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1.  Extracellular Matrix Proteins in Hemostasis and Thrombosis 
The adhesion and aggregation of platelets during hemostasis and thrombosis represents one of the best-understood examples of cell–matrix adhesion. Platelets are exposed to a wide variety of extracellular matrix (ECM) proteins once blood vessels are damaged and basement membranes and interstitial ECM are exposed. Platelet adhesion to these ECM proteins involves ECM receptors familiar in other contexts, such as integrins. The major platelet-specific integrin, αIIbβ3, is the best-understood ECM receptor and exhibits the most tightly regulated switch between inactive and active states. Once activated, αIIbβ3 binds many different ECM proteins, including fibrinogen, its major ligand. In addition to αIIbβ3, there are other integrins expressed at lower levels on platelets and responsible for adhesion to additional ECM proteins. There are also some important nonintegrin ECM receptors, GPIb-V-IX and GPVI, which are specific to platelets. These receptors play major roles in platelet adhesion and in the activation of the integrins and of other platelet responses, such as cytoskeletal organization and exocytosis of additional ECM ligands and autoactivators of the platelets.
Platelets express a variety of receptors (e.g., the major platelet-specific integrin αIIbβ3) that bind to a diverse array of extracellular matrix proteins exposed on injury (e.g., fibrinogen). Drugs targeting these receptor-ligand interactions are effective.
doi:10.1101/cshperspect.a005132
PMCID: PMC3281566  PMID: 21937733
2.  Migfilin and Filamin as Regulators of Integrin Activation in Endothelial Cells and Neutrophils 
PLoS ONE  2011;6(10):e26355.
Cell adhesion and migration depend on engagement of extracellular matrix ligands by integrins. Integrin activation is dynamically regulated by interactions of various cytoplasmic proteins, such as filamin and integrin activators, talin and kindlin, with the cytoplasmic tail of the integrin β subunit. Although filamin has been suggested to be an inhibitor of integrin activation, direct functional evidence for the inhibitory role of filamin is limited. Migfilin, a filamin-binding protein enriched at cell-cell and cell-extracellular matrix contact sites, can displace filamin from β1 and β3 integrins and promote integrin activation. However, its role in activation and functions of different β integrins in human vascular cells is unknown. In this study, using flow cytometry, we demonstrate that filamin inhibits β1 and αIIbβ3 integrin activation, and migfilin can overcome its inhibitory effect. Migfilin protein is widely expressed in different adherent and circulating blood cells and can regulate integrin activation in naturally-occurring vascular cells, endothelial cells and neutrophils. Migfilin can activate β1, β2 and β3 integrins and promote integrin mediated responses while migfilin depletion impairs the spreading and migration of endothelial cells. Thus, filamin can act broadly as an inhibitor and migfilin is a promoter of integrin activation.
doi:10.1371/journal.pone.0026355
PMCID: PMC3197140  PMID: 22043318
3.  Recreation of the terminal events in physiological integrin activation 
The Journal of Cell Biology  2010;188(1):157-173.
In vitro analysis confirms talin binding is sufficient for activation and extension of membrane-embedded integrin.
Increased affinity of integrins for the extracellular matrix (activation) regulates cell adhesion and migration, extracellular matrix assembly, and mechanotransduction. Major uncertainties concern the sufficiency of talin for activation, whether conformational change without clustering leads to activation, and whether mechanical force is required for molecular extension. Here, we reconstructed physiological integrin activation in vitro and used cellular, biochemical, biophysical, and ultrastructural analyses to show that talin binding is sufficient to activate integrin αIIbβ3. Furthermore, we synthesized nanodiscs, each bearing a single lipid-embedded integrin, and used them to show that talin activates unclustered integrins leading to molecular extension in the absence of force or other membrane proteins. Thus, we provide the first proof that talin binding is sufficient to activate and extend membrane-embedded integrin αIIbβ3, thereby resolving numerous controversies and enabling molecular analysis of reconstructed integrin signaling.
doi:10.1083/jcb.200908045
PMCID: PMC2812850  PMID: 20048261
4.  Integrin adhesions 
Cell Adhesion & Migration  2012;6(4):302-306.
