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.     

Keratinocyte motility induced by TGF-beta1 is accompanied by dramatic changes in cellular interactions with laminin 5

Transforming growth factor-beta1 (TGF-beta1) has the ability to induce epithelial cell migration while stopping proliferation. In this study, we show that, concomitant to promoting migration of normal human keratinocytes in vitro, TGF-beta1 induced a marked decrease in their adhesion capacity to processed alpha3-containing laminin 5-coated surfaces. Indeed, the expression levels of alpha3 and alpha6 integrin subunit mRNA and protein, as well as the cell surface alpha3beta1 and alpha6beta4 integrins, were down-regulated. Recent studies showed that keratinocytes over express and deposit laminin 5 during migration and we have shown that laminin 5 found in the matrix of TGF-beta1 induced migrating keratinocytes is present in its unprocessed form [Décline and Rousselle, 2001: J. Cell Sci. 114:811-823]. We show here that TGF-beta1 treatment of the cells promoted a significant increase in their adhesion to the alpha3 chain carboxy-terminal LG4/5 subdomain and that this interaction is likely to be mediated by a heparan sulfate proteoglycan type of receptor. Our results indicate that alpha6beta4 and alpha3beta1 integrin interactions with laminin 5 are diminished during migration while a specific interaction occurs between an additional cellular receptor and the alpha3 LG4/5 module present on unprocessed laminin 5.     

Expression of beta 1, beta 3, beta 4, and beta 5 integrins by human epidermal keratinocytes and non-differentiating keratinocytes

We have compared the adhesive properties and integrin expression profiles of cultured human epidermal keratinocytes and a strain of nondifferentiating keratinocytes (ndk). Both cell types adhered to fibronectin, laminin, and collagen types I and IV, but ndk adhered more rapidly and at lower coating concentrations of the proteins. Antibody blocking experiments showed that adhesion of both cell types to fibronectin was mediated by the alpha 5 beta 1 integrin and to laminin by alpha 3 beta 1 in synergy with alpha 2 beta 1. Keratinocytes adhered to collagen with alpha 2 beta 1, but an antibody to alpha 2 did not inhibit adhesion of ndk to collagen. Both cell types adhered to vitronectin by alpha v-containing integrins. Immunoprecipitation of surface-iodinated and metabolically labeled cells showed that in addition to alpha 2 beta 1, alpha 3 beta 1, and alpha 5 beta 1, both keratinocytes and ndk expressed alpha 6 beta 4 and alpha v beta 5. ndk expressed all these integrins at higher levels than normal keratinocytes. ndk, but not normal keratinocytes, expressed alpha v beta 1 and alpha v beta 3; they also expressed alpha 1 beta 1, an integrin that was not consistently detected on normal keratinocytes. Immunofluorescence experiments showed that in stratified cultures of normal keratinocytes integrin expression was confined to cells in the basal layer; terminally differentiating cells were unstained. In contrast, all cells in the ndk population were integrin positive. Our observations showed that the adhesive properties of ndk differ from normal keratinocytes and reflect differences in the type of integrins expressed, the level of expression and the distribution of integrins on the cell surface. ndk thus have a number of characteristics that distinguish them from normal basal keratinocytes.     

Cell adhesion and migration properties of beta 2-integrin negative polymorphonuclear granulocytes on defined extracellular matrix molecules. Relevance for leukocyte extravasation

