Jonathan D. Humphries

Integrin ligands at a glance

Integrins are one of the major families of cell adhesion receptors ([Humphries, 2000][1]; [Hynes, 2002][2]). All integrins are non-covalently linked, heterodimeric molecules containing an α and a β subunit. Both subunits are type I transmembrane proteins, containing large extracellular domains and

Vinculin controls focal adhesion formation by direct interactions with talin and actin.

Focal adhesions (FAs) regulate cell migration. Vinculin, with its many potential binding partners, can interconnect signals in FAs. Despite the well-characterized structure of vinculin, the molecular mechanisms underlying its action have remained unclear. Here, using vinculin mutants, we separate the vinculin head and tail regions into distinct functional domains. We show that the vinculin head regulates integrin dynamics and clustering and the tail regulates the link to the mechanotransduction force machinery. The expression of vinculin constructs with unmasked binding sites in the head and tail regions induces dramatic FA growth, which is mediated by their direct interaction with talin. This interaction leads to clustering of activated integrin and an increase in integrin residency time in FAs. Surprisingly, paxillin recruitment, induced by active vinculin constructs, occurs independently of its potential binding site in the vinculin tail. The vinculin tail, however, is responsible for the functional link of FAs to the actin cytoskeleton. We propose a new model that explains how vinculin orchestrates FAs.

Proteomic Analysis of Integrin-Associated Complexes Identifies RCC2 as a Dual Regulator of Rac1 and Arf6

Regulator of chromosome condensation–2 is a component of fibronectin-activated signaling pathways that regulate cell migration. Integrin Interactors Integrins mediate cell-cell adhesion, as well as cell adhesion to the extracellular matrix. Identification of the intracellular signaling networks associated with integrins is of interest because integrins are involved in processes such as invasion of tumor cells during metastasis and leukocyte infiltration during inflammation. Humphries et al. developed a method of isolating protein complexes associated with α5β1 integrin, which binds to fibronectin, and with α4β1 integrin, which binds to vascular cell adhesion molecule–1. Although a subset of proteins was detected in both the α5β1 and α4β1 networks, there were several receptor-specific proteins. In particular, regulator of chromosome condensation–2 (RCC2) was identified as a component of the α5β1 integrin–associated signaling network. RCC2 promoted fibronectin-dependent migration by inhibiting two different subnetworks (Rac1 and Arf6). These techniques provide the means to investigate the composition and function of adhesion complexes under different physiological conditions. The binding of integrin adhesion receptors to their extracellular matrix ligands controls cell morphology, movement, survival, and differentiation in various developmental, homeostatic, and disease processes. Here, we report a methodology to isolate complexes associated with integrin adhesion receptors, which, like other receptor-associated signaling complexes, have been refractory to proteomic analysis. Quantitative, comparative analyses of the proteomes of two receptor-ligand pairs, α4β1–vascular cell adhesion molecule–1 and α5β1–fibronectin, defined both core and receptor-specific components. Regulator of chromosome condensation–2 (RCC2) was detected in the α5β1–fibronectin signaling network at an intersection between the Rac1 and adenosine 5′-diphosphate ribosylation factor 6 (Arf6) subnetworks. RCC2 knockdown enhanced fibronectin-induced activation of both Rac1 and Arf6 and accelerated cell spreading, suggesting that RCC2 limits the signaling required for membrane protrusion and delivery. Dysregulation of Rac1 and Arf6 function by RCC2 knockdown also abolished persistent migration along fibronectin fibers, indicating a functional role for RCC2 in directional cell movement. This proteomics workflow now opens the way to further dissection and systems-level analyses of adhesion signaling.

Fibrillin microfibrils are reduced in skin exhibiting striae distensae

Striae distensae (striae: stretch marks) are a common disfiguring condition associated with continuous and progressive stretching of the skin—as occurs during pregnancy. The pathogenesis of striae is unknown but probably relates to changes in those structures that provide skin with its tensile strength and elasticity. Such structures are components of the extracellular matrix, including fibrillin, elastin and collagens. Using a variety of histological techniques, we assessed the distribution of these extracellular matrix components in skin affected by striae. Pregnant women were assessed for the presence of striae, and punch biopsies were obtained from lesional striae and adjacent normal skin. Biopsies were processed for electron microscopy, light microscopy and immunohistochemistry. For histological examination, 7 μm frozen sections were stained so as to identify the elastic fibre network and glycosaminoglycans. Biopsies were also examined with a panel of polyclonal antibodies against collagens I and III, and fibrillin and elastin. Ultrastructural analysis revealed alterations in the appearance of skin affected by striae compared with that of normal skin in that the dermal matrix of striae was looser and more floccular. Light microscopy revealed an increase in glycosaminoglycan content in striae. Furthermore, the number of vertical fibrillin fibres subjacent to the dermal–epidermal junction (DEJ) and elastin fibres in the papillary dermis was significantly reduced in striae compared with normal skin. The orientation of elastin and fibrillin fibres in the deep dermis showed realignment in that the fibres ran parallel to the DEJ. However, no significant alterations were observed in any other extracellular matrix components. This study identifies a reorganization and diminution of the elastic fibre network of skin affected by striae. Continuous strain on the dermal extracellular matrix, as occurs during pregnancy, may remodel the elastic fibre network in susceptible individuals and manifest clinically as striae distensae.

