The ß3-integrin endothelial adhesome regulates microtubule-dependent cell migration.

Integrin ß3 is seen as a key anti-angiogenic target for cancer treatment due to its expression on neovasculature, but the role it plays in the process is complex; whether it is pro- or anti-angiogenic depends on the context in which it is expressed. To understand precisely ß3's role in regulating integrin adhesion complexes in endothelial cells, we characterised, by mass spectrometry, the ß3-dependent adhesome. We show that depletion of ß3-integrin in this cell type leads to changes in microtubule behaviour that control cell migration. ß3-integrin regulates microtubule stability in endothelial cells through Rcc2/Anxa2-driven control of active Rac1 localisation. Our findings reveal that angiogenic processes, both in vitro and in vivo, are more sensitive to microtubule targeting agents when ß3-integrin levels are reduced.

Chlorhexidine hexametaphosphate as a wound care material coating: antimicrobial efficacy, toxicity and effect on healing.

AIM: In this study, chlorhexidine hexametaphosphate (CHX-HMP) is investigated as a persistent antimicrobial coating for wound care materials. MATERIALS & METHODS: CHX-HMP was used as a wound care material coating and compared with chlorhexidine digluconate materials with respect to antimicrobial efficacy, toxicity and wound closure. RESULTS: Antimicrobial efficacy at day 1, 3 and 7 was observed with experimental and commercial materials. CHX-HMP coated materials had less toxic effect on human placental cells than commercial chlorhexidine dressings. CHX-HMP in pluronic gel did not delay healing but reduced wound colonization by E. faecalis. CONCLUSION: CHX-HMP could become a useful component of wound care materials with sustained antimicrobial efficacy, lower toxicity than chlorhexidine digluconate materials, and reduction in wound colonization without affecting closure.

Comparing the Affinity of GTPase-binding Proteins using Competition Assays.

In this protocol we demonstrate a method for comparing the competition between GTPase-binding proteins. Such an approach is important for determining the binding capabilities of GTPases for two reasons: The fact that all interactions involve the same face of the GTPases means that binding events must be considered in the context of competitors, and the fact that the bound nucleotide must also be controlled means that conventional approaches such as immunoprecipitation are unsuitable for GTPase biochemistry. The assay relies on the use of purified proteins. Purified Rac1 immobilized on beads is used as the bait protein, and can be loaded with GDP, a non-hydrolyzable version of GTP or left nucleotide free, so that the signaling stage to be investigated can be controlled. The binding proteins to be investigated are purified from mammalian cells, to allow correct folding, by means of a GFP tag. Use of the same tag on both proteins is important because not only does it allow rapid purification and elution, but also allows detection of both competitors with the same antibody during elution. This means that the relative amounts of the two bound proteins can be determined accurately.

Ultrasonic Stimulation of Mouse Skin Reverses the Healing Delays in Diabetes and Aging by Activation of Rac1.

Chronic skin-healing defects are one of the leading challenges to lifelong well-being, affecting 2-5% of populations. Chronic wound formation is linked to age and diabetes and frequently leads to major limb amputation. Here we identify a strategy to reverse fibroblast senescence and improve healing rates. In healthy skin, fibronectin activates Rac1 in fibroblasts, causing migration into the wound bed, and driving wound contraction. We discover that mechanical stimulation of the skin with ultrasound can overturn healing defects by activating a calcium/CamKinaseII/Tiam1/Rac1 pathway that substitutes for fibronectin-dependent signaling and promotes fibroblast migration. Treatment of diabetic and aged mice recruits fibroblasts to the wound bed and reduces healing times by 30%, restoring healing rates to those observed in young, healthy animals. Ultrasound treatment is equally effective in rescuing the healing defects of animals lacking fibronectin receptors, and can be blocked by pharmacological inhibition of the CamKinaseII pathway. Finally, we discover that the migration defects of fibroblasts from human venous leg ulcer patients can be reversed by ultrasound, demonstrating that the approach is applicable to human chronic samples. By demonstrating that this alternative Rac1 pathway can substitute for that normally operating in the skin, we identify future opportunities for management of chronic wounds.

Coronin-1C Protein and Caveolin Protein Provide Constitutive and Inducible Mechanisms of Rac1 Protein Trafficking.

