Biomedical subjects
Publications and source records attributed to D R Critchley.
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.
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.
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 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.
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.
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.
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.
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.
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.
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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.
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.
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.
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.
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.
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.
Whenever there is heavy proteinuria, the glomerular epithelial cells, the podocytes, show dramatic morphological changes which clearly demonstrate changes in cell adhesion. However, there is little information on the types of cell adhesion molecules expressed in the normal human glomerulus. Assessments of changes in cell adhesion molecules in human proteinuria have been confined to semi-quantitative immunostaining for integrins, and the results have not been entirely consistent. This study sought first to define which cell adhesion molecules are present in the normal glomerulus, using indirect immunofluorescence and a panel of antibodies directed against transmembrane adhesion proteins and against several cytoplasmic proteins which are known to be involved in adhesion. A wide variety of integrins were detected, the dominant form being alpha 3 beta 1. The cytoplasmic focal adhesion proteins vinculin, talin, paxillin, p130CAS, and pp125FAK were detected, although vinculin appeared to be confined mainly to the mesangium. The only intercellular adhesion molecule detected in the vicinity of the slit diaphragm was ZO-1; the results imply that the slit diaphragm does not bear a close relationship to any other form of intercellular junction. Changes in these adhesion components were also studied in proteinuria, using 18 cases each of minimal change nephropathy, 'early' membranous nephropathy, and normal controls. Fluorescence intensity was measured by image capture using a low light video camera and subsequent digital image analysis, an approach which demonstrated acceptable reproducibility. The most striking changes were an increase in phosphotyrosine and p130CAS in the nephrotic patients. Contrary to previous reports, little change was found in the expression of the most abundant integrins, nor did overall glomerular staining for ZO-1 alter. These results imply a controlled alteration in glomerular cell adhesion in nephrotic states in man, probable representing increased turnover of cell adhesion structures rather than the decrease which has been reported in short-term animal models. This is the first report of increased glomerular phosphotyrosine in man, which is associated with less stable adhesions and may be related to the loss of foot processes. Using human biopsy material, it was not possible to determine which proteins were phosphorylated, but the probable relationships to changes in cytoskeletal structure and slit diaphragm permeability justify further study.
We have characterized a panel of 6 monoclonal antibodies raised against human platelet talin by Western blotting, immune precipitation, and immunofluorescence, and shown that antibodies TA205 and TD77 disrupt actin stress fibers and focal adhesions, and inhibit cell motility when microinjected into human fibroblasts. Using a series of chick talin fusion proteins spanning the entire length of the molecule, we have mapped the epitopes recognized by these antibodies to the conserved N- and C-terminal regions of the protein. TA205 bound to an epitope contained within residues 139-433, a region which overlaps an F-actin binding site, and which shows homology with the ezrin/radixin/moesin family of cytoskeletal proteins. The epitope recognized by TD77 was located within the C-terminal region of the protein (residues 2269-2541) which also contains an F-actin binding site homologous to that in the yeast actin-binding protein SIa2p. To investigate the possibility that TD77 disrupts actin stress fibers by binding directly to the C-terminal actin binding site, additional talin fusion proteins were generated and analyzed for TD77 and actin binding. Fusion proteins containing residues 2269-2541, 2304-2541, and 2304-2463 all cosedimented with F-actin, whereas TD77 did not recognize the latter fusion protein. These results show that the C-terminal actin-binding site is distinct from the region recognized by the anti-functional antibody TD77, raising the possibility that it binds to a novel functionally important ligand-binding site in the talin molecule.