Nano-stenciled RGD-gold patterns that inhibit focal contact maturation induce lamellipodia formation in fibroblasts

Cultured fibroblasts adhere to extracellular substrates by means of cell-matrix adhesions that are assembled in a hierarchical way, thereby gaining in protein complexity and size. Here we asked how restricting the size of cell-matrix adhesions affects cell morphology and behavior. Using a nanostencil technique, culture substrates were patterned with gold squares of a width and spacing between 250 nm and 2 μm. The gold was functionalized with RGD peptide as ligand for cellular integrins, and mouse embryo fibroblasts were plated. Limiting the length of cell-matrix adhesions to 500 nm or less disturbed the maturation of vinculin-positive focal complexes into focal contacts and fibrillar adhesions, as indicated by poor recruitment of α5-integrin. We found that on sub-micrometer patterns, fibroblasts spread extensively, but did not polarize. Instead, they formed excessive numbers of lamellipodia and a fine actin meshwork without stress fibers. Moreover, these cells showed aberrant fibronectin fibrillogenesis, and their speed of directed migration was reduced significantly compared to fibroblasts on 2 μm square patterns. Interference with RhoA/ROCK signaling eliminated the pattern-dependent differences in cell morphology. Our results indicate that manipulating the maturation of cell-matrix adhesions by nanopatterned surfaces allows to influence morphology, actin dynamics, migration and ECM assembly of adhering fibroblasts.     

Pertubation of beta1 integrin function using anti-sense or function-blocking antibodies on corneal cells grown on fibronectin and tenascin

During corneal development, neural crest derivatives from the periocular mesenchyme migrate into the cornea and differentiate into corneal fibroblasts. During this time, these cells interact with a variety of extracellular matrices for proper orientation and development. In the present studies, we have examined the interaction of beta(1) integrins on periocular mesenchyme cells (POM) and corneal fibroblasts (CF) with fibronectin and tenascin by perturbing the function of this integrin. POM and CF attached and spread to a much greater extent on fibronectin than on tenascin. An antibody against beta(1) integrin, CSAT, decreased spreading and attachment, and resulted in a lack of immuno-detectable beta(1) integrin in focal adhesions on fibronectin; few beta(1) positive focal adhesions were observed in cells grown on tenascin. An anti-sense retroviral construct decreased endogenous levels of beta(1) integrin protein, and caused decreased attachment and spreading as well as sparse, disorganized focal adhesions. These data indicate that in vitro, both POM and CF have beta(1) integrins that interact with fibronectin and allow them to attach and spread, while tenascin is anti-adhesive. Further studies using both of these experimental paradigms will clarify whether these interactions also occur in vivo.     

Rac regulates integrin-mediated endothelial cell adhesion and migration on laminin-8

Blood vessel formation requires endothelial cell interactions with the extracellular matrix through cell surface receptors, and signaling events that control endothelial cell adhesion, migration, and lumen formation. Laminin-8 (alpha4beta1gamma1) is present in all basement membranes of blood vessels in fetal and adult tissues, but despite its importance in vessel formation, its role in endothelial cell adhesion and migration remains undefined. We examined adhesion and migration of HMEC-1 human microvascular endothelial cells on laminin-8 with an emphasis on the integrin-mediated signaling events, as compared with those on laminin-10/11 and fibronectin. We found that laminin-8 was less potent in HMEC-1 cell adhesion than laminin-1, laminin-10/11, and fibronectin, and mediated cell adhesion through alpha6beta1 integrin. Despite its weak cell-adhesive activity, laminin-8 was as potent as laminin-10/11 in promoting cell migration. Cells adhering to laminin-8 displayed streaks of thin actin filaments and formed lamellipodia at the leading edge of the cells, as observed with cells adhering to laminin-10/11, while cells on fibronectin showed thick actin stress fibers and large focal adhesions. Pull-down assays of GTP-loaded Rho, Rac, and Cdc42 demonstrated that Rac, but not Rho or Cdc42, was preferentially activated on laminin-8 and laminin-10/11, when compared with fibronectin. Furthermore, a dominant-negative mutant of Rac suppressed cell spreading, lamellipodial formation, and migration on laminin-8, but not on fibronectin. These results, taken together, indicate that Rac is activated during endothelial cell adhesion to laminin-8, and is pivotal for alpha6beta1 integrin-mediated cell spreading and migration on laminin-8.     