Sustained directional fibroblast migration requires both polarized activation of the protrusive signal, Rac1, and redistribution of inactive Rac1 from the rear of the cell so that it can be redistributed or degraded. In this work, we determine how alternative endocytic mechanisms dictate the fate of Rac1 in response to the extracellular matrix environment. We discover that both coronin-1C and caveolin retrieve Rac1 from similar locations at the rear and sides of the cell. We find that coronin-1C-mediated extraction, which is responsible for Rac1 recycling, is a constitutive process that maintains Rac1 protein levels within the cell. In the absence of coronin-1C, the effect of caveolin-mediated endocytosis, which targets Rac1 for proteasomal degradation, becomes apparent. Unlike constitutive coronin-1C-mediated trafficking, caveolin-mediated Rac1 endocytosis is induced by engagement of the fibronectin receptor syndecan-4. Such an inducible endocytic/degradation mechanism would predict that, in the presence of fibronectin, caveolin defines regions of the cell that are resistant to Rac1 activation but, in the absence of fibronectin leaves more of the membrane susceptible to Rac1 activation and protrusion. Indeed, we demonstrate that fibronectin-stimulated activation of Rac1 is accelerated in the absence of caveolin and that, when caveolin is knocked down, polarization of active Rac1 is lost in FRET experiments and culminates in shunting migration in a fibrous fibronectin matrix. Although the concept of polarized Rac1 activity in response to chemoattractants has always been apparent, our understanding of the balance between recycling and degradation explains how polarity can be maintained when the chemotactic gradient has faded.

Integration of the Rac1- and actin-binding properties of Coronin-1C.

The coronin family of actin-binding proteins regulate actin branching by inhibiting Arp2/3. We recently reported 2 interactions that were unique to coronin-1C: binding of a Rac1 inhibitor, RCC2, to the unique linker region and Rac1 itself to the propeller domain in a manner that differs from that proposed for other coronins. Through these interactions coronin-1C redistributes Rac1 from the back of the cell to the leading edge for either activation or sequestration by the associated Rac1-inhibitor, RCC2. Here we investigate the relationship between the Rac1- and actin-binding properties of coronin-1C and find that, although actin appears to be involved in the retrafficking of Rac1, signaling by Rac1 lies upstream of the stress fiber-formation, for which the coronins were originally characterized.

Coronin-1C and RCC2 guide mesenchymal migration by trafficking Rac1 and controlling GEF exposure.

Sustained forward migration through a fibrillar extracellular matrix requires localization of protrusive signals. Contact with fibronectin at the tip of a cell protrusion activates Rac1, and for linear migration it is necessary to dampen Rac1 activity in off-axial positions and redistribute Rac1 from non-protrusive membrane to the leading edge. Here, we identify interactions between coronin-1C (Coro1C), RCC2 and Rac1 that focus active Rac1 to a single protrusion. Coro1C mediates release of inactive Rac1 from non-protrusive membrane and is necessary for Rac1 redistribution to a protrusive tip and fibronectin-dependent Rac1 activation. The second component, RCC2, attenuates Rac1 activation outside the protrusive tip by binding to the Rac1 switch regions and competitively inhibiting GEF action, thus preventing off-axial protrusion. Depletion of Coro1C or RCC2 by RNA interference causes loss of cell polarity that results in shunting migration in 1D or 3D culture systems. Furthermore, morpholinos against Coro1C or RCC2, or mutation of any of the binding sites in the Rac1-RCC2-Coro1C complex delays the arrival of neural crest derivatives at the correct location in developing zebrafish, demonstrating the crucial role in migration guidance in vivo.

Resolution mediator chemerin15 reprograms the wound microenvironment to promote repair and reduce scarring.

Disorders of cutaneous repair can cause disability or death given that skin functions as a protective barrier against the external environment. The inflammatory response triggered by tissue damage is thought to play both positive (e.g., pathogen-killing) and negative (e.g., scarring) roles in repair. Inflammatory resolution mediators such as chemerin15 (C15) control the magnitude and duration of the inflammatory response; however, their role in wound repair and scarring is unknown. Here, we show that the C15 precursor, chemerin, and its receptor, ChemR23, are both upregulated after skin damage and that the receptor is expressed by macrophages, neutrophils, and keratinocytes. Dynamic live-imaging studies of murine cutaneous wounds demonstrate that C15 delivery dampens the immediate intravascular inflammatory events, including platelet adhesion to neutrophils, an important event in driving leukocyte recruitment. C15 administration indirectly accelerates wound closure while altering fibroblast-mediated collagen deposition and alignment to reduce scarring. Macrophage recruitment is restricted to the immediate wound site rather than spilling extensively into the adjacent tissue as in control wounds, and macrophage phenotype in C15-treated wounds is skewed toward a less inflammatory phenotype with reduced iNOS, increased Arginase-1, and lower wound tumor necrosis factor α (TNF-α) expression. Modulation of inflammatory resolution pathways in acute and chronic wounds may therefore provide a novel therapeutic avenue to improve repair and reduce scarring.