Cell migration requires the coordination of adhesion site assembly and turnover. Canonical models for nascent adhesion formation postulate that integrin binding to extracellular matrix (ECM) proteins results in the rapid recruitment of cytoskeletal proteins such as talin and paxillin to integrin cytoplasmic domains. It is thought that integrin-talin clusters recruit and activate tyrosine kinases such as focal adhesion kinase (FAK). However, the molecular connections of this linkage remain unresolved. Our recent findings support an alternative model whereby FAK recruits talin to new sites of β1 integrin-mediated adhesion in mouse embryonic fibroblasts and human ovarian carcinoma cells. This is dependent on a direct binding interaction between FAK and talin and occurs independently of direct talin binding to β1 integrin. Herein, we discuss differences between nascent and mature adhesions, interactions between FAK, talin and paxillin, possible mechanisms of FAK activation and how this FAK-talin complex may function to promote cell motility through increased adhesion turnover.
doi:10.4161/cam.20488
PMCID: PMC3478250  PMID: 22983197
adhesion assembly; focal adhesion kinase; focal adhesion turnover; motility; nascent adhesion; recruitment; talin; β1 integrin
5.  Integrin activation 
Biochemical Society transactions  2008;36(Pt 2):229-234.
Agonist stimulation of integrin receptors, composed of transmembrane α and β subunits, leads cells to regulate integrin affinity (‘activation’), a process that controls cell adhesion and migration, and extracellular matrix assembly. A final step in integrin activation is the binding of talin to integrin β cytoplasmic domains. We used forward, reverse and synthetic genetics to engineer and order integrin activation pathways of a prototypic integrin, platelet αIIbβ3. PMA activated αIIbβ3 only after expression of both PKCα (protein kinase Cα) and talin at levels approximating those in platelets. Inhibition of Rap1 GTPase reduced αIIbβ3 activation, whereas expression of constitutively active Rap1A(G12V) bypassed the requirement for PKCα. Overexpression of a Rap effector, RIAM (Rap1-GTP-interacting adaptor molecule), activated αIIbβ3 and bypassed the requirement for PKCα and Rap1. In addition, shRNA (short hairpin RNA)-mediated knockdown of RIAM blocked talin interaction with and activation of integrin αIIbβ3. Rap1 activation caused the formation of an ‘activation complex’ containing talin and RIAM that redistributed to the plasma membrane and activated αIIbβ3. The central finding was that this Rap1-induced formation of an ’integrin activation complex’ leads to the unmasking of the integrin-binding site on talin, resulting in integrin activation.
doi:10.1042/BST0360229
PMCID: PMC2588347  PMID: 18363565
cell adhesion; integrin; Rap1; signal transduction; talin; thrombosis
6.  Integrin Clustering in Two and Three Dimensions 
Langmuir  2012;28(12):5379-5386.
Integrins are transmembrane proteins that allow cells to bind to their external environment. They are the primary regulators of cell-matrix interactions, with direct roles in cell motility and signaling, which in turn regulate numerous physiological processes. Under common experimental conditions, integrins tend to cluster for sturdy and effective binding to extra-cellular matrix molecules. These clusters often evolve into focal adhesions, which regulate downstream signaling. However, integrin clusters are more pronounced and have longer lifetimes in two-dimensional assays than in more realistic three-dimensional environments. While a number of models and theoretical approaches have focused on integrin binding and diffusion, the reasons for the differences between two- and three-dimensional clustering have remained elusive. In this study, we model an individual cluster attached to a two-dimensional collagen film and attached to collagen fibers of various sizes in three-dimensional matrices. We then discuss how our results explain differences in size and lifetime, and how they hint at reasons for other differences between the two environments. Further, we make predictions regarding the stability of clusters based on different overall intracellular conditions. Our results show good agreement with experiments and provide a quantitative basis for understanding how matrix dimensionality and structure regulate integrin behavior in environments that mimic in vivo conditions.
doi:10.1021/la203725a
PMCID: PMC3314142  PMID: 22204631
7.  Why Integrin as a Primary Target for Imaging and Therapy 
Theranostics  2011;1:30-47.