Tissue transglutaminase is an integrin-binding adhesion coreceptor for fibronectin

The protein cross-linking enzyme tissue transglutaminase binds in vitro with high affinity to fibronectin via its 42-kD gelatin-binding domain. Here we report that cell surface transglutaminase mediates adhesion and spreading of cells on the 42-kD fibronectin fragment, which lacks integrin-binding motifs. Overexpression of tissue transglutaminase increases its amount on the cell surface, enhances adhesion and spreading on fibronectin and its 42-kD fragment, enlarges focal adhesions, and amplifies adhesion-dependent phosphorylation of focal adhesion kinase. These effects are specific for tissue transglutaminase and are not shared by its functional homologue, a catalytic subunit of factor XIII. Adhesive function of tissue transglutaminase does not require its cross-linking activity but depends on its stable noncovalent association with integrins. Transglutaminase interacts directly with multiple integrins of beta1 and beta3 subfamilies, but not with beta2 integrins. Complexes of transglutaminase with integrins are formed inside the cell during biosynthesis and accumulate on the surface and in focal adhesions. Together our results demonstrate that tissue transglutaminase mediates the interaction of integrins with fibronectin, thereby acting as an integrin-associated coreceptor to promote cell adhesion and spreading.     

Mena binds α5 integrin directly and modulates α5β1 function

Mena is an Ena/VASP family actin regulator with roles in cell migration, chemotaxis, cell-cell adhesion, tumor cell invasion, and metastasis. Although enriched in focal adhesions, Mena has no established function within these structures. We find that Mena forms an adhesion-regulated complex with α5β1 integrin, a fibronectin receptor involved in cell adhesion, motility, fibronectin fibrillogenesis, signaling, and growth factor receptor trafficking. Mena bound directly to the carboxy-terminal portion of the α5 cytoplasmic tail via a 91-residue region containing 13 five-residue "LERER" repeats. In fibroblasts, the Mena-α5 complex was required for "outside-in" α5β1 functions, including normal phosphorylation of FAK and paxillin and formation of fibrillar adhesions. It also supported fibrillogenesis and cell spreading and controlled cell migration speed. Thus, fibroblasts require Mena for multiple α5β1-dependent processes involving bidirectional interactions between the extracellular matrix and cytoplasmic focal adhesion proteins.     

The N-terminal 70-kDa fragment of fibronectin binds to cell surface fibronectin assembly sites in the absence of intact fibronectin.

Binding of the N-terminal 70-kDa (70K) fragment of fibronectin to fibroblasts blocks assembly of intact fibronectin and is an accurate indicator of the ability of various agents to enhance or inhibit fibronectin assembly. Such binding is widely thought to be to already assembled fibronectin. We evaluated this hypothesis with fibronectin-null mouse fibroblasts plated on laminin-1 in the absence of intact fibronectin. As a proteolytic fragment or recombinant protein, 70K bound fibronectin-null cells specifically in linear arrays that extended outwards from the periphery of spread cells. At early time points, these arrays were similar to those formed by intact fibronectin. 70K arrays formed within 5 min following ligand addition at concentrations as low as 5 nM, indicating rapid and high affinity binding. Bound 70K was extractable with Triton X-100 or deoxycholate but became insoluble when cross-linked with a membrane-impermeable agent into large SDS-stable complexes. Intact fibronectin, in contrast, became progressively non-extractable in the absence of cross-linking. The detergent-resistant arrays of cross-linked 70K localized to tips of cellular extensions and partially overlapped with alpha6 and beta1 integrin subunits at the base of the extensions. alpha5 did not localize with 70K arrays, but became progressively co-localized with assemblies of intact fibronectin over time. These results support a model in which the 70-kDa region of fibronectin binds to linearly arrayed cell surface molecules of adherent cells to initiate assembly, display of the arrays is controlled by the integrin that mediates adhesion, and fibronectin-binding integrins promote fibronectin-fibronectin interactions during progression of assembly.     

