymerization in association with attached bundles, leading to assembly of stress fibers and maturation of focal adhesions.

Reconstitution of in vivo macrophage-tumor cell pairing and streaming motility on one-dimensional micro-patterned substrates

In mammary tumors, intravital imaging techniques have uncovered an essential role for macrophages during tumor cell invasion and metastasis mediated by an epidermal growth factor (EGF) / colony stimulating factor-1 (CSF-1) paracrine loop. It was previously demonstrated that mammary tumors in mice derived from rat carcinoma cells (MTLn3) exhibited high velocity migration on extracellular matrix (ECM) fibers. These cells form paracrine loop-dependent linear assemblies of alternating host macrophages and tumor cells known as "streams." Here, we confirm by intravital imaging that similar streams form in close association with ECM fibers in a highly metastatic patient-derived orthotopic mammary tumor (TN1). To understand the in vivo cell motility behaviors observed in streams, an in vitro model of fibrillar tumor ECM utilizing adhesive 1D micropatterned substrates was developed. MTLn3 cells on 1D fibronectin or type I collagen substrates migrated with higher velocity than on 2D substrates and displayed enhanced lamellipodial protrusion and increased motility upon local interaction and pairing with bone marrow-derived macrophages (BMMs). Inhibitors of EGF or CSF-1 signaling disrupted this interaction and reduced tumor cell velocity and protrusion, validating the requirement for an intact paracrine loop. Both TN1 and MTLn3 cells in the presence of BMMs were capable of co-assembling into linear arrays of alternating tumor cells and BMMs that resembled streams in vivo, suggesting the stream assembly is cell autonomous and can be reconstituted on 1D substrates. Our results validate the use of 1D micropatterned substrates as a simple and defined approach to study fibrillar ECM-dependent cell pairing, migration and relay chemotaxis as a complementary tool to intravital imaging.

Cell crawling mediates collective cell migration to close undamaged epithelial gaps

Fundamental biological processes such as morphogenesis and wound healing involve the closure of epithelial gaps. Epithelial gap closure is commonly attributed either to the purse-string contraction of an intercellular actomyosin cable or to active cell migration, but the relative contribution of these two mechanisms remains unknown. Here we present a model experiment to systematically study epithelial closure in the absence of cell injury. We developed a pillar stencil approach to create well-defined gaps in terms of size and shape within an epithelial cell monolayer. Upon pillar removal, cells actively respond to the newly accessible free space by extending lamellipodia and migrating into the gap. The decrease of gap area over time is strikingly linear and shows two different regimes depending on the size of the gap. In large gaps, closure is dominated by lamellipodium-mediated cell migration. By contrast, closure of gaps smaller than 20 μm was affected by cell density and progressed independently of Rac, myosin light chain kinase, and Rho kinase, suggesting a passive physical mechanism. By changing the shape of the gap, we observed that low-curvature areas favored the appearance of lamellipodia, promoting faster closure. Altogether, our results reveal that the closure of epithelial gaps in the absence of cell injury is governed by the collective migration of cells through the activation of lamellipodium protrusion.

Gadkin negatively regulates cell spreading and motility via sequestration of the actin-nucleating ARP2/3 complex

Regulation of actin dynamics is key to many cell physiological processes, ranging from protrusion formation and control of cell shape to cellular motility, endocytosis, and vesicle movement. The actin-related protein (ARP)2/3 complex is a major actin nucleator organizing branched filament networks in lamellipodial protrusions and during cell migration downstream of nucleation-promoting factors (NPFs). Although many NPFs have been characterized in detail, only few ARP2/3 inhibitors are known. Here, we identify the trans-Golgi network (TGN)/endosomally localized adaptor protein (AP)-1-associated adaptor protein Gadkin as a negative regulator of ARP2/3 function. Loss of Gadkin is associated with a partial redistribution of ARP2/3 to the plasma membrane and with increased cell spreading and migration, phenotypes that depend on the presence of a functional ARP2/3 complex. Gadkin directly binds to ARP2/3 via a conserved tryptophan-based acidic cluster motif reminiscent of ARP2/3-binding sequences of NPFs but fails to facilitate ARP2/3-mediated actin assembly. Consistent with an inhibitory role of Gadkin on ARP2/3 function, ARP2/3 is found on motile Gadkin-containing endosomal vesicles under migration-inhibiting conditions from where it relocalizes to the plasma membrane following activation of NPFs. Together with the observation that Gadkin-mediated inhibition of cell spreading requires its binding to ARP2/3, these data indicate that Gadkin is a negative regulator of ARP2/3 function present on intracellular membranes.

