Quantifying cell-matrix adhesion dynamics in living cells using interference reflection microscopy.

Focal adhesions and podosomes are integrin-mediated cell-substratum contacts that can be visualized using interference reflection microscopy (IRM). Here, we have developed automated image-processing procedures to quantify adhesion turnover from IRM images of live cells. Using time sequences of images, we produce adhesion maps that reveal the spatial changes of adhesions and contain additional information on the time sequence of these changes. Such maps were used to characterize focal adhesion dynamics in mouse embryo fibroblasts lacking one or both alleles of the vinculin gene. Loss of vinculin expression resulted in increased assembly, disassembly and/or in increased translocation of focal adhesions, suggesting that vinculin is important for stabilizing focal adhesions. This method is also useful for studying the rapid dynamics of podosomes as observed in primary mouse dendritic cells.

Structural model and functional significance of pH-dependent talin-actin binding for focal adhesion remodeling.

Actin filament binding by the focal adhesion (FA)-associated protein talin stabilizes cell-substrate adhesions and is thought to be rate-limiting in cell migration. Although F-actin binding by talin is known to be pH-sensitive in vitro, with lower affinity at higher pH, the functional significance of this pH dependence is unknown. Because increased intracellular pH (pH(i)) promotes cell migration and is a hallmark of metastatic carcinomas, we asked whether it increases FA remodeling through lower-affinity talin-actin binding. Talin contains several actin binding sites, but we found that only the COOH-terminal USH-I/LWEQ module showed pH-dependent actin binding, with lower affinity and decreased maximal binding at higher pH. Molecular dynamics simulations and NMR of this module revealed a structural mechanism for pH-dependent actin binding. A cluster of titratable amino acids with upshifted pK(a) values, including His-2418, was identified at one end of the five-helix bundle distal from the actin binding site. Protonation of His-2418 induces changes in the conformation and dynamics of the remote actin binding site. Structural analyses of a mutant talin-H2418F at pH 6.0 and 8.0 suggested changes different from the WT protein, and we confirmed that actin binding by talin-H2418F was relatively pH-insensitive. In motile fibroblasts, increasing pH(i) decreased FA lifetime and increased the migratory rate. However, expression of talin-H2418F increased lifetime 2-fold and decreased the migratory rate. These data identify a molecular mechanism for pH-sensitive actin binding by talin and suggest that FA turnover is pH-dependent and in part mediated by pH-dependent affinity of talin for binding actin.

Genetic, biochemical and structural approaches to talin function.

The cytoskeletal protein talin plays a key role in coupling the integrin family of cell adhesion molecules to the actin cytoskeleton. In this paper I present a brief review on talin and summarize our recent studies, in which we have taken both genetic and structural approaches to further elucidate the function of the protein.

Cytoskeletal proteins talin and vinculin in integrin-mediated adhesion.

The cytoskeletal proteins talin and vinculin form part of a macromolecular complex on the cytoplasmic face of integrin-mediated cellular junctions with the extracellular matrix. Recent genetic, biochemical and structural data show that talin is essential for the assembly of such junctions, whereas vinculin appears to be important in regulating adhesion dynamics and cell migration.

Analysis of the mammalian talin2 gene TLN2.

We have utilised genomic and EST databases to assemble the sequence of the human talin2 (TLN2) gene. Talin2 protein is similar in size and sequence to talin1 throughout its length (74% identity, 86% similarity). The major differences are in (i) the size of the genes, the TLN2 gene is >200 kb compared with approximately 30 kb for TLN1 due to a difference in intron size, although intron/exon boundaries, with the exception of two, are strictly conserved; (ii) the expression patterns, TLN1 gives rise to an approximately 8-kb mRNA which is observed in all tissues, whereas TLN2 gives rise to multiple transcripts with the highest levels in heart.

Synemin may function to directly link muscle cell intermediate filaments to both myofibrillar Z-lines and costameres.