The Adaptor Protein Paxillin Is Essential for Normal Development in the Mouse and Is a Critical Transducer of Fibronectin Signaling

The integrin family of cell adhesion receptors are important for a diverse set of biological responses during development. Although many integrins have been shown to engage a similar set of cytoplasmic effector proteins in vitro, the importance of these proteins in the biological events mediated by different integrin receptors and ligands is uncertain. We have examined the role of one of the best-characterized integrin effectors, the focal adhesion protein paxillin, by disruption of the paxillin gene in mice. Paxillin was found to be critically involved in regulating the development of mesodermally derived structures such as heart and somites. The phenotype of the paxillin(-/-) mice closely resembles that of fibronectin(-/-) mice, suggesting that paxillin is a critical transducer of signals from fibronectin receptors during early development. Paxillin was also found to play a critical role in fibronectin receptor biology ex vivo since cultured paxillin-null fibroblasts display abnormal focal adhesions, reduced cell migration, inefficient localization of focal adhesion kinase (FAK), and reduced fibronectin-induced phosphorylation of FAK, Cas, and mitogen-activated protein kinase. In addition, we found that paxillin-null fibroblasts show some defects in the cortical cytoskeleton and cell spreading on fibronectin, raising the possibility that paxillin could play a role in structures distinct from focal adhesions. Thus, paxillin and fibronectin regulate some common embryonic developmental events, possibly due to paxillin modulation of fibronectin-regulated focal adhesion dynamics and organization of the membrane cytoskeletal structures that regulate cell migration and spreading.     

Syndecan 4 heparan sulfate proteoglycan is a selectively enriched and widespread focal adhesion component.

Focal adhesion formation in fibroblasts results from complex transmembrane signaling processes initiated by extracellular matrix molecules. Although a role for integrins with attendant tyrosine kinases has been established, there is evidence that cell surface heparan sulfate proteoglycans (HSPGs) are also involved with an associated role of protein kinase C. The identity of the proteoglycan has remained elusive, but we now report that syndecan 4 (ryudocan/amphiglycan) is present in focal adhesions of a number of cell types. Affinity-purified antibodies raised against a unique portion of the cytoplasmic domain of syndecan 4 core protein recognized an HSPG of similar characteristics to those of syndecan 4. These antibodies stained focal adhesions only after cell permeabilization and recognized differing mammalian species. Syndecan 4 was associated with focal adhesions that contained either beta 1 or beta 3 integrin subunits and those that formed on substrates of fibronectin, laminin, vitronectin, or type I collagen. No focal adhesions were found that were vinculin-containing but lacked syndecan 4. In contrast, syndecan 2, whose cytoplasmic domain is closely homologous to syndecan 4, does not appear to be a focal adhesion component. Thus, syndecan 4 represents a new transmembrane focal adhesion component, probably involved in their assembly.     

Release of integrin macroaggregates as a mechanism of rear detachment during keratinocyte migration

Cell-substrate adhesion can be mediated by the relatively short-lived focal complexes and focal adhesions and by the more stable hemidesmosomes. During cell migration both types of cell-substrate adhesions must be disrupted allowing the cell rear to detach. Rear detachment has been described to be accompanied by membrane ripping and the loss of cellular material in a variety of cell types including fibroblasts and chondrocytes, but also in fast moving cells such as keratinocytes. Here we show that migrating keratinocytes leave behind "migration tracks" of cellular remnants that can be classified due to their size, distribution and molecular composition. Type I macroaggregates appeared as spherical and tubular structures with a diameter of about 50-100 nm that were arranged like "pearls on a string". These structures apparently derived from fragmentation of long tubular extensions, the retracting fibers, at the cell rear and contained high amounts of beta1 integrin and different alpha integrins that are components of fibronectin and laminin receptors in migrating keratinocytes usually found in focal adhesions. Type II macroaggregates were recognized as spherical structures with a diameter of about 30 - 50 nm that were arranged in clusters scattered over the gaps between type I, macroaggregates. In contrast to type I type II macroaggregates contained high amounts of beta4 integrin and seemed to derive from former hemidesmosomes. Both types of macroaggregates were completely membrane covered, impermeable compartments devoid of cytosolic proteins. Our observations strongly support the concept that the release of macroaggregates represents a distinct cellular mechanism of rear detachment based on the loss of adhesive receptors embedded in membrane-covered cellular remnants.     

The inner lives of focal adhesions

In focal adhesions of eukaryotic cells, transmembrane receptors of the integrin family and a large set of adaptor proteins form the physical link between the extracellular substrate and the actin cytoskeleton. During cell migration, nascent focal adhesions within filopodia and lamellipodia make the initial exploratory contacts with the cellular environment, whereas maturing focal adhesions pull the cell forward against the resistance of 'sliding' focal adhesions at the cell rear. Experimental approaches are now available for analysing the dynamics and interior structure of these different focal adhesions. Analysing focal-adhesion dynamics using green-fluorescent-protein-linked integrin leads us to propose that the acto-myosin-controlled density and turnover of integrins in focal adhesions is used to sense the elasticity and spacing of extracellular ligands, regulating cell migration by mechanically transduced signaling.     