PWE-007 Paracrine prostaglandin-E signalling modulates canine gastric epithelial cell migration

IntroductionGastric ulceration is a limiting complication of therapy with cyclooxygenase (COX) antagonists, widely used anti-inflammatory/analgesic drugs in both humans and dogs. COX-derived prostaglandin E (PGE) has an important role in...

Role of Coronin 1B in PDGF-Induced Migration of Vascular Smooth Muscle Cells

The type I subclass of coronins, a family of actin-binding proteins, regulates various actin-dependent cellular processes, including migration. However, the existence and role of coronins in vascular smooth muscle cell (VSMC) migration has yet to be determined. The goal of the present study was to define the mechanism by which coronins regulate platelet-derived growth factor (PDGF)-induced VSMC migration. Coronin 1B (Coro1B) and 1C (Coro1C) were both found to be expressed in VSMCs at the mRNA and protein levels. Downregulation of Coro1B by siRNA increases PDGF-induced migration, while downregulation of Coro1C has no effect. We confirmed through kymograph analysis that the Coro1B-downregulation-mediated increase in migration is directly linked to increased lamellipodial protraction rate and protrusion distance in VSMC. In other cell types, coronins exert their effects on lamellipodia dynamics by an inhibitory interaction with the ARP2/3 complex, which is disrupted by the phosphorylation of Coro1B. We found that PDGF induces phosphorylation of Coro1B on serine-2 via PKCε, leading to a decrease in the interaction of Coro1B with the ARP2/3 complex. VSMCs transfected with a phosphodeficient S2A Coro1B mutant showed decreased migration in response to PDGF, suggesting that the phosphorylation of Coro1B is required for the promotion of migration by PDGF. In both the rat and mouse, Coro1B phosphorylation was increased in response to vessel injury in vivo. Our data suggest that phosphorylation of Coro1B and the subsequent reduced interaction with ARP2/3 complex participate in PDGF-induced VSMC migration, an important step in vascular lesion formation.

FMNL2 Drives Actin-Based Protrusion and Migration Downstream of Cdc42

SummaryCell migration entails protrusion of lamellipodia, densely packed networks of actin filaments at the cell front. Filaments are generated by nucleation, likely mediated by Arp2/3 complex and its activator Scar/WAVE [1]. It is unclear whether formins contribute to lamellipodial actin filament nucleation or serve as elongators of filaments nucleated by Arp2/3 complex [2]. Here we show that the Diaphanous-related formin FMNL2, also known as FRL3 or FHOD2 [3], accumulates at lamellipodia and filopodia tips. FMNL2 is cotranslationally modified by myristoylation and regulated by interaction with the Rho-guanosine triphosphatase Cdc42. Abolition of myristoylation or Cdc42 binding interferes with proper FMNL2 activation, constituting an essential prerequisite for subcellular targeting. In vitro, C-terminal FMNL2 drives elongation rather than nucleation of actin filaments in the presence of profilin. In addition, filament ends generated by Arp2/3-mediated branching are captured and efficiently elongated by the formin. Consistent with these biochemical properties, RNAi-mediated silencing of FMNL2 expression decreases the rate of lamellipodia protrusion and, accordingly, the efficiency of cell migration. Our data establish that the FMNL subfamily member FMNL2 is a novel elongation factor of actin filaments that constitutes the first Cdc42 effector promoting cell migration and actin polymerization at the tips of lamellipodia.