Synemin is a large intermediate filament (IF) protein that has been identified in all types of muscle cells in association with desmin- and/or vimentin-containing IFs. Our previous studies (Bellin, R. M., Sernett, S. W., Becker, B., Ip, W., Huiatt, T. W., and Robson, R. M. (1999) J. Biol. Chem. 274, 29493-29499) demonstrated that synemin forms heteropolymeric IFs with major IF proteins and contains a binding site for the myofibrillar Z-line protein alpha-actinin. By utilizing blot overlay assays, we show herein that synemin also interacts with the costameric protein vinculin. Furthermore, extensive assays utilizing the Gal4 yeast two-hybrid system demonstrate interactions of synemin with desmin and vimentin and additionally define more precisely the protein subdomains involved in the synemin/alpha-actinin and synemin/vinculin interactions. The C-terminal approximately 300-amino acid region of synemin binds to the N-terminal head and central rod domains of alpha-actinin and the approximately 150-amino acid C-terminal tail of vinculin. Overall, these interactions indicate that synemin may anchor IFs to myofibrillar Z-lines via interactions with alpha-actinin and to costameres at the sarcolemma via interactions with vinculin and/or alpha-actinin. These linkages would enable the IFs to directly link all cellular myofibrils and to anchor the peripheral layer of myofibrils to the costameres.

Disruption of the talin gene arrests mouse development at the gastrulation stage.

Studies on cultured cells show that the cytoskeletal protein talin plays a key role in cell spreading and the assembly of cell-extracellular matrix junctions. To examine the role of talin in vivo, we have generated mice with a targeted disruption of the talin gene. Heterozygotes are normal, but no surviving homozygous mutant animals were obtained, proving that talin is required for embryogenesis. Mutant embryos develop normally to the blastocyst stage and implant, but there is a gross disorganization of the embryos at gastrulation (6.5-7.5 days post coitum), and they die around 8.5-9.5 days post coitum. The embryonic ectoderm is reduced in size, with fewer cells, and is incompletely organised compared with wild-type embryos. The mutant embryos show disorganised extraembryonic tissues, and the ectoplacental and excocoelomic cavities are not formed. This seems to be because embryonic mesoderm accumulates as a mass on the posterior side of the embryos and fails to migrate to extraembryonic regions, although mesodermal cells are evident in the embryo proper. Spreading of trophoblast cells derived from cultured mutant blastocysts on fibronectin and laminin is also considerably reduced. Therefore, the fundamental deficit in these embryos seems to be a failure of cell migration at gastrulation.

Focal adhesions - the cytoskeletal connection.

Cellular contacts with the extracellular matrix are regulated by the Rho family of GTPases through their effects on both the actin and the microtubule cytoarchitecture. Recent genetic, biochemical and structural data have highlighted the role played by a subset of actin-binding proteins in coupling integrins to cytoskeletal actin and in assembling signalling complexes that are important for cell motility and cell proliferation.

Crystal structure of the vinculin tail suggests a pathway for activation.

Vinculin plays a dynamic role in the assembly of the actin cytoskeleton. A strong interaction between its head and tail domains that regulates binding to other cytoskeletal components is disrupted by acidic phospholipids. Here, we present the crystal structure of the vinculin tail, residues 879-1066. Five amphipathic helices form an antiparallel bundle that resembles exchangeable apolipoproteins. A C-terminal arm wraps across the base of the bundle and emerges as a hydrophobic hairpin surrounded by a collar of basic residues, adjacent to the N terminus. We show that the C-terminal arm is required for binding to acidic phospholipids but not to actin, and that binding either ligand induces conformational changes that may represent the first step in activation.

Talin contains three similar vinculin-binding sites predicted to form an amphipathic helix.

Using recombinant talin polypeptides and an SDS/PAGE-blot overlay assay, we have previously identified three regions of talin that are involved in binding to vinculin [Gilmore, Wood, Ohanian, Jackson, Patel, Rees, Hynes and Critchley (1993) J. Cell Biol. 122, 337-347]. We have confirmed these observations by using a yeast two-hybrid assay and shown that talin residues 498-656, 852-950 and 1929-2029 are each capable of binding to vinculin residues 1-258. We have further defined the three vinculin-binding sites in talin to residues 607-636, 852-876 and 1944-1969; alignment of these sequences shows 59% similarity, although there are only two identical residues. Predictions of secondary structure indicate that this vinculin-binding motif forms an amphipathic alpha-helix. The hydrophobic face of helix 607-636 contains three aligned leucines (residues 608, 615 and 622), which show conservative substitutions in the other two sites. To test the possibility that this might constitute a leucine zipper involved in vinculin binding, we mutated each leucine residue to an alanine. The results showed that this leucine repeat is not essential to the interaction between talin and vinculin. We also used the yeast two-hybrid system to define further the talin-binding site within vinculin residues 1-258. C-terminal deletions made in accordance with exon boundaries showed that vinculin residues 1-167 are capable of interacting with each of the three vinculin-binding sites in talin. However, all N-terminal deletions abolished binding. The results suggest that the talin-binding site in vinculin has a relatively complex fold, whereas the vinculin-binding motif in talin is contained within a short linear peptide sequence that is repeated three times in the talin rod domain.

