Nanoscale architecture of integrin-based cell adhesions

Cell adhesions to the extracellular matrix (ECM) are necessary for morphogenesis, immunity and wound healing. Focal adhesions are multifunctional organelles that mediate cell-ECM adhesion, force transmission, cytoskeletal regulation and signalling. Focal adhesions consist of a complex network of trans-plasma-membrane integrins and cytoplasmic proteins that form a?<200-nm plaque linking the ECM to the actin cytoskeleton. The complexity of focal adhesion composition and dynamics implicate an intricate molecular machine. However, focal adhesion molecular architecture remains unknown. Here we used three-dimensional super-resolution fluorescence microscopy (interferometric photoactivated localization microscopy) to map nanoscale protein organization in focal adhesions. Our results reveal that integrins and actin are vertically separated by a ～40-nm focal adhesion core region consisting of multiple protein-specific strata: a membrane-apposed integrin signalling layer containing integrin cytoplasmic tails, focal adhesion kinase and paxillin; an intermediate force-transduction layer containing talin and vinculin; and an uppermost actin-regulatory layer containing zyxin, vasodilator-stimulated phosphoprotein and α-actinin. By localizing amino- and carboxy-terminally tagged talins, we reveal talin's polarized orientation, indicative of a role in organizing the focal adhesion strata. The composite multilaminar protein architecture provides a molecular blueprint for understanding focal adhesion functions.     

Cell-surface regulation of beta 1-integrin activity on developing retinal neurons.

Integrins are a family of alpha beta heterodimeric receptors that mediate cell-cell and cell-substratum interactions. Integrin binding to extracellular ligands regulates cell adhesion, shape, motility, intracellular signalling and gene expression. Mechanisms that regulate integrin function are, therefore, central to the participation of integrins in a diverse set of cellular events. Here we report the identification of TASC, a monoclonal antibody to a novel epitope on the integrin beta 1 subunit, which inhibits cell adhesion to vitronectin but promotes adhesion to laminin and collagen types I and IV. We show that developing retinal neurons that have lost responsiveness to laminin regain the ability to bind laminin in the presence of TASC. Thus, beta 1-class integrins are likely to occupy multiple affinity states that can be modulated at the cell surface.     

Integrin cytoplasmic tyrosine motif is required for outside-in alphaIIbbeta3 signalling and platelet function.

Integrins not only bind adhesive ligands, they also act as signalling receptors. Both functions allow the integrin alphaIIbbeta3 to mediate platelet aggregation. Platelet agonists activate alphaIIbbeta3 (inside-out signalling) to allow the binding of soluble fibrinogen. Subsequent platelet aggregation leads to outside-in alphaIIbbeta3 signalling, which results in calcium mobilization, tyrosine phosphorylation of numerous proteins including beta3 itself, increased cytoskeletal reorganisation and further activation of alphaIIbbeta3. Thus, outside-in signals enhance aggregation, although the mechanisms and functional consequences of specific signalling events remain unclear. Here we describe a mouse that expresses an alphaIIbbeta3 in which the tyrosines in the integrin cytoplasmic tyrosine motif have been mutated to phenylalanines. These mice are selectively impaired in outside-in alphaIIbbeta3 signalling, with defective aggregation and clot-retraction responses in vitro, and an in vivo bleeding defect which is characterized by a pronounced tendency to rebleed. These data provide evidence for an important role of outside-in signalling in platelet physiology. Furthermore, they identify the integrin cytoplasmic tyrosine motif as a key mediator of beta-integrin signals and a potential target for new therapeutic agents.     

Helicobacter exploits integrin for type IV secretion and kinase activation

Integrins are important mammalian receptors involved in normal cellular functions as well as pathogenesis of chronic inflammation and cancer. We propose that integrins are exploited by the gastric pathogen and type-1 carcinogen Helicobacter pylori for injection of the bacterial oncoprotein cytotoxin-associated gene A (CagA) into gastric epithelial cells. Virulent H. pylori express a type-IV secretion pilus that injects CagA into the host cell; CagA then becomes tyrosine-phosphorylated by Src family kinases. However, the identity of the host cell receptor involved in this process has remained unknown. Here we show that the H. pylori CagL protein is a specialized adhesin that is targeted to the pilus surface, where it binds to and activates integrin alpha5beta1 receptor on gastric epithelial cells through an arginine-glycine-aspartate motif. This interaction triggers CagA delivery into target cells as well as activation of focal adhesion kinase and Src. Our findings provide insights into the role of integrins in H.-pylori-induced pathogenesis. CagL may be exploited as a new molecular tool for our further understanding of integrin signalling.     