Anti-integrin monoclonal antibodies

Integrins are a family of 24 heterodimeric transmembrane receptors that support cell-cell and cell-ECM (extracellular matrix) interactions in a multitude of physiological and disease situations ([Humphries, 2000][1]; [Hynes, 2002][2]). Adhesion that is mediated by integrins is controlled dynamically

Definition of a consensus integrin adhesome and its dynamics during adhesion complex assembly and disassembly

Integrin receptor activation initiates the formation of integrin adhesion complexes (IACs) at the cell membrane that transduce adhesion-dependent signals to control a multitude of cellular functions. Proteomic analyses of isolated IACs have revealed an unanticipated molecular complexity; however, a global view of the consensus composition and dynamics of IACs is lacking. Here, we have integrated several IAC proteomes and generated a 2,412-protein integrin adhesome. Analysis of this data set reveals the functional diversity of proteins in IACs and establishes a consensus adhesome of 60 proteins. The consensus adhesome is likely to represent a core cell adhesion machinery, centred around four axes comprising ILK–PINCH–kindlin, FAK–paxillin, talin–vinculin and α-actinin–zyxin–VASP, and includes underappreciated IAC components such as Rsu-1 and caldesmon. Proteomic quantification of IAC assembly and disassembly detailed the compositional dynamics of the core cell adhesion machinery. The definition of this consensus view of integrin adhesome components provides a resource for the research community.

A syndecan-4 hair trigger initiates wound healing through caveolin- and RhoG-regulated integrin endocytosis.

Summary Cell migration during wound healing requires adhesion receptor turnover to enable the formation and disassembly of cell-extracellular matrix contacts. Although recent advances have improved our understanding of integrin trafficking pathways, it is not known how extracellular ligand engagement controls receptor dynamics. Using atomic force microscopy, we have measured cell avidity for fibronectin and defined a mechanism for the outside-in regulation of α5β1-integrin. Surprisingly, adhesive strength was attenuated by the syndecan-4-binding domain of fibronectin due to a rapid triggering of α5β1-integrin endocytosis. Association of syndecan-4 with PKCα was found to trigger RhoG activation and subsequent dynamin- and caveolin-dependent integrin uptake. Like disruption of syndecan-4 or caveolin, gene disruption of RhoG in mice was found to retard closure of dermal wounds due to a migration defect of the fibroblasts and keratinocytes of RhoG null mice. Thus, this syndecan-4-regulated integrin endocytic pathway appears to play a key role in tissue repair.

Quantification of integrin receptor agonism by fluorescence lifetime imaging

Both spatiotemporal analyses of adhesion signalling and the development of pharmacological inhibitors of integrin receptors currently suffer from the lack of an assay to measure integrin-effector binding and the response of these interactions to antagonists. Indeed, anti-integrin compounds have failed in the clinic because of secondary side effects resulting from agonistic activity. Here, we have expressed integrin-GFP and effector-mRFP pairs in living cells and quantified their association using fluorescence lifetime imaging microscopy (FLIM) to measure fluorescence resonance energy transfer (FRET). Association of talin with β1 integrin and paxillin with α4 integrin was dependent on both the ligand and receptor activation state, and was sensitive to inhibition with small molecule RGD and LDV mimetics, respectively. An adaptation of the assay revealed the agonistic activity of these small molecules, thus demonstrating that these compounds may induce secondary effects in vivo via integrin activation. This study provides insight into the dependence of the activity of small molecule anti-integrin compounds upon receptor conformation, and provides a novel quantitative assay for the validation of potential integrin antagonists.

An unraveling tale of how integrins are activated from within

Integrin cytoplasmic tail domains are short, but are essential for normal receptor function because of their key role in relaying bidirectional signals across the plasma membrane. Although it is well established that the cytoplasmic tails both initiate signalling pathways inside the cell and control the transition of integrins from a resting to a ligand-binding competent state, until recently the structural basis of these changes has been unclear. In the past year, however, a series of structural studies has revealed certain features of cytoplasmic domain function, and in this review we focus on how these advances have enlightened our understanding of integrin tail structure and function.

Defining the extracellular matrix using proteomics

The cell microenvironment has a profound influence on the behaviour, growth and survival of cells. The extracellular matrix (ECM) provides not only mechanical and structural support to cells and tissues but also binds soluble ligands and transmembrane receptors to provide spatial coordination of signalling processes. The ability of cells to sense the chemical, mechanical and topographical features of the ECM enables them to integrate complex, multiparametric information into a coherent response to the surrounding microenvironment. Consequently, dysregulation or mutation of ECM components results in a broad range of pathological conditions. Characterization of the composition of ECM derived from various cells has begun to reveal insights into ECM structure and function, and mechanisms of disease. Proteomic methodologies permit the global analysis of subcellular systems, but extracellular and transmembrane proteins present analytical difficulties to proteomic strategies owing to the particular biochemical properties of these molecules. Here, we review advances in proteomic approaches that have been applied to furthering our understanding of the ECM microenvironment. We survey recent studies that have addressed challenges in the analysis of ECM and discuss major outcomes in the context of health and disease. In addition, we summarize efforts to progress towards a systems‐level understanding of ECM biology.