Fibulin-5 binds human smooth-muscle cells through alpha5beta1 and alpha4beta1 integrins, but does not support receptor activation

Fibulin-5, an extracellular matrix glycoprotein expressed in elastin-rich tissues, regulates vascular cell behaviour and elastic fibre deposition. Recombinant full-length human fibulin-5 supported primary human aortic SMC (smooth-muscle cell) attachment through alpha5beta1 and alpha4beta1 integrins. Cells on fibulin-5 spread poorly and displayed prominent membrane ruffles but no stress fibres or focal adhesions, unlike cells on fibronectin that also binds these integrins. Cell migration and proliferation were significantly lower on fibulin-5 than on fibronectin. Treatment of cells on fibulin-5 with a beta1 integrin-activating antibody induced stress fibres, increased attachment, migration and proliferation, and stimulated signalling of epidermal growth factor receptor and platelet-derived growth factor receptors alpha and beta. Fibulin-5 also modulated fibronectin-mediated cell spreading and morphology. We have thus identified the beta1 integrins on primary SMCs that fibulin-5 interacts with, and have shown that failure of fibulin-5 to activate these receptors limits cell spreading, migration and proliferation.     

Reduced cell migration and disruption of the actin cytoskeleton in calpain-deficient embryonic fibroblasts

The physiological functions and substrates of the calcium-dependent protease calpain remain only partly understood. The mu- and m-calpains consist of a mu- or m-80-kDa large subunit (genes Capn1 and Capn2), and a common 28-kDa small subunit (Capn4). To assess the role of calpain in migration, we used fibroblasts obtained from Capn4(-/-) mouse embryos. The cells lacked calpain activity on casein zymography and did not generate the characteristic calpain-generated spectrin breakdown product that is observed in wild-type cells. Capn4(-/-) cells had decreased migration rates and abnormal organization of the actin cytoskeleton with a loss of central stress fibers. Interestingly, these cells extended numerous thin projections and displayed delayed retraction of membrane protrusions and filopodia. The number of focal adhesions was decreased in Capn4(-/-) cells, but the cells had prominent vinculin-containing focal complexes at the cell periphery. The levels of the focal adhesion proteins, alpha-actinin, focal adhesion kinase (FAK), spectrin, talin, and vinculin, were the same in Capn4(+/+) and Capn4(-/-) cells. FAK, alpha-actinin, and vinculin were not cleaved in either cell type plated on fibronectin. However, proteolysis of the focal complex component, talin, was detected in the wild-type cells but not in the Capn4(-/-) cells, suggesting that calpain cleavage of talin is important during cell migration. Moreover, talin cleavage was again observed when calpain activity was partially restored in Capn4(-/-) embryonic fibroblasts by stable transfection with a vector expressing the rat 28-kDa calpain small subunit. The results demonstrate unequivocally that calpain is a critical regulator of cell migration and of the organization of the actin cytoskeleton and focal adhesions.     

Syndecan-4 binding to the high affinity heparin-binding domain of fibronectin drives focal adhesion formation in fibroblasts

Cell adhesion to extracellular matrix involves signaling mechanisms which control attachment, spreading and the formation of focal adhesions and stress fibers. Fibronectin can provide sufficient signals for all three processes, even when protein synthesis is prevented by cycloheximide. Primary fibroblasts attach and spread following integrin ligation, but do not form focal adhesions unless treated with a heparin-binding fragment of fibronectin (HepII), a peptide from this domain, or phorbol esters to activate protein kinase C. Syndecan-4 heparan sulfate proteoglycan is a transmembrane component present together with integrins in focal adhesions. Syndecan-4 binds and activates protein kinase Calpha, whose activity is needed for focal adhesion formation. We now report that the glycosaminoglycan chains of syndecan-4 bind recombinant HepII and it is incorporated into forming focal adhesions.     

Syndecan-4 associates with alpha-actinin

Cell adhesion to the extracellular matrix influences many cellular functions. The integrin family of matrix receptors plays major roles in the formation of adhesions, but other proteins modulate integrin signaling. Syndecan-4, a transmembrane proteoglycan, cooperatively signals with integrins during the formation of focal adhesions. To date, a direct link between syndecan-4 and the cytoskeleton has remained elusive. We now demonstrate by Triton X-100 extraction immunoprecipitation and in vitro binding assays that the focal adhesion component alpha-actinin interacts with syndecan-4 in a beta-integrin-independent manner.     

