Abstract: Integrin receptors regulate cell fate by coupling the binding of extracellular adhesion proteins to the assembly of intracellular cytoskeletal and signaling complexes. A detailed, integrative view of adhesion complexes will provide insight into the molecular mechanisms that control cell morphology, survival, movement, and differentiation. To date, membrane receptor-associated signaling complexes have been refractory to proteomic analysis because of their inherent lability and inaccessibility. We developed a methodology to isolate ligand-induced integrin adhesion complexes, and we used this technique to analyze the composition of complexes associated with multiple receptor-ligand pairs and define core and receptor-specific subnetworks. In particular, we identified regulator of chromosome condensation-2 (RCC2) as a component of fibronectin-activated signaling pathways that regulate directional cell movement. The development of this proteomics pipeline provides the means to investigate the molecular composition and function of various adhesion complexes.
Abstract: Cell adhesion to the extracellular matrix (ECM) is necessary for fundamental cellular processes such as survival, migration, and differentiation. Adhesion is mediated by integrin receptors, which recruit multiprotein adhesion complexes to sites of attachment to the ECM. Adhesion complexes provide a structural connection between the ECM and cytoskeleton, transmit mechanical force, and act as signaling hubs to control cell behavior. Recent high-resolution imaging studies of adhesion sites reveal some aspects of their spatial organization and provide insights into their function at the molecular level.
Abstract: 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 α(5)β(1)-integrin. Surprisingly, adhesive strength was attenuated by the syndecan-4-binding domain of fibronectin due to a rapid triggering of α(5)β(1)-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.
Abstract: Intercellular communication in metazoa not only requires autocrine, paracrine and exocrine signalling systems, but it also relies on the structural and positional information encoded in extracellular matrices (ECMs). Most cells in tissues are structurally and functionally integrated with their surrounding ECM in a highly organised manner involving thousands of dynamic connections. On the intracellular face of these linkages, adhesion receptors - principally integrins and syndecans - link the cytoskeleton to the plasma membrane and compartmentalise cytoplasmic signalling events, whereas at the extracellular face the same receptors direct and organise the deposition of the ECM itself. Adhesion receptors transduce mechanical force bidirectionally across the plasma membrane by tethering variably deformable ECMs to the contractile cytoskeleton (Figure 1), and they translate the topography and composition of the ECM into chemical signals that determine behaviour. The membrane-proximal functions of adhesion receptors in turn trigger distal processes within cells, such as alterations in the direction of cell movement and the regulation of gene transcription, and long-range effects outside cells, such as the construction of ECM networks and consequent shaping of higher-order tissue structure. Given the diverse and fundamental roles attributed to adhesion, it is understandable that adhesion receptor engagement has been reported to alter the flux through virtually all major signalling pathways.
Abstract: Integrin alpha5beta1 is a key receptor for the extracellular matrix protein fibronectin. Antagonists of human integrin alpha5beta1 have therapeutic potential as anti-angiogenic agents in cancer and diseases of the eye. However, the structure of the integrin is unsolved and the atomic basis of fibronectin and antagonist binding by integrin alpha5beta1 is poorly understood. In the present study, we demonstrate that zebrafish alpha5beta1 integrins do not interact with human fibronectin or the human alpha5beta1 antagonists JSM6427 and cyclic peptide CRRETAWAC. Zebrafish alpha5beta1 integrins do bind zebrafish fibronectin-1, and mutagenesis of residues on the upper surface and side of the zebrafish alpha5 subunit beta-propeller domain shows that these residues are important for the recognition of the Arg-Gly-Asp (RGD) motif and the synergy sequence [Pro-His-Ser-Arg-Asn (PHSRN)] in fibronectin. Using a gain-of-function analysis involving swapping regions of the zebrafish integrin alpha5 subunit with the corresponding regions of human alpha5 we show that blades 1-4 of the beta-propeller are required for human fibronectin recognition, suggesting that fibronectin binding involves a broad interface on the side and upper face of the beta-propeller domain. We find that the loop connecting blades 2 and 3 of the beta-propeller, the D3-A3 loop, contains residues critical for antagonist recognition, with a minor role played by residues in neighbouring loops. A new homology model of human integrin alpha5beta1 supports an important function for D3-A3 loop residues Trp157 and Ala158 in the binding of antagonists. These results will aid the development of reagents that block integrin alpha5beta1 functions in vivo.
Abstract: The formation, maturation, and dissolution of focal adhesions are basic prerequisites of cell migration and rely on the recruitment, signalling, and endocytosis of integrins. In many instances, extracellular matrix molecules are recognised by a number of integrins, and it is the sequential involvement of different integrins that allows establishment of cell polarity and migration towards a matrix stimulus. In this review, we consider both the similarities and differences between two key fibronectin receptors, alpha(v)beta(3) and alpha(5)beta(1) integrin. By considering the GTPase and kinase signalling and trafficking of two such closely-related receptors, we begin to understand how cell migration is coordinated.
Abstract: 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, alpha(4)beta(1)-vascular cell adhesion molecule-1 and alpha(5)beta(1)-fibronectin, defined both core and receptor-specific components. Regulator of chromosome condensation-2 (RCC2) was detected in the alpha(5)beta(1)-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.
Abstract: Knowledge of the uptake, membrane translocation, refolding and ribosome interaction of the ribosome-inactivating toxin ricin is incomplete at the present time. Ricin A chain (RTA) is the catalytic subunit of holotoxin and is also of particular interest as a vaccine candidate. For many studies into the uptake and immunological applications of ricin, it is essential to have inactive variants. Here, following error-prone polymerase chain reaction of the RTA open reading frame, we have used a modified gap-repair protocol in Saccharomyces cerevisiae to show that it is possible to rapidly generate a panel of inactive RTA mutants. Since yeast cells have ribosomes that are highly sensitive to RTA, we utilized a genetic selection based on the viability of transformants. This enabled the recovery of a number of mutations, some not previously identified, which permitted production of full-length but non-toxic RTA proteins. Such disarmed toxins may have utility as tools to study the cytosolic entry and action of RTA, and as potential vaccine candidates.