Regulated adhesion of leukocytes to the extracellular matrix is essential for transmigration of blood vessels and subsequent migration into the stroma of inflamed tissues. Although beta(2)-integrins play an indisputable role in adhesion of polymorphonuclear granulocytes (PMN) to endothelium, we show here that beta(1)- and beta(3)-integrins but not beta(2)-integrin are essential for the adhesion to and migration on extracellular matrix molecules of the endothelial cell basement membrane and subjacent interstitial matrix. Mouse wild type and beta(2)-integrin null PMN and the progranulocytic cell line 32DC13 were employed in in vitro adhesion and migration assays using extracellular matrix molecules expressed at sites of extravasation in vivo, in particular the endothelial cell laminins 8 and 10. Wild type and beta(2)-integrin null PMN showed the same pattern of ECM binding, indicating that beta(2)-integrins do not mediate specific adhesion of PMN to the extracellular matrix molecules tested; binding was observed to the interstitial matrix molecules, fibronectin and vitronectin, via integrins alpha(5)beta(1) and alpha(v)beta(3), respectively; to laminin 10 via alpha(6)beta(1); but not to laminins 1, 2, and 8, collagen type I and IV, perlecan, or tenascin-C. PMN binding to laminins 1, 2, and 8 could not be induced despite surface expression of functionally active integrin alpha(6)beta(1), a major laminin receptor, demonstrating that expression of alpha(6)beta(1) alone is insufficient for ligand binding and suggesting the involvement of accessory factors. Nevertheless, laminins 1, 8, and 10 supported PMN migration, indicating that differential cellular signaling via laminins is independent of the extent of adhesion. The data demonstrate that adhesive and nonadhesive interactions with components of the endothelial cell basement membrane and subjacent interstitium play decisive roles in controlling PMN movement into sites of inflammation and illustrate that beta(2)-integrins are not essential for such interactions.     

Laminin 5 Deposition Promotes Keratinocyte Motility

We examined the role of individual integrins in promoting human keratinocyte migration. In short-term assays on collagen type I- or fibronectin-coated substrates, migration was blocked by antibody to the α2 integrin and the α5 integrin, respectively. Unexpectedly, antibodies to integrin α3 also significantly inhibited cell locomotion on both ligands. Time-course immunofluorescence staining revealed that keratinocyte migration was accompanied by deposition of endogenous laminin 5. Since α3β1 is a known receptor for this ligand, this observation suggested that migrating keratinocytes use freshly deposited laminin 5 in locomotion. Indeed, further investigation showed that anti-laminin 5 blocking antibodies effectively inhibited keratinocyte motility on both collagen and fibronectin substrates. Furthermore, cell migration on laminin 5-coated substrates was blocked by both anti-α3 and anti-laminin 5 antibodies. Laminin 5 did not appear important in the initial attachment of keratinocytes, since adhesion of cells to collagen type I- or fibronectin-coated surfaces was not blocked by antibody to α3 integrin or to laminin 5, but could be inhibited by antibody to α2 or α5, respectively. Using anin vitrowound assay, blocking antibodies to α3 integrin and to laminin 5 also blocked reepithelization of the denuded monolayer. These results show that α3β1 integrin plays an important role in the migration of keratinocytes via their interaction with laminin 5. Furthermore, they suggest that cell migration is dependent not only on exogenous ligands but, importantly, on endogenously secreted laminin 5. Finally, the data are consistent with our earlier finding that laminin 5 is the first extracellular matrix component to be expressed and deposited by migrating keratinocytes during wound healingin vivo[1].     

Ligation of integrin alpha 3beta 1 by laminin 5 at the wound edge activates Rho-dependent adhesion of leading keratinocytes on collagen

Wounding of the epidermis signals the transition of keratinocytes from quiescent anchorage on endogenous basement membrane laminin 5 to migration on exposed dermal collagen. In this study, we attempt to characterize activation signals that transform quiescent keratinocytes into migratory leading cells at the wound edge. Previously, we reported that adhesion and spreading on collagen via integrin alpha(2)beta(1) by cultured human foreskin keratinocytes (HFKs) requires RhoGTP, a regulator of actin stress fibers. In contrast, adhesion and spreading on laminin 5 requires integrins alpha(3)beta(1) and alpha(6)beta(4) and is dependent on phosphoinositide 3-hydroxykinase (Nguyen, B. P., Gil, S. G., and Carter, W. G. (2000) J. Biol. Chem. 275, 31896-31907). Here, we report that quiescent HFKs do not adhere to collagen but adhere and spread on laminin 5. By using collagen adhesion as one criterion for conversion to a "leading wound cell," we found that activation of collagen adhesion requires elevation of RhoGTP. Adhesion of quiescent HFKs to laminin 5 via integrin alpha(3)beta(1) and alpha(6)beta(4) is sufficient to increase levels of RhoGTP required for adhesion and spreading on collagen. Consistently, adhesion of quiescent HFKs to laminin 5, but not collagen, also promotes expression of the precursor form of laminin 5, a characteristic of leading keratinocytes in the epidermal outgrowth. We suggest that wounding of quiescent epidermis initiates adhesion and spreading of keratinocytes at the wound edge on endogenous basement membrane laminin 5 via alpha(3)beta(1) and alpha(6)beta(4) in a Rho-independent mechanism. Spreading on endogenous laminin 5 via alpha(3)beta(1) is necessary but not sufficient to elevate expression of precursor laminin 5 and RhoGTP, allowing for subsequent collagen adhesion via alpha(2)beta(1), all characteristics of leading keratinocytes in the epidermal outgrowth.     