Integrin-mediated cell adhesion is involved in many essential normal cellular and pathological functions including cell survival, growth, differentiation, migration, inflammatory responses, platelet aggregation, tissue repair and tumor invasion. 24 different heterodimerized transmembrane integrin receptors are combined from 18 different α and 8 different β subunits. Each integrin subunit contains a large extracellular domain, a single transmembrane domain and a usually short cytoplasmic domain. Integrins bind extracellular matrix (ECM) proteins through their large extracellular domain, and engage the cytoskeleton via their short cytoplasmic tails. These integrin-mediated linkages on either side of the plasma membrane are dynamically linked. Thus, integrins communicate over the plasma membrane in both directions, i.e., outside-in and inside-out signaling. In outside-in signaling through integrins, conformational changes of integrin induced by ligand binding on the extracellular domain altered the cytoplasmic domain structures to elicit various intracellular signaling pathways. Inside-out signaling originates from non-integrin cell surface receptors or cytoplasmic molecules and it activates signaling pathways inside the cells, ultimately resulting in the activation/deactivation of integrins. Integrins are one of key family proteins for cell adhesion regulation through binding to a large number of ECM molecules and cell membrane proteins. Lack of expression of integrins may result in a wide variety of effects ranging from blockage in pre-implantation to embryonic or perinatal lethality and developmental defects. Based on both the key role they played in angiogenesis, leukocytes function and tumor development and easy accessibility as cell surface receptors interacting with extracellular ligands, the integrin superfamily represents the best opportunity of targeting both antibodies and small-molecule antagonists for both therapeutic and diagnostic utility in various key diseases so far.
PMCID: PMC3086610  PMID: 21544229
Integrin; inside-out signaling; outside-in signaling; cell adhesion molecule; angiogenesis.
8.  Integrin Clustering Is Driven by Mechanical Resistance from the Glycocalyx and the Substrate 
PLoS Computational Biology  2009;5(12):e1000604.
Integrins have emerged as key sensory molecules that translate chemical and physical cues from the extracellular matrix (ECM) into biochemical signals that regulate cell behavior. Integrins function by clustering into adhesion plaques, but the molecular mechanisms that drive integrin clustering in response to interaction with the ECM remain unclear. To explore how deformations in the cell-ECM interface influence integrin clustering, we developed a spatial-temporal simulation that integrates the micro-mechanics of the cell, glycocalyx, and ECM with a simple chemical model of integrin activation and ligand interaction. Due to mechanical coupling, we find that integrin-ligand interactions are highly cooperative, and this cooperativity is sufficient to drive integrin clustering even in the absence of cytoskeletal crosslinking or homotypic integrin-integrin interactions. The glycocalyx largely mediates this cooperativity and hence may be a key regulator of integrin function. Remarkably, integrin clustering in the model is naturally responsive to the chemical and physical properties of the ECM, including ligand density, matrix rigidity, and the chemical affinity of ligand for receptor. Consistent with experimental observations, we find that integrin clustering is robust on rigid substrates with high ligand density, but is impaired on substrates that are highly compliant or have low ligand density. We thus demonstrate how integrins themselves could function as sensory molecules that begin sensing matrix properties even before large multi-molecular adhesion complexes are assembled.
Author Summary
Critical cell decisions, including whether to live, proliferate, or assemble into tissue structures, are directed by cues from the extracellular matrix, the external protein scaffold that surrounds cells. Integrin receptors on the cell surface bind to the extracellular matrix and cluster into complexes that translate matrix cues into the set of instructions a cell follows. Using a newly developed model of the cell-matrix interface, in this work we detail a simple yet efficient mechanism by which integrins could “sense” important matrix properties, including chemical composition and mechanical stiffness, and cluster appropriately. This mechanism relies on mechanical resistance to integrin-matrix interaction provided by the glycocalyx, the slimy sugar and protein coating on the cell, as well as the stiffness of the matrix and the cell itself. In general, the resistance alters integrin-ligand reaction rates, such that integrin clustering is favored for many physiologically relevant conditions. Interestingly, the mechanical properties of the cell and ECM are altered in many prevalent diseases, such as cancer, and our work suggests how these mechanical perturbations might adversely influence integrin function.
doi:10.1371/journal.pcbi.1000604
PMCID: PMC2782178  PMID: 20011123
9.  Bimolecular integrin–ligand interactions quantified using peptide-functionalized dextran-coated microparticles 
Integrative Biology  2011;4(1):84-92.