Integrin-mediated cell adhesion: the cytoskeletal connection.

Members of the integrin family of cell adhesion molecules play a pivotal role in the interaction between animal cells and the extracellular matrix. This article reviews the evidence (i) that the integrin beta-subunit cytoplasmic domain is important in the localization of integrins to focal adhesions, and for integrin-mediated cell adhesion/spreading; and (ii) that the integrin beta-subunit can be linked to F-actin via the actin-binding proteins talin, alpha-actinin and filamin. Talin has two or more actin-binding sites, and three binding sites for the cytoskeletal protein vinculin. Because vinculin can also bind F-actin, it may cross-link talin and actin, thereby stabilizing the interaction. In addition, vinculin contains a binding site for VASP (vasodilator-stimulated phospho-protein), a protein which may serve to recruit a profilin/G-actin complex to talin, which has actin-nucleating activity. Evidence that talin, vinculin and alpha-actinin are important in the assembly of focal adhesions, obtained using antisense technology and protein microinjection, is reviewed. To analyse the role of talin in focal adhesions, we have disrupted both copies of the talin gene in mouse embryonic stem (ES) cells. Undifferentiated talin (-/-) ES cell mutants are unable to assemble focal adhesions when plated on fibronectin, whereas vinculin (-/-) ES cells are able to do so. Finally, the role of small GTP-binding proteins in the assembly of focal adhesions is discussed, along with our recent studies using streptolysin-O-permeabilized Swiss 3T3 cells which suggest that the GTP-binding protein ADP-ribosylation factor-1 (ARF-1) is important in targeting the protein paxillin to focal adhesions.

A cell-free system to study regulation of focal adhesions and of the connected actin cytoskeleton.

Assembly and modulation of focal adhesions during dynamic adhesive processes are poorly understood. We describe here the use of ventral plasma membranes from adherent fibroblasts to explore mechanisms regulating integrin distribution and function in a system that preserves the integration of these receptors into the plasma membrane. We find that partial disruption of the cellular organization responsible for the maintenance of organized adhesive sites allows modulation of integrin distribution by divalent cations. High Ca2+ concentrations induce quasi-reversible diffusion of beta1 integrins out of focal adhesions, whereas low Ca2+ concentrations induce irreversible recruitment of beta1 receptors along extracellular matrix fibrils, as shown by immunofluorescence and electron microscopy. Both effects are independent from the presence of actin stress fibers in this system. Experiments with cells expressing truncated beta1 receptors show that the cytoplasmic portion of beta1 is required for low Ca2+-induced recruitment of the receptors to matrix fibrils. Analysis with function-modulating antibodies indicates that divalent cation-mediated receptor distribution within the membrane correlates with changes in the functional state of the receptors. Moreover, reconstitution experiments show that purified alpha-actinin colocalizes and redistributes with beta1 receptors on ventral plasma membranes depleted of actin, implicating binding of alpha-actinin to the receptors. Finally, we found that recruitment of exogenous actin is specifically restricted to focal adhesions under conditions in which new actin polymerization is inhibited. Our data show that the described system can be exploited to investigate the mechanisms of integrin function in an experimental setup that permits receptor redistribution. The possibility to uncouple, under cell-free conditions, events involved in focal adhesion and actin cytoskeleton assembly should facilitate the comprehension of the underlying molecular mechanisms.

ARF1 mediates paxillin recruitment to focal adhesions and potentiates Rho-stimulated stress fiber formation in intact and permeabilized Swiss 3T3 fibroblasts.

Focal adhesion assembly and actin stress fiber formation were studied in serum-starved Swiss 3T3 fibroblasts permeabilized with streptolysin-O. Permeabilization in the presence of GTPgammaS stimulated rho-dependent formation of stress fibers, and the redistribution of vinculin and paxillin from a perinuclear location to focal adhesions. Addition of GTPgammaS at 8 min after permeabilization still induced paxillin recruitment to focal adhesion-like structures at the ends of stress fibers, but vinculin remained in the perinuclear region, indicating that the distributions of these two proteins are regulated by different mechanisms. Paxillin recruitment was largely rho-independent, but could be evoked using constitutively active Q71L ADP-ribosylation factor (ARF1), and blocked by NH2-terminally truncated Delta17ARF1. Moreover, leakage of endogenous ARF from cells was coincident with loss of GTPgammaS- induced redistribution of paxillin to focal adhesions, and the response was recovered by addition of ARF1. The ability of ARF1 to regulate paxillin recruitment to focal adhesions was confirmed by microinjection of Q71LARF1 and Delta17ARF1 into intact cells. Interestingly, these experiments showed that V14RhoA- induced assembly of actin stress fibers was potentiated by Q71LARF1. We conclude that rho and ARF1 activate complimentary pathways that together lead to the formation of paxillin-rich focal adhesions at the ends of prominent actin stress fibers.