Deposition of laminin 5 in epidermal wounds regulates integrin signaling and adhesion

Adhesion of keratinocytes in a wound outgrowth to laminin 5 in the basement membrane via integrins alpha6beta4 and alpha3beta1 is distinct from adhesion to dermal collagen via alpha2beta1 or to fibronectin via alpha5beta1. Leading cells in the outgrowth are distinguished from following keratinocytes by deposition of laminin 5, failure to communicate via gap junctions and sensitivity to toxin B, an inhibitor of RhoGTPase. Laminin 5 deposited by leading keratinocytes onto dermal collagen dominates over dermal ligands and changes the cell signals required for adhesion from collagen-dependent to laminin-5-dependent. Thus, deposition of laminin 5 can instruct keratinocytes to switch from an activated phenotype to a quiescent and integrated epithelial phenotype.     

Changes in keratinocyte adhesion during terminal differentiation: reduction in fibronectin binding precedes alpha 5 beta 1 integrin loss from the cell surface

During terminal differentiation keratinocytes move out of the basal layer of the epidermis and thereby lose contact with the basement membrane. We show that terminal differentiation in culture involves loss of adhesiveness to fibronectin, laminin, and collagen types I and IV. The adhesive changes precede, by several hours, loss of the alpha 2 beta 1, alpha 3 beta 1, and alpha 5 beta 1 integrins from the cell surface. Keratinocyte adhesion to fibronectin is mediated by the alpha 5 beta 1 integrin, and the decrease in adhesion of intact cells to fibronectin is correlated with a decrease in the ability of alpha 5 beta 1 receptors to bind fibronectin. Thus modulation of integrin function early in terminal differentiation may be an early event determining cell migration out of the basal layer.     

Keratinocyte apoptosis on type I collagen gel caused by lack of laminin 5/10/11 deposition and Akt signaling

Cultured human foreskin keratinocytes (HFKs) adhere to and grow on nonfibrous collagen via integrin alpha2beta1. During incubation, the receptors used for adhesion are changed to integrins alpha3beta1 and alpha6beta4 and those receptors bind to laminin 5 which is deposited by keratinocytes themselves. In this report, we examined the behaviors of HFKs and transformed keratinocytes on collagen fibril gels. These cells adhered to and spread on collagen gels using integrin alpha2beta1. After several hours on collagen gels, however, cells became round and apoptosis occurred. The behavior of keratinocytes contrasted to that of fibroblasts that grew well even on collagen gel. At the point of apoptosis, integrins alpha2beta1 and alpha3beta1 were not found in the contact region of HFKs. Also, deposition of laminin 5 on collagen gel was not found despite the synthesis of mRNA for laminin 5 and laminin 10/11, while soluble laminin 5 protein is readily detectable. Phosporylation of Akt, which is known as a survival signal, was detected in HFKs cultured on coated collagen; however, the protein level and signals of Akt were dramatically decreased on collagen gel after 1 day of culture. These results indicate that collagen gel has different effects than nonfibrous collagen on HFKs and transformed keratinocytes and the interactions of integrin alpha3beta1 and laminin 5/10/11 are indispensable for maintenance of keratinocyte adhesion and survival.     