Integrins play a key role in cell–cell and cell–matrix interactions. Artificial surfaces grafted with integrin ligands, mimicking natural interfaces, have been used to study integrin-mediated cell adhesion. Here we report the use of a new chemical engineering technology in combination with single-molecule nanomechanical measurements to quantify peptide binding to integrins. We prepared latex beads with covalently-attached dextran. The beads were then functionalized with the bioactive peptides, cyclic RGDFK (cRGD) and the fibrinogen γC-dodecapeptide (H12), corresponding to the active sites for fibrinogen binding to the platelet integrin αIIbβ3. Using optical tweezers-based force spectroscopy to measure non-specific protein–protein interactions, we found the dextran-coated beads nonreactive towards fibrinogen, thus providing an inert platform for biospecific modifications. Using periodate oxidation followed by reductive amination, we functionalized the bead-attached dextran with either cRGD or H12 and used the peptide-grafted beads to measure single-molecule interactions with the purified αIIbβ3. Bimolecular force spectroscopy revealed that the peptide-functionalized beads were highly and specifically reactive with the immobilized αIIbβ3. Further, the cRGD- and H12-functionalized beads displayed a remarkable interaction profile with a bimodal force distribution up to 90 pN. The cRGD–αIIbβ3 interactions had greater binding strength than that of H12–αIIbβ3, indicating that they are more stable and resistant mechanically, consistent with the platelet reactivity of RGD-containing ligands. Thus, the results reported here describe the mechanistic characteristics of αIIbβ3–ligand interactions, confirming the utility of peptide-functionalized latex beads for the quantitative analysis of molecular recognition.
doi:10.1039/c1ib00085c
PMCID: PMC3337774  PMID: 22120019
10.  αV-Integrins Are Required for Mechanotransduction in MDCK Epithelial Cells 
PLoS ONE  2013;8(8):e71485.
The properties of epithelial cells within tissues are regulated by their immediate microenvironment, which consists of neighboring cells and the extracellular matrix (ECM). Integrin heterodimers orchestrate dynamic assembly and disassembly of cell-ECM connections and thereby convey biochemical and mechanical information from the ECM into cells. However, the specific contributions and functional hierarchy between different integrin heterodimers in the regulation of focal adhesion dynamics in epithelial cells are incompletely understood. Here, we have studied the functions of RGD-binding αV-integrins in a Madin Darby Canine Kidney (MDCK) cell model and found that αV-integrins regulate the maturation of focal adhesions (FAs) and cell spreading. αV-integrin-deficient MDCK cells bound collagen I (Col I) substrate via α2β1-integrins but failed to efficiently recruit FA components such as talin, focal adhesion kinase (FAK), vinculin and integrin-linked kinase (ILK). The apparent inability to mature α2β1-integrin-mediated FAs and link them to cellular actin cytoskeleton led to disrupted mechanotransduction in αV-integrin deficient cells seeded onto Col I substrate.
doi:10.1371/journal.pone.0071485
PMCID: PMC3747215  PMID: 23977051
11.  G protein subunit Gα13 binds to integrin αIIbβ3 and mediates integrin “outside-in” signaling 
Science (New York, N.Y.)  2010;327(5963):340-343.
Integrins mediate cell adhesion to the extracellular matrix and transmit signals within the cell that stimulate cell spreading, retraction, migration, and proliferation. The mechanism of integrin outside-in signaling has been unclear. We found that the heterotrimeric guanine nucleotide-binding protein (G protein), Gα13, directly bound to the integrin β3 cytoplasmic domain, and that Gα13-integrin interaction was promoted by ligand binding to the integrin αIIbβ3 and by guanosine triphosphate (GTP)-loading of Gα13. Interference of Gα13 expression or a myristoylated fragment of Gα13 that inhibited interaction of αIIbβ3 with Gα13 diminished activation of protein kinase c-Src and stimulated the small GTPase RhoA, consequently inhibiting cell spreading and accelerating cell retraction. We conclude that integrins are non-canonical Gα13-coupled receptors that provide a mechanism for dynamic regulation of RhoA.
doi:10.1126/science.1174779
PMCID: PMC2842917  PMID: 20075254
12.  c-Abl Kinase Is a Regulator of αvβ3 Integrin Mediated Melanoma A375 Cell Migration 
PLoS ONE  2013;8(6):e66108.