Disruption of the talin gene compromises focal adhesion assembly in undifferentiated but not differentiated embryonic stem cells.

We have used gene disruption to isolate two talin (-/-) ES cell mutants that contain no intact talin. The undifferentiated cells (a) were unable to spread on gelatin or laminin and grew as rounded colonies, although they were able to spread on fibronectin (b) showed reduced adhesion to laminin, but not fibronectin (c) expressed much reduced levels of beta1 integrin, although levels of alpha5 and alphaV were wild-type (d) were less polarized with increased membrane protrusions compared with a vinculin (-/-) ES cell mutant (e) were unable to assemble vinculin or paxillin-containing focal adhesions or actin stress fibers on fibronectin, whereas vinculin (-/-) ES cells were able to assemble talin-containing focal adhesions. Both talin (-/-) ES cell mutants formed embryoid bodies, but differentiation was restricted to two morphologically distinct cell types. Interestingly, these differentiated talin (-/-) ES cells were able to spread and form focal adhesion-like structures containing vinculin and paxillin on fibronectin. Moreover, the levels of the beta1 integrin subunit were comparable to those in wild-type ES cells. We conclude that talin is essential for beta1 integrin expression and focal adhesion assembly in undifferentiated ES cells, but that a subset of differentiated cells are talin independent for both characteristics.

The focal adhesion phosphoprotein, VASP.

Vasodilator-stimulated phosphoprotein (VASP) is associated with focal adhesions and areas of dynamic membrane activity, where it is thought to have an important role in actin filament assembly and cell motility. VASP contains a central proline-rich sequence which recruits the G-actin binding protein profilin. Localization of VASP to the leading edge of a migrating cell can lead to local accumulation of profilin, which in turn can supply actin monomers to growing filament ends. VASP binds to the focal adhesion proteins vinculin and zyxin and this probably directs the phosphoprotein to focal adhesions and the leading edge of stimulated cells. VASP functions as a binding intermediate between profilin and focal adhesion proteins. Intracellular pathogens, including Listeria monocytogenes, have coat proteins which bind VASP. This is one way in which these pathogens use VASP, and other proteins from the host cell, to assemble the actin filaments they require to move around the cytoplasm of infected cells and enter neighbouring cells. Understanding the role of VASP and other proteins in cell and bacterial motility is likely to lead to development of new therapeutic strategies for diseases including atherosclerosis and tumour growth, and for limiting the spread of intracellular pathogens.

Characterisation of the promoter which regulates expression of a phosphoglucomutase-related protein, a component of the dystrophin/utrophin cytoskeleton predominantly expressed in smooth muscle.

We have recently characterised a 60-kDa muscle-specific phosphoglucomutase-related protein (PGM-RP) which is expressed predominantly in adult visceral and vascular smooth muscle. Here we show that the adult vascular smooth muscle cell line PAC1, which retains the capacity to synthesise metavinculin (a marker of the contractile phenotype) also expressed PGM-RP. However, an embryonic smooth muscle cell line A10, which lacks metavinculin, expressed low levels of PGM-RP. Levels of PGM-RP increased in quiescent PAC1 and A10 cells, and were elevated in response to angiotensin II. PGM-RP is therefore a good marker of the contractile/differentiated smooth muscle phenotype. We have sequenced 1.8 kb of the human PGM-RP promoter and shown that it lacks a conventional TATA box. There are multiple transcription start sites, the most predominant of which are inside an initiator sequence (Inr), which is close to two CT boxes and a GATA element. A minimal promoter-CAT construct (p57-CAT) containing the Inr, a CT box and GATA element directed high-level chloramphenicol acetyltransferase (CAT) expression in the differentiated smooth muscle cell line PAC1, and low-level expression in the embryonic smooth muscle cell line A10. This fits well with the pattern of expression of the endogenous gene. A construct (p146-CAT) containing all of the mRNA initiation sites directed a reduced level of CAT expression, and constructs containing 1.8 kb and 3.3 kb upstream of the major transcription start site displayed even lower activity. Sequence comparisons suggest that the PGM-RP promoter evolved from the main phosphoglucomutase promoter which is active in wide range of cell types. The PGM-RP promoter may have acquired negative regulatory elements as expression of the gene became muscle-specific.