Pro-collagenase-1 (matrix metalloproteinase-1) binds the alpha(2)beta(1) integrin upon release from keratinocytes migrating on type I collagen

In injured skin, collagenase-1 (matrix metalloproteinase-1 (MMP-1)) is induced in migrating keratinocytes. This site-specific expression is regulated by binding of the alpha(2)beta(1) integrin with dermal type I collagen, and the catalytic activity of MMP-1 is required for keratinocyte migration. Because of this functional association among substrate/ligand, receptor, and proteinase, we assessed whether the integrin also directs the compartmentalization of MMP-1 to its matrix target. Indeed, pro-MMP-1 co-localized to sites of alpha(2)beta(1) contacts in migrating keratinocytes. Furthermore, pro-MMP-1 co-immunoprecipitated with alpha(2)beta(1) from keratinocytes, and alpha(2)beta(1) co-immunoprecipitated with pro-MMP-1. No other MMPs bound alpha(2)beta(1), and no other integrins interacted with MMP-1. Pro-MMP-1 also provided a substrate for alpha(2)beta(1)-dependent adhesion of platelets. Complex formation on keratinocytes was most efficient on native type I collagen and reduced or ablated on denatured or cleaved collagen. Competition studies suggested that the alpha(2) I domain interacts with the linker and hemopexin domains of pro-MMP-1, not with the pro-domain. These data indicate that the interaction of pro-MMP-1 with alpha(2)beta(1) confines this proteinase to points of cell contact with collagen and that the ternary complex of integrin, enzyme, and substrate function together to drive and regulate keratinocyte migration.     

Marching at the front and dragging behind: differential alphaVbeta3-integrin turnover regulates focal adhesion behavior.

Integrins are cell-substrate adhesion molecules that provide the essential link between the actin cytoskeleton and the extracellular matrix during cell migration. We have analyzed alphaVbeta3-integrin dynamics in migrating cells using a green fluorescent protein-tagged beta3-integrin chain. At the cell front, adhesion sites containing alphaVbeta3-integrin remain stationary, whereas at the rear of the cell they slide inward. The integrin fluorescence intensity within these different focal adhesions, and hence the relative integrin density, is directly related to their mobility. Integrin density is as much as threefold higher in sliding compared with stationary focal adhesions. High intracellular tension under the control of RhoA induced the formation of high-density contacts. Low-density adhesion sites were induced by Rac1 and low intracellular tension. Photobleaching experiments demonstrated a slow turnover of beta3-integrins in low-density contacts, which may account for their stationary nature. In contrast, the fast beta3-integrin turnover observed in high-density contacts suggests that their apparent sliding may be caused by a polarized renewal of focal contacts. Therefore, differential acto-myosin-dependent integrin turnover and focal adhesion densities may explain the mechanical and behavioral differences between cell adhesion sites formed at the front, and those that move in the retracting rear of migrating cells.     

Decreased expression of alpha3 and beta1 integrin subunits is responsible for differentiation-associated changes in cells behavior in terminally differentiated human oral keratinocytes

Primary normal human oral keratinocytes (NHOKs) terminally differentiate in serial subculture. To investigate whether this subculture-induced differentiation of NHOKs affects integrin expression and cell-matrix interaction, we studied the expression levels of integrin subunits and cellular response to the extracellular matrix (ECM) proteins in NHOKs at different population doublings. The phosphorylation statuses of focal adhesion kinase (FAK), extracellular signal regulated kinase (ERK), p38, and c-Jun amino-terminal kinase (JNK) were also determined in NHOK cells cultured on ECM proteins, to evaluate the functions of integrins with respect to cellular responses to ECM proteins. The expression levels of alpha3 and beta1 integrin subunits progressively decreased in NHOKs undergoing terminal differentiation. The ability of NHOKs to spread upon laminin and type I collagen significantly decreased in terminally differentiated oral keratinocytes. Keratinocyte migration was significantly increased on type I collagen for terminally differentiated NHOKs. Similar results were seen following preincubation of rapidly proliferating NHOKs with function-blocking antibodies to alpha3 or beta1 integrin subunit. In contrast, fibronectin had no effect on cellular responses in NHOKs, which were almost negligible in the expression levels of alpha5 integrin subunits. The extent of FAK phosphorylation in terminally differentiated NHOKs was notably lower than that of rapidly proliferating cells, but was enhanced in terminally differentiated cells that were cultured on type I collagen. Our results indicate that decreased expression of alpha3 and beta1 integrin subunits is responsible for differentiation-associated changes in cells behavior in terminally differentiated oral keratinocytes. Our data also show that the abrogation of the alpha5beta1 integrin function caused by omitting alpha5 subunit is linked to the loss of a cell-fibronectin interaction in human oral keratinocytes.     