Integrins are heterodimeric transmembrane receptors that physically link the extracellular matrix (ECM) to the intracellular actin cytoskeleton, and are also signaling molecules that transduce signals bi-directionally across the plasma membrane. Integrin regulation is essential for tumor cell migration in response to growth factors. c-Abl kinase is a nonreceptor tyrosine kinase and is critical for signaling transduction from various receptors. Here we show that c-Abl kinase is involved in A375 cell migration mediated by αvβ3 integrin in response to PDGF stimulation. c-Abl kinase colocalizes with αvβ3 integrin dynamically and affects αvβ3 integrin affinity by regulating its cluster. The interaction between c-Abl kinase and αvβ3 integrin was dependent on the activity of c-Abl kinase induced by PDGF stimulation, but was not dependent on the binding of αvβ3 integrin with its ligands, suggesting that c-Abl kinase is not involved in the outside-in signaling of αvβ3 integrin. Talin head domain was required for the interaction between c-Abl kinase and αvβ3 integrin, and the SH3 domain of c-Abl kinase was involved in its interaction with talin and αvβ3 integrin. Taken together, we have uncovered a novel and critical role of c-Abl kinase in αvβ3 integrin mediated melanoma cell migration.
doi:10.1371/journal.pone.0066108
PMCID: PMC3689700  PMID: 23805201
13.  Regulation of axonal outgrowth and pathfinding by integrin-ECM interactions 
Developmental neurobiology  2011;71(11):901-923.
Developing neurons use a combination of guidance cues to assemble a functional neural network. A variety of proteins immobilized within the extracellular matrix (ECM) provide specific binding sites for integrin receptors on neurons. Integrin receptors on growth cones associate with a number of cytosolic adaptor and signaling proteins that regulate cytoskeletal dynamics and cell adhesion. Recent evidence suggests that soluble growth factors and classic axon guidance cues may direct axon pathfinding by controlling integrin-based adhesion. Moreover, since classic axon guidance cues themselves are immobilized within the ECM and integrins modulate cellular responses to many axon guidance cues, interactions between activated receptors modulate cell signals and adhesion. Ultimately, growth cones control axon outgrowth and pathfinding behaviors by integrating distinct biochemical signals to promote the proper assembly of the nervous system. In this review, we discuss our current understanding how ECM proteins and their associated integrin receptors control neural network formation.
doi:10.1002/dneu.20931
PMCID: PMC3192254  PMID: 21714101
axon pathfinding; actin; microtubules; laminin
14.  Focal Adhesion Kinase Regulation of Neovascularization 
Microvascular research  2011;83(1):64-70.
In this review, we discuss the role of focal adhesion kinase (FAK), an intracellular tyrosine kinase, in endothelial cells in relation to neovascularization. Genetic and in vitro studies have identified critical factors, receptor systems, and their intracellular signaling components that regulate the neovasculogenic phenotypes of endothelial cells. Among these factors, FAK appears to regulate several aspects of endothelial cellular behavior, including migration, survival, cytoskeletal organization, as well as cell proliferation. Upon adhesion of endothelial cells to extracellular matrix (ECM) ligands, integrins cluster on the plane of plasma-membrane, while cytoplasmic domains of integrins interact with cytoskeletal proteins and signaling molecules including FAK. However, FAK not only serves as a critical component of integrin signaling, but is also a downstream element of the VEGF/VEGF-receptor and other ligand-receptor systems that regulate neovascularization. A complete understanding of FAK-mediated neovascularization, therefore, should address the molecular and cellular mechanisms that regulate the biology of FAK. Continued research on FAK may, therefore, yield novel therapies to improve treatment modalities for the pathological neovascularization associated with diseases.
doi:10.1016/j.mvr.2011.05.002
PMCID: PMC3186864  PMID: 21616084
Angiogenesis; bFGF; FAK; neovascularization; Src; VEGF
15.  ECM regulates MT1-MMP localization with β1 or αvβ3 integrins at distinct cell compartments modulating its internalization and activity on human endothelial cells 
The Journal of Cell Biology  2002;159(3):509-521.