Type I gamma phosphatidylinositol phosphate kinase targets and regulates focal adhesions

The ability of cells to form cell contacts, adhere to the extracellular matrix, change morphology, and migrate is essential for development, wound healing, metastasis, cell survival and the immune response. These events depend on the binding of integrin to the extracellular matrix, and assembly of focal adhesions, which are complexes comprising scaffolding and signalling proteins organized by adhesion to the extracellular matrix. Phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)) regulates interactions between these proteins, including the interaction of vinculin with actin and talin. The binding of talin to beta-integrin is strengthened by PtdIns(4,5)P(2), suggesting that the basis of focal adhesion assembly is regulated by this lipid mediator. Here we show that the type I phosphatidylinositol phosphate kinase isoform-gamma 661 (PIPKI gamma 661), an enzyme that makes PtdIns(4,5)P(2), is targeted to focal adhesions by an association with talin. PIPKI gamma 661 is tyrosine phosphorylated by focal adhesion associated kinase signalling, increasing both the activity of phosphatidylinositol phosphate kinase and its association with talin. This defines a mechanism for spatial generation of PtdIns(4,5)P(2) at focal adhesions.     

The SH2/SH3 adaptor Grb4 transduces B-ephrin reverse signals

Bidirectional signals mediated by membrane-anchored ephrins and Eph receptor tyrosine kinases have important functions in cell-cell recognition events, including those that occur during axon pathfinding and hindbrain segmentation. The reverse signal that is transduced into B-ephrin-expressing cells is thought to involve tyrosine phosphorylation of the signal's short, conserved carboxy-terminal cytoplasmic domain. The Src-homology-2 (SH2) domain proteins that associate with activated tyrosine-phosphorylated B-subclass ephrins have not been identified, nor has a defined cellular response to reverse signals been described. Here we show that the SH2/SH3 domain adaptor protein Grb4 binds to the cytoplasmic domain of B ephrins in a phosphotyrosine-dependent manner. In response to B-ephrin reverse signalling, cells increase FAK catalytic activity, redistribute paxillin, lose focal adhesions, round up, and disassemble F-actin-containing stress fibres. These cellular responses can be blocked in a dominant-negative fashion by expression of the isolated Grb4 SH2 domain. The Grb4 SH3 domains bind a unique set of other proteins that are implicated in cytoskeletal regulation, including the Cbl-associated protein (CAP/ponsin), the Abl-interacting protein-1 (Abi-1), dynamin, PAK1, hnRNPK and axin. These data provide a biochemical pathway whereby cytoskeletal regulators are recruited to Eph-ephrin bidirectional signalling complexes.     

Regulation of focal adhesion-associated protein tyrosine kinase by both cellular adhesion and oncogenic transformation.

Increasing evidence indicates that the integrin family of cell adhesion receptors can transduce biochemical signals from the extracellular matrix to the cell interior to modulate cell growth and differentiation. We have shown that integrin/ligand interactions can trigger tyrosine phosphorylation of a protein of M(r) 120,000 (pp120), so it is possible that signal transduction by integrins might involve activation of intracellular protein tyrosine kinases as an early event in cell binding to the extracellular matrix. Here we report that pp120 is identical to the focal adhesion-associated protein tyrosine kinase pp125FAK (refs 3, 4). We show that tyrosine phosphorylation of this protein is modulated both by cell adhesion and transformation by pp60v-src, and that these changes in phosphorylation are correlated with increased pp125FAK tyrosine kinase activity. A model is proposed to relate these findings to the molecular basis of anchorage-independent growth of transformed cells.     