Anti-integrin antibodies induce type IV collagenase expression in keratinocytes

During wound healing, pericellular proteolysis is thought to be essential for the detachment of keratinocytes from basement membrane and in their migration into the wound bed. We have characterized integrin-type cell adhesion/migration receptors in human mucosal keratinocytes and examined their function in the regulation of type IV collagenase gene expression. Two major integrins of the β1 class, α2β1 and αβ1, were found to function as collagen and fibronectin receptors, respectively. Antibodies against β1 and α3 integrin subunits were found to stimulate the expression of the 92 kDa type IV collagenase severalfold in a dosedependent manner. Keratinocytes expressed also the 72 kDa type IV collagenase, the synthesis of which remained, however, unchanged in keratinocytes treated with anti-integrin antibodies. Stimulation of 92 kDa enzyme was found to be caused directly by antibody binding to integrins, since Fab-fragments of anti-β1 antibodies alone were able to induce collagenase expression in the absence of secondary, clustering antibodies. Antibodies against α2β1 integrin caused no stimulation. Keratinocytes seeded on different substrata (plastic, collagen, fibronectin, laminin, or vitronectin) showed equal induction of type IV collagenase expression. Expression of 92 kDa type IV collagenase could not be induced by peptides (GRGDS, GRGES), proteins (fibronectin, laminin, fibrinogen., albumin), or antibodies to fibronectin. We suggest that proteolytic processes around keratinocytes can be regulated by extracellular factors signalling through integrin-type receptors. © 1993 Wiley-Liss, Inc.     

Beta(1)-integrins are involved in migration of human fetal tracheal epithelial cells and tubular morphogenesis

Development of human fetal airways requires interaction of the respiratory epithelium and the extracellular matrix through integrins. Nevertheless, the specific roles of beta(1)-integrins during development and tubular morphogenesis are still unknown. To analyze beta(1)-integrin localization and influence during migration, we developed a model of human fetal tracheal explants growing on collagen and overlaid with a second layer of collagen to form a sandwich. In this configuration, cord and tubule formation proceeded normally but were inhibited by incubation with anti-beta(1)-integrin subunit antibodies. On a collagen matrix, beta(1)-integrins were immunolocalized on the entire plasma membrane of migrating epithelial cells and almost exclusively on the basal plasma membrane of nonmigratory epithelial cells. In a sandwich configuration, beta(1)-integrins became detectable in the cytoplasm of epithelial cells. Coating cultures with collagen transiently altered the morphology of migrating cells and their speed and direction of migration, whereas incubation with anti-beta(1)-integrin subunit antibodies irreversibly altered these parameters. These observations suggest that the matrix environment, by modulating beta(1)-integrin expression patterns, plays a key role during tubular morphogenesis of human fetal tracheal epithelium, principally by modulating epithelial cell migration.     

Polarized expression of integrin receptors (alpha 6 beta 4, alpha 2 beta 1, alpha 3 beta 1, and alpha v beta 5) and their relationship with the cytoskeleton and basement membrane matrix in cultured human keratinocytes