Modulation of myoepithelial-associated alpha6beta4 integrin in a breast cancer cell line alters invasive potential.

In normal breast, cell-stromal contact is mediated by myoepithelial cells which strongly express alpha2beta1, alpha3beta1, and alpha6beta4 integrins, while epithelial cells exhibit alpha2beta1 and alpha3beta1 integrins at cell-cell borders, but do not express alpha6beta4 integrin. Breast carcinomas consistently show down-regulation of all integrins. We have investigated the modulatory effect of stromal proteins, hormones, and transforming growth factor beta (TGF-beta) on integrin expression in breast cancer cell lines MCF-7, T47-D, and MDA-MB 231 using indirect immunofluorescence and confocal laser scanning microscopy. MCF-7 and T47-D cells displayed low levels of both alpha2beta1 and alpha3beta1 integrins, and no alpha6beta4 integrin, and this profile remained unchanged by modulatory agents. The MDA-MB 231 cells exhibited stronger staining for alpha2beta1 and alpha3beta1 integrins and focal staining for alpha6beta4 integrin under control conditions, but markedly enhanced reactivity for the alpha6beta4 complex in the presence of TGF-beta. This was associated with acquisition of a spread cellular morphology and localization of alpha6beta4 at the cell periphery in a discrete punctate distribution. There was associated enhanced expression of epiligrin, the ligand for alpha6beta4, with similar localization to the cell periphery. Cell invasion assays through a Matrigel barrier revealed significantly reduced invasive potential of TGF-beta-treated cells, an effect largely reversed following preincubation of the treated cells with anti-beta4 integrin antibody. We conclude that alpha6beta4 integrin can be up-regulated by TGF-beta and has an anti-invasive effect on MDA-MB 231 cells. In addition to alpha6beta4, MDA-MB 231 cells exhibit other myoepithelial markers including cytokeratin 14, vimentin, and weak expression of CALLA. These findings support the concept of a subgroup of breast carcinomas displaying features of myoepithelial differentiation.

Requirement for Rho in integrin signalling.

Overnight culture of Swiss 3T3 cells in serum-free medium leads to loss of focal adhesions and associated actin stress fibres, although the cells remain well spread. The small GTP-binding protein Rho is required for the formation of stress fibres and focal adhesions induced by growth factors such as lysophosphatidic acid (LPA) in serum-starved Swiss 3T3 cells, and for the LPA-induced tyrosine phosphorylation of several focal adhesion proteins. Plating of cells on extracellular matrix proteins also stimulates protein tyrosine phosphorylation and the formation of stress fibres and focal adhesions in the absence of added growth factors. These responses were inhibited in cells scrape-loaded with the Rho inhibitor C3 transferase. Focal adhesion and stress fibre formation was also triggered by addition of a peptide GRGDS, which is recognised by a number of integrins and is contained within the cell binding domain of a variety of extracellular matrix proteins. The activity of the GRGDS peptide was blocked by microinjecting cells with C3 transferase, suggesting that peptide binding to integrins stimulates a Rho-dependent assembly of focal adhesions. These experiments indicate that Rho is involved in signalling downstream of integrins.

Further analysis of the role of spectrin repeat motifs in alpha-actinin dimer formation.

Protein constructs consisting of repeats 1-4, repeats 1-3 and repeats 2-4 of the rod domain of chicken alpha-actinin were expressed as fusion proteins in Escherichia coli. Based on the evidence of circular dichroism spectra and cooperative thermal unfolding profiles both truncated rod fragments were judged to have assumed the native structural fold. The thermal stabilities were in both cases significantly lower than that of the intact rod (repeats 1-4). Analyses by sedimentation equilibrium and velocity provided further evidence to show that fragment 1-4 is entirely dimeric in the concentration range of these experiments, resembling therefore the rod domain isolated by proteolytic digestion of native alpha-actinin. Fragment 2-4, and probably also 1-3, show concentration-dependent association, with dissociation constants, estimated by sedimentation equilibrium, in the 1-10 microM range. Thus, in confirmation of earlier work, all four repeats are required to generate a maximally stable anti-parallel dimer (Kd approximately 10 pM), suggesting the presence of binding sites in all of them to allow for aligned pairing.