Vinculin activation by talin through helical bundle conversion

Vinculin is a conserved component and an essential regulator of both cell-cell (cadherin-mediated) and cell-matrix (integrin-talin-mediated focal adhesions) junctions, and it anchors these adhesion complexes to the actin cytoskeleton by binding to talin in integrin complexes or to alpha-actinin in cadherin junctions. In its resting state, vinculin is held in a closed conformation through interactions between its head (Vh) and tail (Vt) domains. The binding of vinculin to focal adhesions requires its association with talin. Here we report the crystal structures of human vinculin in its inactive and talin-activated states. Talin binding induces marked conformational changes in Vh, creating a novel helical bundle structure, and this alteration actively displaces Vt from Vh. These results, as well as the ability of alpha-actinin to also bind to Vh and displace Vt from pre-existing Vh-Vt complexes, support a model whereby Vh functions as a domain that undergoes marked structural changes that allow vinculin to direct cytoskeletal assembly in focal adhesions and adherens junctions. Notably, talin's effects on Vh structure establish helical bundle conversion as a signalling mechanism by which proteins direct cellular responses.     

Recruitment and regulation of phosphatidylinositol phosphate kinase type 1 gamma by the FERM domain of talin

Membrane phosphoinositides control a variety of cellular processes through the recruitment and/or regulation of cytosolic proteins. One mechanism ensuring spatial specificity in phosphoinositide signalling is the targeting of enzymes that mediate their metabolism to specific subcellular sites. Phosphatidylinositol phosphate kinase type 1 gamma (PtdInsPKI gamma) is a phosphatidylinositol-4-phosphate 5-kinase that is expressed at high levels in brain, and is concentrated at synapses. Here we show that the predominant brain splice variant of PtdInsPKI gamma (PtdInsPKI gamma-90) binds, by means of a short carboxy-terminal peptide, to the FERM domain of talin, and is strongly activated by this interaction. Talin, a principal component of focal adhesion plaques, is also present at synapses. PtdInsPKI gamma-90 is expressed in non-neuronal cells, albeit at much lower levels than in neurons, and is concentrated at focal adhesion plaques, where phosphatidylinositol-4,5-bisphosphate has an important regulatory role. Overexpression of PtdInsPKI gamma-90, or expression of its C-terminal domain, disrupts focal adhesion plaques, probably by local disruption of normal phosphoinositide balance. These findings define an interaction that has a regulatory role in cell adhesion and suggest new similarities between molecular interactions underlying synaptic junctions and general mechanisms of cell adhesion.     

Critical role for the p110alpha phosphoinositide-3-OH kinase in growth and metabolic regulation

The eight catalytic subunits of the mammalian phosphoinositide-3-OH kinase (PI(3)K) family form the backbone of an evolutionarily conserved signalling pathway; however, the roles of most PI(3)K isoforms in organismal physiology and disease are unknown. To delineate the role of p110alpha, a ubiquitously expressed PI(3)K involved in tyrosine kinase and Ras signalling, here we generated mice carrying a knockin mutation (D933A) that abrogates p110alpha kinase activity. Homozygosity for this kinase-dead p110alpha led to embryonic lethality. Mice heterozygous for this mutation were viable and fertile, but displayed severely blunted signalling via insulin-receptor substrate (IRS) proteins, key mediators of insulin, insulin-like growth factor-1 and leptin action. Defective responsiveness to these hormones led to reduced somatic growth, hyperinsulinaemia, glucose intolerance, hyperphagia and increased adiposity in mice heterozygous for the D933A mutation. This signalling function of p110alpha derives from its highly selective recruitment and activation to IRS signalling complexes compared to p110beta, the other broadly expressed PI(3)K isoform, which did not contribute to IRS-associated PI(3)K activity. p110alpha was the principal IRS-associated PI(3)K in cancer cell lines. These findings demonstrate a critical role for p110alpha in growth factor and metabolic signalling and also suggest an explanation for selective mutation or overexpression of p110alpha in a variety of cancers.     

Transformation of cell adhesion properties by exogenously introduced E-cadherin cDNA

E-cadherin is a cell surface glycoprotein responsible for Ca2+-dependent intercellular adhesion between epithelial cells; it is also called uvomorulin, L-CAM (ref. 3), cell-CAM 120/80 (ref.4) or Arc-1 (ref. 5). Because blocking the action of E-cadherin by monoclonal antibodies causes dispersion of compact cell colonies, this molecule is thought to be an important factor for maintenance of multicellular systems. To demonstrate directly that E-cadherin is involved in cell-cell adhesion, we cloned full-length cDNA encoding E-cadherin from F9 cells and introduced it into L fibroblasts deficient in E-cadherin. These L cells acquire strong Ca2+-dependent aggregating activity by expressing the E-cadherin derived from the introduced cDNA and were morphologically transformed so as to form colonies in which cells were tightly connected to each other.     