In human keratinocytes cultured in conditions which allow differentiation and stratification and are suitable to reconstitute a fully functional epidermis, alpha 6 beta 4 and two members of the beta 1 integrin family (alpha 2 beta 1 and alpha 3 beta 1) were respectively polarized to the basal and lateral domains of the plasmamembrane both in growing colonies and in the reconstituted epidermis. Conversely, the alpha v integrin subunit, presumably in association with beta 5, was expressed at the basal surface in growing and migrating but not in stationary keratinocytes. The integrin alpha 6 beta 4: (a) was organized in typical patches which often showed a "leopard skin" pattern where spots corresponded to microfilament-free areas; (b) was not associated with focal contacts containing vinculin and talin but rather corresponded to relatively removed contact areas of the basal membrane as shown by interference reflection microscopy; and (c) was coherent to patches of laminin secreted and deposited underneath the ventral membrane of individual cells. The two beta 1 integrins (alpha 2 beta 1 and alpha 3 beta 1), both endowed with laminin receptor properties, were not associated with focal adhesions under experimental conditions allowing full epidermal maturation but matched the lateral position of vinculin (but not talin), cingulin, and desmoplakin, all makers of intercellular junctions. Often thin strips of laminin were observed in between the lateral aspects of individual basal keratinocytes. The integrin complex alpha v beta 5 had a topography similar to that of talin- and vinculin-containing focal adhesions mostly in the peripheral cells of expanding keratinocyte colonies and in coincidence with fibronectin strands. The discrete topography of beta 1 and beta 4 integrins has a functional role in the maintenance of the state of aggregation of cultured keratinocytes since lateral aggregation was impaired by antibodies to beta 1 whereas antibodies to beta 4 prevented cell-matrix adhesion (De Luca, M., R. N. Tamura, S. Kajiji, S. Bondanza, P. Rossino, R. Cancedda, P. C. Marchisio, and V. Quaranta. Proc. Natl. Acad. Sci. USA. 87:6888-6892). Moreover, the surface polarization of integrins followed attachment and depended both on the presence of Ca2+ in the medium and on the integrity of the cytoskeleton. We conclude that our in vitro functional tests and structural data suggest a correlation between the pattern of integrin expression on defined plasmamembrane domains and the mechanism of epidermal assembly.     

Dynamics of beta 1 integrin-mediated adhesive contacts in motile fibroblasts.

Motile chick skeletal fibroblasts adhere to a laminin substrate by means of clustered beta 1 integrins. These integrin "macroaggregates" are similar to classic focal contacts but do not appear dark under interference-reflection microscopy. They contain alpha 5 integrin and are associated with extracellular fibronectin. To study their behavior during cell movement, time-lapse, low-light video microscopy was used to image integrins on living cells tagged with a fluorescent anti-beta 1 integrin antibody. Integrin macroaggregates remain fixed with respect to the substratum, despite the fact that they fluctuate in size, density, and shape over a period of minutes. Upon detachment of the cell rear, as much as 85% of the beta 1 integrin density of a macroaggregate remains behind on the substrate, along with both alpha 5 integrin and fibronectin. Release of the cell rear does not involve cleavage of the beta 1 integrin cytoplasmic domain from the remainder of the protein. These results indicate that cell motility does not require regulated detachment of integrin receptors from the substrate. On the other hand, cytoskeletal components and a variable fraction of the integrins are carried forward with the cell during detachment, suggesting that some type of cortical disassembly process does occur. Integrin macroaggregate structures are not recycled intact after detachment of the cell rear from the substrate. They do not persist on the cell surface, nor can they be seen to be engulfed by vesicles; yet, some of the individual integrins that make up these macroaggregates are eventually transported forward by both vesicular and cell-surface routes. Antibody-tagged integrins accumulate in dense patches at the lateral edges and dorsal surface of the cell, and move forward on the cell surface. The tagged integrins also enter cytoplasmic vesicles, which move forward within the cytoplasm. Macroaggregates generally form and grow at the cell front; however, application of fluorescent antibody causes integrins to disappear from the leading edge. Therefore, it has not been possible to directly visualize the recycling of the forward moving tagged integrins into new macroaggregates at the cell front. Surprisingly, under these conditions cells move normally despite the absence of any delivery of tagged integrin to the leading edge, indicating that recycling of integrins to the lamella is not required for apparently normal motility.     

Beta-1 and beta-4 integrins, and laminin 67 kDa receptors in interaction between A431 cells and laminin isoforms

Laminins, as basal membrane glycoproteins, are able to stimulate cell adhesion and migration, and to influence gene expression. The laminin molecule has a set of bioactive sites that interact with different integrin and nonintegrin receptors, and, as a result, the reaction of the same cell type to different laminin isoforms may be different. The aim of this study was to determine the contributions of both integrins with beta 1 and beta 4 chains and 67 kDa laminin receptor in the interaction of A431 cells with two laminin isoforms: laminin-1 and laminin-2/4. The obtained data show that integrin alpha 6 beta 4 is more specific for interaction with laminin-2/4 than with laminin-1 and takes part in the stage of attachment of A431 cells to laminin. 67 kDa receptor promotes cell spreading on laminin-2/4 and inhibits cell spreading on laminin-1. An assumption was made about the complex action of receptors for interaction of A431 cells with laminins ("integrin alpha 6 beta 4--67 kDa receptors" complex).     