Localization of urokinase type plasminogen activator to focal adhesions requires ligation of vitronectin integrin receptors

Previous studies have shown that the adhesion protein, vitronectin, directs the localization of urokinase-type plasminogen activator (uPA) to areas of cell-substrate adhesion, where uPA is thought to regulate cell migration as well as pericellular proteolysis. In the present study, HT-1080 cell lines expressing either wild-type vitronectin or vitronectin containing a single amino-acid substitution in the integrin binding domain were used to assess whether ligation of the alphavbeta5 integrin was required for uPA localization to focal adhesions. The synthesis of wild-type vitronectin by HT-1080 cells adherent to either collagen or fibronectin resulted in the redistribution of both the alphavbeta5 integrin as well as uPA to focal adhesion structures. In contrast, cells synthesizing mutant vitronectin, containing the amino-acid substitution in the integrin binding domain, were unable to direct the redistribution of either alphavbeta5 or uPA to focal adhesions. Recombinant forms of wild-type and mutant vitronectin were prepared in a baculovirus system and compared for their ability to direct the redistribution of vitronectin integrin receptors as well as uPA on human skin fibroblasts. In the absence of vitronectin, fibroblast cells adherent to fibronectin assemble focal adhesions which contain the beta1 integrin but do not contain uPA. Addition of recombinant wild-type, but not mutant, vitronectin to fibroblasts adherent to fibronectin resulted in the redistribution of alphavbeta3, alphavbeta5, and uPA into focal adhesions. However, when cells were plated directly onto antibodies directed against either the alphavbeta3 or alphavbeta5 integrins, uPA was not localized on the cell surface. These data indicate that ligation of vitronectin integrin receptors is necessary but not sufficient for the localization of uPA to areas of cell matrix adhesion, and suggest that vitronectin may promote cell migration by recruiting vitronectin integrin receptors and components of the plasminogen activator system to areas of cell matrix contact.     

Tyrosine phosphatase SHP-2 regulates IL-1 signaling in fibroblasts through focal adhesions

Interleukin-1beta (IL-1beta) mediates destruction of matrix collagens in diverse inflammatory diseases including arthritis, periodontitis, and pulmonary fibrosis by activating fibroblasts, cells that interact with matrix proteins through integrin-based adhesions. In vitro, IL-1beta signaling is modulated by focal adhesions, supramolecular protein complexes that are enriched with tyrosine kinases and phosphatases. We assessed the importance of tyrosine phosphatases in regulating cell-matrix interactions and IL-1beta signaling. In human gingival fibroblasts plated on fibronectin, IL-1beta enhanced the maturation of focal adhesions as defined by morphology and enrichment with paxillin and alpha-actinin. IL-1beta also induced activation of ERK and recruitment of phospho-ERK to focal complexes/adhesions. Treatment with the potent tyrosine phosphatase inhibitor pervanadate, in the absence of IL-1beta, recapitulated many of these responses indicating the importance of tyrosine phosphatases. Immunoblotting of collagen bead-associated complexes revealed that the tyrosine phosphatase, SHP-2, was also enriched in focal complexes/adhesions. Depletion of SHP-2 by siRNA or by homologous recombination markedly altered IL-1beta-induced ERK activation and maturation of focal adhesions. IL-1beta-induced tyrosine phosphorylation of SHP-2 on residue Y542 promoted focal adhesion maturation. Association of Gab1 with SHP-2 in focal adhesions correlated temporally with activation of ERK and was abrogated in cells expressing mutant (Y542F) SHP-2. We conclude that IL-1beta mediated maturation of focal adhesions is dependent on tyrosine phosphorylation of SHP-2 at Y542, leading to recruitment of Gab1, a process that may influence the downstream activation of ERK.     

TGF-beta 1 regulates adhesion of mucosal mast cell homologues to laminin-1 through expression of integrin alpha 7