Syndecan-4 phosphorylation is a control point for integrin recycling.

Precise spatiotemporal coordination of integrin adhesion complex dynamics is essential for efficient cell migration. For cells adherent to fibronectin, differential engagement of α5ß1 and αVß3 integrins is used to elicit changes in adhesion complex stability, mechanosensation, matrix assembly, and migration, but the mechanisms responsible for receptor regulation have remained largely obscure. We identify phosphorylation of the membrane-intercalated proteoglycan syndecan-4 as an essential switch controlling integrin recycling. Src phosphorylates syndecan-4 and, by driving syntenin binding, leads to suppression of Arf6 activity and recycling of αVß3 to the plasma membrane at the expense of α5ß1. The resultant elevation in αVß3 engagement promotes stabilization of focal adhesions. Conversely, abrogation of syndecan-4 phosphorylation drives surface expression of α5ß1, destabilizes adhesion complexes, and disrupts cell migration. These data identify the dynamic spatiotemporal regulation of Src-mediated syndecan-4 phosphorylation as an essential switch controlling integrin trafficking and adhesion dynamics to promote efficient cell migration.

Syndecan and integrin interactomes: large complexes in small spaces.

The syndecan family of transmembrane proteoglycans cooperate with integrins to regulate both early and late events in adhesion formation. The heparan sulphate chains substituted on to the syndecan ectodomains are capable of engaging ligands over great distance, while the protein core spans the plasma membrane and initiates cytoplasmic signals through a short cytoplasmic tail. These properties create a spatial paradox. The volume of the heparan sulphate chains greatly exceeds that of the integrins with which it cooperates, while the short cytodomain must bind to multiple cytoplasmic factors, despite being long enough to bind only one or two. In this review we consider the structural rearrangements that a cell undertakes to overcome spatial restrictions and compare the interactomes of syndecans and integrins to gain insight into the composition of adhesions and how they are regulated over time.

Induction of adhesion-dependent signals using low-intensity ultrasound.

In multicellular organisms, cell behavior is dictated by interactions with the extracellular matrix. Consequences of matrix-engagement range from regulation of cell migration and proliferation, to secretion and even differentiation. The signals underlying each of these complex processes arise from the molecular interactions of extracellular matrix receptors on the surface of the cell. Integrins are the prototypic receptors and provide a mechanical link between extracellular matrix and the cytoskeleton, as well as initiating some of the adhesion-dependent signaling cascades. However, it is becoming increasingly apparent that additional transmembrane receptors function alongside the integrins to regulate both the integrin itself and signals downstream. The most elegant of these examples is the transmembrane proteoglycan, syndecan-4, which cooperates with α(5)ß(1)-integrin during adhesion to fibronectin. In vivo models demonstrate the importance of syndecan-4 signaling, as syndecan-4-knockout mice exhibit healing retardation due to inefficient fibroblast migration. In wild-type animals, migration of fibroblasts toward a wound is triggered by the appearance of fibronectin that leaks from damaged capillaries and is deposited by macrophages in injured tissue. Therefore there is great interest in discovering strategies that enhance fibronectin-dependent signaling and could accelerate repair processes. The integrin-mediated and syndecan-4-mediated components of fibronectin-dependent signaling can be separated by stimulating cells with recombinant fibronectin fragments. Although integrin engagement is essential for cell adhesion, certain fibronectin-dependent signals are regulated by syndecan-4. Syndecan-4 activates the Rac1 protrusive signal, causes integrin redistribution, triggers recruitment of cytoskeletal molecules, such as vinculin, to focal adhesions, and thereby induces directional migration. We have looked for alternative strategies for activating such signals and found that low-intensity pulsed ultrasound (LIPUS) can mimic the effects of syndecan-4 engagement. In this protocol we describe the method by which 30 mW/cm(2), 1.5 MHz ultrasound, pulsed at 1 kHz (Fig. 1) can be applied to fibroblasts in culture (Fig. 2) to induce Rac1 activation and focal adhesion formation. Ultrasound stimulation is applied for a maximum of 20 minutes, as this combination of parameters has been found to be most efficacious for acceleration of clinical fracture repair. The method uses recombinant fibronectin fragments to engage α(5)ß(1)-integrin, without engagement of syndecan-4, and requires inhibition of protein synthesis by cycloheximide to block deposition of additional matrix by the fibroblasts. The positive effect of ultrasound on repair mechanisms is well documented, and by understanding the molecular effect of ultrasound in culture we should be able to refine the therapeutic technique to improve clinical outcomes.