Beta1 integrins are distributed in adhesion structures with fibronectin and caveolin and in coated pits.

Integrins are found in adhesion structures, which link the extracellular matrix to cytoskeletal proteins. Here, we attempt to further define the distribution of beta1 integrins in the context of their association with matrix proteins and other cell surface molecules relevant to the endocytic process. We find that beta1 integrins colocalize with fibronectin in fibrillar adhesion structures. A fraction of caveolin is also organized along these adhesion structures. The extracellular matrix protein laminin is not concentrated in these structures. The alpha4beta1 integrin exhibits a distinct distribution from other beta1 integrins after cells have adhered for 1 h to extracellular matrix proteins but is localized in adhesion structures after 24 h of adhesion. There are differences between the fibronectin receptors: alpha5beta1 integrins colocalize with adaptor protein-2 in coated pits, while alpha4beta1 integrins do not. This parallels our earlier observation that of the two laminin receptors, alpha1beta1 and alpha6beta1, only alpha1beta1 integrins colocalize with adaptor protein-2 in coated pits. Calcium chelation or inhibition of mitogen-activated protein kinase kinase, protein kinase C, or src did not affect localization of alpha1beta1 and alpha5beta1 integrins in coated pits. Likewise, the integrity of coated-pit structures or adhesion structures is not required for integrin and adaptor protein-2 colocalization. This suggests a robust and possibly constitutive interaction between these integrins and coated pits.     

A crucial role of beta 1 integrins for keratinocyte migration in vitro and during cutaneous wound repair

Integrins are ubiquitous transmembrane receptors that play crucial roles in cell-cell and cell-matrix interactions. In this study, we have determined the effects of the loss of beta 1 integrins in keratinocytes in vitro and during cutaneous wound repair. Flow cytometry of cultured beta 1-deficient keratinocytes confirmed the absence of beta 1 integrins and showed downregulation of alpha 6 beta 4 but not of alpha v integrins. beta 1-null keratinocytes were characterised by poor adhesion to various substrates, by a reduced proliferation rate and by a strongly impaired migratory capacity. In vivo, the loss of beta 1 integrins in keratinocytes caused a severe defect in wound healing. beta 1-null keratinocytes showed impaired migration and were more densely packed in the hyperproliferative epithelium. Surprisingly, their proliferation rate was not reduced in early wounds and even increased in late wounds. The failure in re-epithelialisation resulted in a prolonged inflammatory response, leading to dramatic alterations in the expression of important wound-regulated genes. Ultimately, beta 1-deficient epidermis did cover the wound bed, but the epithelial architecture was abnormal. These findings demonstrate a crucial role of beta 1 integrins in keratinocyte migration and wound re-epithelialisation. Movies available on-line     

The Rac activator Tiam1 is required for (alpha)3(beta)1-mediated laminin-5 deposition, cell spreading, and cell migration

The Rho-like guanosine triphosphatase Rac1 regulates various signaling pathways, including integrin-mediated adhesion and migration of cells. However, the mechanisms by which integrins signal toward Rac are poorly understood. We show that the Rac-specific guanine nucleotide exchange factor Tiam1 (T-lymphoma invasion and metastasis 1) is required for the integrin-mediated laminin (LN)-5 deposition, spreading, and migration of keratinocytes. In contrast to wild-type keratinocytes, Tiam1-deficient (Tiam1-/-) keratinocytes are unable to adhere to and spread on a glass substrate because they are unable to deposit their own LN5 substrate. Both Tiam1 and V12Rac1 can rescue the defects of Tiam1-/- keratinocytes, indicating that these deficiencies are caused by impaired Tiam1-mediated Rac activation. Tiam1-/- cells are unable to activate Rac upon alpha3beta1-mediated adhesion to an exogenous LN5 substrate. Moreover, Tiam1 deficiency impairs keratinocyte migration in vitro and reepithelialization of excision wounds in mouse skin. Our studies indicate that Tiam1 is a key molecule in alpha3beta1-mediated activation of Rac, which is essential for proper production and secretion of LN5, a requirement for the spreading and migration of keratinocytes.