Mucosal mast cells (MMC) or their precursors migrate through the intestinal lamina propria to reside intraepithelially, where expression of mouse mast cell protease-1 indicates the mature phenotype. Alterations in expression of integrins that govern cell adhesion to the extracellular matrix may regulate this process. As the key cytokine mediating differentiation of mouse mast cell protease-1-expressing MMC homologues in vitro, TGF-beta1 was considered a likely candidate for regulation of the integrins that facilitate intraepithelial migration of MMC. Therefore, we examined adhesion of bone marrow-derived mast cells cultured with and without TGF-beta1 to laminin-1, fibronectin, and vitronectin along with expression of integrins likely to regulate this adhesion. Adhesion of PMA-stimulated cultured mast cells to laminin-1 increased from 5.3 +/- 3.6% (mean +/- SEM) in the absence of TGF-beta1 to 58.7 +/- 4.0% (p < 0.05) when cultured mast cells had differentiated into MMC homologues in the presence of TGF-beta1. Increased adhesion of MMC homologues to laminin-1 was also stimulated by FcepsilonRI cross-linking and the calcium ionophore A23187. Expression of the laminin-binding integrin alpha(7) by MMC homologues grown in the presence of TGF-beta1 was demonstrated by RT-PCR and flow cytometry, and preincubation of MMC homologues with the alpha(7)-neutralizing Ab 6A11 inhibited adhesion to laminin-1 by 98% (p < 0.05), demonstrating a novel role for this molecule in adhesion of a hemopoietic cell to laminin-1.     

Physical state of the extracellular matrix regulates the structure and molecular composition of cell-matrix adhesions

This study establishes that the physical state of the extracellular matrix can regulate integrin-mediated cytoskeletal assembly and tyrosine phosphorylation to generate two distinct types of cell-matrix adhesions. In primary fibroblasts, alpha(5)beta(1) integrin associates mainly with fibronectin fibrils and forms adhesions structurally distinct from focal contacts, independent of actomyosin-mediated cell contractility. These "fibrillar adhesions" are enriched in tensin, but contain low levels of the typical focal contact components paxillin, vinculin, and tyrosine-phosphorylated proteins. However, when the fibronectin is covalently linked to the substrate, alpha(5)beta(1) integrin forms highly tyrosine-phosphorylated, "classical" focal contacts containing high levels of paxillin and vinculin. These experiments indicate that the physical state of the matrix, not just its molecular composition, is a critical factor in defining cytoskeletal organization and phosphorylation at adhesion sites. We propose that molecular organization of adhesion sites is controlled by at least two mechanisms: 1) specific integrins associate with their ligands in transmembrane complexes with appropriate cytoplasmic anchor proteins (e.g., fibronectin-alpha(5)beta(1) integrin-tensin complexes), and 2) physical properties (e.g., rigidity) of the extracellular matrix regulate local tension at adhesion sites and activate local tyrosine phosphorylation, recruiting a variety of plaque molecules to these sites. These mechanisms generate structurally and functionally distinct types of matrix adhesions in fibroblasts.     

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)β(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)β(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.     

Localization of Cranin (Dystroglycan) at Sites of Cell-Matrix and Cell-Cell Contact: Recruitment to Focal Adhesions Is Dependent upon Extracellular Ligands

We report that cranin (dystroglycan) can become recruited to focal adhesions of cultured rat REF 52 fibroblasts and human aortic smooth muscle cells. Within mature focal adhesions, cranin was present within the plaque region defined by β1 integrin, vinculin and phosphotyrosine staining, but occupied a larger domain corresponding to, the terminal segments of stress fibers that was more precisely co-extensive with the cytoskeletal proteins alpha-actinin, utrophin and aciculin. When REF 52 fibroblasts were plated on different substrata in the absence of protein synthesis and secretion in serum-free medium, focal clusters of cranin readily formed within 2 hours on matrix proteins that bind cranin directly (laminin or agrin) which were maintained as the focal adhesions became mature. In contrast, cranin failed to become targeted to cell-substratum attachment sites, either at early or later times. when cells were plated on a variety of other substrata that elicit formation of focal adhesions but do not bind cranin directly (fibronectin, vitronectin, collagen type IV, or anti-β integrin antibody TS2/16). These data strongly suggest that targeting of cranin to focal adhesions was dependent upon the presence of an extracellular ligand capable of binding cranin directly. How-ever, some cultured nonmuscle cell lines (e.g., human umbilical vein endothelial cells, NIH 3T3 and CHO cells) failed to localize cranin to focal adhesions, even when plated on laminin. Cranin was also enriched at cell-cell adherens-type junctions of human normal breast MCF-10 epithelial cells, and at growth cones of E17 rat hippocampal axons. That cranin can become targeted to sites of cell-cell and cell-substratum contact in diverse cell types supports the hypothesis that cranin may be involved in mediating or regulating cell adhesion. The absence of muscle-specific and synapse-specific proteins within fibroblasts and epithelial cells provides a different context for thinking about cranin (dystroglycan) that may aid in discerning general principles of its structure and function.