SNX17 protects integrins from degradation by sorting between lysosomal and recycling pathways.

The FERM-like domain-containing sorting nexins of the SNX17/SNX27/SNX31 family have been proposed to mediate retrieval of transmembrane proteins from the lysosomal pathway. In this paper, we describe a stable isotope labeling with amino acids in culture-based quantitative proteomic approach that allows an unbiased, global identification of transmembrane cargoes that are rescued from lysosomal degradation by SNX17. This screen revealed that several integrins required SNX17 for their stability, as depletion of SNX17 led to a loss of ß1 and ß5 integrins and associated a subunits from HeLa cells as a result of increased lysosomal degradation. SNX17 bound to the membrane distal NPXY motif in ß integrin cytoplasmic tails, thereby preventing lysosomal degradation of ß integrins and their associated a subunits. Furthermore, SNX17-dependent retrieval of integrins did not depend on the retromer complex. Consistent with an effect on integrin recycling, depletion of SNX17 also caused alterations in cell migration. Our data provide mechanistic insight into the retrieval of internalized integrins from the lysosomal degradation pathway, a prerequisite for subsequent recycling of these matrix receptors.

SHARPINing integrin inhibition.

The activity state of integrins is crucial for cell adhesion, migration and differentiation, and is regulated predominantly by protein interactions of the integrin ß cytoplasmic domain. SHARPIN is now shown to negatively regulate integrin activation by binding the α-integrin subunit and interfering with the association of the ß cytodomain with activating proteins.

A syndecan-4 hair trigger initiates wound healing through caveolin- and RhoG-regulated integrin endocytosis.

Cell migration during wound healing requires adhesion receptor turnover to enable the formation and disassembly of cell-extracellular matrix contacts. Although recent advances have improved our understanding of integrin trafficking pathways, it is not known how extracellular ligand engagement controls receptor dynamics. Using atomic force microscopy, we have measured cell avidity for fibronectin and defined a mechanism for the outside-in regulation of α(5)ß(1)-integrin. Surprisingly, adhesive strength was attenuated by the syndecan-4-binding domain of fibronectin due to a rapid triggering of α(5)ß(1)-integrin endocytosis. Association of syndecan-4 with PKCα was found to trigger RhoG activation and subsequent dynamin- and caveolin-dependent integrin uptake. Like disruption of syndecan-4 or caveolin, gene disruption of RhoG in mice was found to retard closure of dermal wounds due to a migration defect of the fibroblasts and keratinocytes of RhoG null mice. Thus, this syndecan-4-regulated integrin endocytic pathway appears to play a key role in tissue repair.

Proteomic analysis of integrin adhesion complexes.

Integrin receptors regulate cell fate by coupling the binding of extracellular adhesion proteins to the assembly of intracellular cytoskeletal and signaling complexes. A detailed, integrative view of adhesion complexes will provide insight into the molecular mechanisms that control cell morphology, survival, movement, and differentiation. To date, membrane receptor-associated signaling complexes have been refractory to proteomic analysis because of their inherent lability and inaccessibility. We developed a methodology to isolate ligand-induced integrin adhesion complexes, and we used this technique to analyze the composition of complexes associated with multiple receptor-ligand pairs and define core and receptor-specific subnetworks. In particular, we identified regulator of chromosome condensation-2 (RCC2) as a component of fibronectin-activated signaling pathways that regulate directional cell movement. The development of this proteomics pipeline provides the means to investigate the molecular composition and function of various adhesion complexes.

Proteomic analysis of integrin-associated complexes identifies RCC2 as a dual regulator of Rac1 and Arf6.