Alternative cellular roles for proteins identified using proteomics

The recent report by Grigera et al. [1] provided interesting new data on the ability of the actin-binding protein cortactin to associate with regulator of chromosome condensation-2 (RCC2; also known as TD-60), a component of the mitotic machinery [2]. Owing to the localisation of cortactin to the equatorial plane of dividing cells, the authors inferred that its interaction with RCC2 was functionally important in mitosis. This may be the case, but the data also support an alternative interpretation that RCC2 may play a role in cortactin-mediated branched actin regulation during cell movement. Cortactin promotes actin nucleation and stabilises F-actin, and thus it regulates many cell motility processes, such as focal adhesion assembly, lamellipodial protrusion, vesicle transport and endocytosis [3], although the full gamut of cortactin function is incompletely understood. Cortactin strongly localises to the leading edge of migrating cells, where it is phosphorylated, and signalling to small guanosine triphosphatases (GTPases), such as Rac1, plays an important part in its function [4,5]. Engagement of integrin adhesion receptors leads to activation of small GTPases, which play a central role in the modulation of cell adhesion complexes [6,7]. Using a proteomic approach, we showed that specific integrin adhesion complexes contain RCC2 (Fig. 1) [8–10]. Moreover, we demonstrated that, through precise restriction of Rac1 and Arf6 GTPase activity, RCC2 controls directional cell motility [8,9,11]. Indeed, our findings suggest that, rather than acting as a putative guanine nucleotide exchange factor and activating Rac1, RCC2 limits the activation of Rac1. Fig. 1 Proteomic analysis of molecular interactions can reveal alternative cellular roles for proteins. RCC2 (red circle, or node, in interaction network) is a passenger protein with a role in cell division [2]. Proteomic and bioinformatic analyses of integrin ... Together with the work of Grigera et al. [1], our data indicate an intriguing overlap between molecules implicated in cell adhesion processes and RCC2 function. Thus, the data reported by Grigera et al. [1] do not exclude the possibility that RCC2 interacts with cortactin as a component of the cell migration machinery [8,9] or indeed of integrin adhesion-regulated mitosis [12,13]. A current lack of data reporting the colocalisation of RCC2 with cortactin prevents a complete understanding of the interaction between these molecules, but further study of their contributions to cell behaviour is warranted. The works discussed above exemplify the discovery of unexpected cellular roles for proteins; such examples continue to increase as more data-driven proteomic studies appear in the literature. The consequence, in the postgenomic era, is that a single gene cannot be correlated with a single protein function. Proteins may possess multifunctionality within a single polypeptide chain, known as “moonlighting” [14], or as different splice variants. Therefore, understanding the context of protein interactions is critical to gaining mechanistic insight into protein function. Many MS-based proteomic studies rely on analysis of cell or tissue lysates, in which cellular context (e.g. subcellular localisation or cell type-specific expression) has been eliminated. Furthermore, many analyses of proteomic data use protein interaction networks as a tool for data interrogation. Such analyses lever the wealth of knowledge about all reported protein interactions and can be incredibly informative, including by suggesting alternative functions for proteins [15], but these datasets should be interpreted in the context of their derivation and the methodological limitations. Reported interactions in large-scale interaction networks have usually been amalgamated from data from multiple conditions, cell types and subcellular locations. With a lack of cellular context, proteins can appear to be promiscuous binders, whereas, in a cell, they may bind to distinct partners under restricted conditions. Accordingly, to achieve mechanistic insight into protein function, the analysis of protein interaction networks should rarely be the final interpretation of MS data, but instead be the springboard for further experimentation. Overall, data-driven MS-based proteomic and bioinformatic analyses, within the framework of hypothesis-driven research, provide a valuable opportunity for the discovery of new cellular roles for proteins, and will continue to provide surprising results about biological systems.

BART Inhibits Pancreatic Cancer Cell Invasion by Rac1 Inactivation through Direct Binding to Active Rac1

We report that Binder of Arl Two (BART) plays a role in inhibiting cell invasion by regulating the activity of the Rho small guanosine triphosphatase protein Rac1 in pancreatic cancer cells. BART was originally identified as a binding partner of ADP-ribosylation factor-like 2, a small G protein implicated as a regulator of microtubule dynamics and folding. BART interacts with active forms of Rac1, and the BART-Rac1 complex localizes at the leading edges of migrating cancer cells. Suppression of BART increases active Rac1, thereby increasing cell invasion. Treatment of pancreatic cancer cells in which BART is stably knocked down with a Rac1 inhibitor decreases invasiveness. Thus, BART-dependent inhibition of cell invasion is likely associated with decreased active Rac1. Suppression of BART induces membrane ruffling and lamellipodial protrusion and increases peripheral actin structures in membrane ruffles at the edges of lamellipodia. The Rac1 inhibitor inhibits the lamellipodia formation that is stimulated by suppression of BART. Our results imply that BART regulates actin-cytoskeleton rearrangements at membrane ruffles through modulation of the activity of Rac1, which, in turn, inhibits pancreatic cancer cell invasion.