Regulated expression and binding of three VLA (beta 1) integrin receptors on T cells

Regulated adhesion of T cells to extracellular matrix (ECM) proteins is likely to be essential in T cell migration. Constitutive binding of various other cell types to ECM components is mediated by members of the VLA (very late antigen) subfamily of integrins. We describe here the regulated binding of resting CD4+ human T cells to ECM through three VLA integrins: VLA-4 and VLA-5 binding to fibronectin (FN), and a novel pathway of VLA-6 binding to laminin (LN). Binding to ECM is regulated in two ways. First, unlike other VLA-mediated interactions, VLA binding activity of the T cells is rapidly and dramatically augmented with cell activation without change in level of expression of the VLA molecules. Second, binding is regulated with T-cell differentiation; memory T cells express three- to four-fold more VLA-4, VLA-5, and VLA-6 than do naive cells, and bind more efficiently through them to FN and LN.     

Acetylation-dependent regulation of endothelial Notch signalling by the SIRT1 deacetylase

Notch signalling is a key intercellular communication mechanism that is essential for cell specification and tissue patterning, and which coordinates critical steps of blood vessel growth. Although subtle alterations in Notch activity suffice to elicit profound differences in endothelial behaviour and blood vessel formation, little is known about the regulation and adaptation of endothelial Notch responses. Here we report that the NAD(+)-dependent deacetylase SIRT1 acts as an intrinsic negative modulator of Notch signalling in endothelial cells. We show that acetylation of the Notch1 intracellular domain (NICD) on conserved lysines controls the amplitude and duration of Notch responses by altering NICD protein turnover. SIRT1 associates with NICD and functions as a NICD deacetylase, which opposes the acetylation-induced NICD stabilization. Consequently, endothelial cells lacking SIRT1 activity are sensitized to Notch signalling, resulting in impaired growth, sprout elongation and enhanced Notch target gene expression in response to DLL4 stimulation, thereby promoting a non-sprouting, stalk-cell-like phenotype. In vivo, inactivation of Sirt1 in zebrafish and mice causes reduced vascular branching and density as a consequence of enhanced Notch signalling. Our findings identify reversible acetylation of the NICD as a molecular mechanism to adapt the dynamics of Notch signalling, and indicate that SIRT1 acts as rheostat to fine-tune endothelial Notch responses.     

Identification of Lps2 as a key transducer of MyD88-independent TIR signalling

In humans, ten Toll-like receptor (TLR) paralogues sense molecular components of microbes, initiating the production of cytokine mediators that create the inflammatory response. Using N-ethyl-N-nitrosourea, we induced a germline mutation called Lps2, which abolishes cytokine responses to double-stranded RNA and severely impairs responses to the endotoxin lipopolysaccharide (LPS), indicating that TLR3 and TLR4 might share a specific, proximal transducer. Here we identify the Lps2 mutation: a distal frameshift error in a Toll/interleukin-1 receptor/resistance (TIR) adaptor protein known as Trif or Ticam-1. Trif(Lps2) homozygotes are markedly resistant to the toxic effects of LPS, and are hypersusceptible to mouse cytomegalovirus, failing to produce type I interferons when infected. Compound homozygosity for mutations at Trif and MyD88 (a cytoplasmic TIR-domain-containing adaptor protein) loci ablates all responses to LPS, indicating that only two signalling pathways emanate from the LPS receptor. However, a Trif-independent cell population is detectable when Trif(Lps2) mutant macrophages are stimulated with LPS. This reveals that an alternative MyD88-dependent 'adaptor X' pathway is present in some, but not all, macrophages, and implies afferent immune specialization.     