The binding of integrin adhesion receptors to their extracellular matrix ligands controls cell morphology, movement, survival, and differentiation in various developmental, homeostatic, and disease processes. Here, we report a methodology to isolate complexes associated with integrin adhesion receptors, which, like other receptor-associated signaling complexes, have been refractory to proteomic analysis. Quantitative, comparative analyses of the proteomes of two receptor-ligand pairs, alpha(4)beta(1)-vascular cell adhesion molecule-1 and alpha(5)beta(1)-fibronectin, defined both core and receptor-specific components. Regulator of chromosome condensation-2 (RCC2) was detected in the alpha(5)beta(1)-fibronectin signaling network at an intersection between the Rac1 and adenosine 5'-diphosphate ribosylation factor 6 (Arf6) subnetworks. RCC2 knockdown enhanced fibronectin-induced activation of both Rac1 and Arf6 and accelerated cell spreading, suggesting that RCC2 limits the signaling required for membrane protrusion and delivery. Dysregulation of Rac1 and Arf6 function by RCC2 knockdown also abolished persistent migration along fibronectin fibers, indicating a functional role for RCC2 in directional cell movement. This proteomics workflow now opens the way to further dissection and systems-level analyses of adhesion signaling.

Giving off mixed signals--distinct functions of alpha5beta1 and alphavbeta3 integrins in regulating cell behaviour.

The formation, maturation, and dissolution of focal adhesions are basic prerequisites of cell migration and rely on the recruitment, signalling, and endocytosis of integrins. In many instances, extracellular matrix molecules are recognised by a number of integrins, and it is the sequential involvement of different integrins that allows establishment of cell polarity and migration towards a matrix stimulus. In this review, we consider both the similarities and differences between two key fibronectin receptors, alpha(v)beta(3) and alpha(5)beta(1) integrin. By considering the GTPase and kinase signalling and trafficking of two such closely-related receptors, we begin to understand how cell migration is coordinated.

An integrin-alpha4-14-3-3zeta-paxillin ternary complex mediates localised Cdc42 activity and accelerates cell migration.

Alpha4 integrins are used by leukocytes and neural crest derivatives for adhesion and migration during embryogenesis, immune responses and tumour invasion. The pro-migratory activity of alpha4 integrin is mediated in part through the direct binding of the cytoplasmic domain to paxillin. Here, using intermolecular FRET and biochemical analyses, we report a novel interaction of the alpha4 integrin cytoplasmic domain with 14-3-3zeta. This interaction depends on serine phosphorylation of alpha4 integrin at a site (S978) distinct from that which regulates paxillin binding (S988). Using a combination of metabolic labelling and targeted mass spectrometry by multiple reaction monitoring we demonstrate the low stoichiometry phosphorylation of S978. The interaction between alpha4 integrin and 14-3-3zeta is enhanced by the direct association between 14-3-3zeta and paxillin, resulting in the formation of a ternary complex that stabilises the recruitment of each component. Although pair-wise interaction between alpha4 integrin and paxillin is sufficient for normal Rac1 regulation, the integrity of the ternary complex is essential for focused Cdc42 activity at the lamellipodial leading edge and directed cell movement. Taken together, these data identify a key signalling nexus mediating alpha4 integrin-dependent migration.

Syndecans shed their reputation as inert molecules.

The syndecan transmembrane proteoglycans synergize with receptors for extracellular matrix molecules and growth factors to initiate cytoplasmic signals in response to a range of extracellular stimuli. Syndecans influence a wide range of physiological processes, but their contribution is most apparent during wound repair. Aspects of syndecan biology that have attracted research interest include extracellular matrix binding, outside-to-inside plasma membrane signal propagation, activation of cytoplasmic signals, and shedding of the syndecan extracellular domain, but the mechanisms by which syndecan cytoplasmic signals modulate extracellular function remain largely unresolved. Hayashida et al. have now discovered that association between an endocytic regulator, Rab5, and the syndecan-1 cytoplasmic domain controlled the shedding of the syndecan-1 extracellular domain. The work describes a mechanistic investigation into inside-to-outside syndecan signaling and highlights several gaps in our understanding of the relation between cell-surface receptors and proteases. In this Perspective, we summarize the current understanding of receptor interplay and identify the challenges that face investigators of adhesion- and growth factor-dependent signaling.