RIP3 mediates the embryonic lethality of caspase-8-deficient mice

Apoptosis and necroptosis are complementary pathways controlled by common signalling adaptors, kinases and proteases; among these, caspase-8 (Casp8) is critical for death receptor-induced apoptosis. This caspase has also been implicated in non-apoptotic pathways that regulate Fas-associated via death domain (FADD)-dependent signalling and other less defined biological processes as diverse as innate immune signalling and myeloid or lymphoid differentiation patterns. Casp8 suppresses RIP3-RIP1 (also known as RIPK3-RIPK1) kinase complex-dependent necroptosis that follows death receptor activation as well as a RIP3-dependent, RIP1-independent necrotic pathway that has emerged as a host defence mechanism against murine cytomegalovirus. Disruption of Casp8 expression leads to embryonic lethality in mice between embryonic days 10.5 and 11.5 (ref. 7). Thus, Casp8 may naturally hold alternative RIP3-dependent death pathways in check in addition to promoting apoptosis. We find that RIP3 is responsible for the mid-gestational death of Casp8-deficient embryos. Remarkably, Casp8(-/-)Rip3(-/-) double mutant mice are viable and mature into fertile adults with a full immune complement of myeloid and lymphoid cell types. These mice seem immunocompetent but develop lymphadenopathy by four months of age marked by accumulation of abnormal T cells in the periphery, a phenotype reminiscent of mice with Fas-deficiency (lpr/lpr; also known as Fas). Thus, Casp8 contributes to homeostatic control in the adult immune system; however, RIP3 and Casp8 are together completely dispensable for mammalian development.     

Anchoring mechanisms for LFA-3 cell adhesion glycoprotein at membrane surface

The manner in which a membrane protein is anchored to the lipid bilayer may have a profound influence on its function. Most cell surface membrane proteins are anchored by a membrane-spanning segment(s) of the polypeptide chain, but another type of anchor has been described for several proteins: a phosphatidyl inositol glycan moiety, attached to the protein C terminus. This type of linkage has been identified on membrane proteins involved in adhesion and transmembrane signalling and could be important in the execution of these functions. We report here that an immunologically important adhesion glycoprotein, lymphocyte function-associated antigen 3 (LFA-3), can be anchored to the membrane by both types of mechanism. These two distinct cell-surface forms of LFA-3 are derived from different biosynthetic precursors. The existence of a phosphatidyl-inositol-linked and a transmembrane anchored form of LFA-3 has important implications for adhesion and transmembrane signalling by LFA-3.     

Semaphorin 7A promotes axon outgrowth through integrins and MAPKs

Striking parallels exist between immune and nervous system cellular signalling mechanisms. Molecules originally shown to be critical for immune responses also serve neuronal functions, and similarly neural guidance cues can modulate immune function. We show here that semaphorin 7A (Sema7A), a membrane-anchored member of the semaphorin family of guidance proteins previously known for its immunomodulatory effects, can also mediate neuronal functions. Unlike many other semaphorins, which act as repulsive guidance cues, Sema7A enhances central and peripheral axon growth and is required for proper axon tract formation during embryonic development. Unexpectedly, Sema7A enhancement of axon outgrowth requires integrin receptors and activation of MAPK signalling pathways. These findings define a previously unknown biological function for semaphorins, identify an unexpected role for integrins and integrin-dependent intracellular signalling in mediating semaphorin responses, and provide a framework for understanding and interfering with Sema7A function in both immune and nervous systems.     

Functional cloning of ICAM-2, a cell adhesion ligand for LFA-1 homologous to ICAM-1

The leukocyte adhesion molecule LFA-1 mediates a wide range of lymphocyte, monocyte, natural killer cell, and granulocyte interactions with other cells in immunity and inflammation. LFA-1 (CD11a/CD18) is a receptor for intercellular adhesion molecule 1 (ICAM-1, CD54), a surface molecule which is constitutively expressed on some tissues and induced on other in inflammation. Induction of ICAM-1 on epithelial cells, endothelial cells and fibroblasts mediates LFA-1-dependent adhesion of lymphocytes. Several lines of evidence have suggested the existence of a second LFA-1 ligand: homotypic adhesion of one cell line was inhibited by a monoclonal antibody to LFA-1, but not by one to ICAM-1; there exists an LFA-1-dependent, ICAM-1-independent pathway of adhesion to endothelial cells; and also, there are some types of target cells in which LFA-1-dependent T-lymphocyte adhesion and lysis are independent of ICAM-1. We have cloned this second ligand, designated ICAM-2, using a novel method for identifying ligands of adhesion molecules. ICAM-2 is an integral membrane protein with two immunoglobulin-like domains, whereas ICAM-1 has five. Remarkably, ICAM-2 is much more closely related to the two most N-terminal domains of ICAM-1 (34% identity) than either ICAM-1 or ICAM-2 is to other members of the immunoglobulin superfamily, demonstrating the existence of a subfamily of immunoglobulin-like ligands that bind the same integrin receptor.