Regulation of membrane-type 1 matrix metalloproteinase (MT1-MMP) by different extracellular matrices (ECMs) on human endothelial cells (ECs) has been investigated. First, MT1-MMP is found at the intercellular contacts of confluent ECs grown on β1 integrin–dependent matrix such as type 1 collagen (COL I), fibronectin (FN), or fibrinogen (FG), but not on gelatin (GEL) or vitronectin (VN). The novel localization of MT1-MMP at cell–cell contacts is assessed by confocal videomicroscopy of MT1-MMP-GFP–transfected ECs. Moreover, MT1-MMP colocalizes with β1 integrins at the intercellular contacts, whereas it is preferentially found with αvβ3 integrin at motility-associated structures on migrating ECs. In addition, clustered integrins recruit MT1-MMP and neutralizing anti-β1 or anti-αv integrin mAb displace MT1-MMP from its specific sites, pointing to a biochemical association that is finally demonstrated by coimmunoprecipitation assays. On the other hand, COL I, FN, or FG up-regulate cell surface MT1-MMP on confluent ECs by an impairment of its internalization, whereas expression and internalization are not modified on GEL or VN. In addition, MT1-MMP activity is diminished in confluent ECs on COL I, FN, or FG. Finally, MT1-MMP participates and cooperates with β1 and αvβ3 integrins in the migration of ECs on different ECM. These data show a novel mechanism by which ECM regulates MT1-MMP association with β1 or αvβ3 integrins at distinct cellular compartments, thus modulating its internalization, activity, and function on human ECs.
doi:10.1083/jcb.200205026
PMCID: PMC2173082  PMID: 12427871
MMP; adhesion; endocytosis; angiogenesis; extracellular matrix
16.  Integrin-dependent Control of Translation: Engagement of Integrin αIIbβ3 Regulates Synthesis of Proteins in Activated Human Platelets  
The Journal of Cell Biology  1999;144(1):175-184.
Integrins are widely expressed plasma membrane adhesion molecules that tether cells to matrix proteins and to one another in cell–cell interactions. Integrins also transmit outside-in signals that regulate functional responses of cells, and are known to influence gene expression by regulating transcription. In previous studies we found that platelets, which are naturally occurring anucleate cytoplasts, translate preformed mRNA transcripts when they are activated by outside-in signals. Using strategies that interrupt engagement of integrin αIIbβ3 by fibrinogen and platelets deficient in this integrin, we found that αIIbβ3 regulates the synthesis of B cell lymphoma 3 (Bcl-3) when platelet aggregation is induced by thrombin. We also found that synthesis of Bcl-3, which occurs via a specialized translation control pathway regulated by mammalian target of rapamycin (mTOR), is induced when platelets adhere to immobilized fibrinogen in the absence of thrombin and when integrin αIIbβ3 is engaged by a conformation-altering antibody against integrin αIIbβ3. Thus, outside-in signals delivered by integrin αIIbβ3 are required for translation of Bcl-3 in thrombin-stimulated aggregated platelets and are sufficient to induce translation of this marker protein in the absence of thrombin. Engagement of integrin α2β1 by collagen also triggered synthesis of Bcl-3. Thus, control of translation may be a general mechanism by which surface adhesion molecules regulate gene expression.
PMCID: PMC2148114  PMID: 9885253
adhesion; integrins; platelets; translation; gene regulation
17.  Combined Integrin Phosphoproteomic Analyses and siRNA-based Functional Screening Identified Key Regulators for Cancer Cell Adhesion and Migration 
Cancer research  2009;69(8):3713-3720.
Integrins interact with extracellular matrix (ECM) and deliver intracellular signaling for cell proliferation, survival and motility. During tumor metastasis, integrin-mediated cell adhesion to and migration on the ECM proteins are required for cancer cell survival and adaptation to the new microenvironment. Using SILAC-MS, we profiled the phosphoproteomic changes induced by the interactions of cell integrins with type I collagen, the most common ECM substratum. Integrin-ECM interactions modulate phosphorylation of 517 serine, threonine, or tyrosine residues in 513 peptides, corresponding to 357 proteins. Among these proteins, 33 key signaling mediators with kinase or phosphatase activity were subjected to siRNA-based functional screening. Three integrin-regulated kinases, DBF4, PAK2 and GRK6, were identified for their critical role in cell adhesion and migration possibly through their regulation of actin cytoskeleton arrangement. Altogether, we not only depict an integrin-modulated phosphorylation network during cell-ECM protein interactions but also reveal novel regulators for cell adhesion and migration.
doi:10.1158/0008-5472.CAN-08-2515
PMCID: PMC2669841  PMID: 19351860
phosphoproteomics; integrin; adhesion; metastasis; signaling
18.  Leukocyte adhesion deficiency-III is caused by mutations in KINDLIN3 affecting integrin activation 
Nature medicine  2009;15(3):306-312.
Integrins are the major adhesion receptors of leukocytes and platelets. β1 and β2 integrin function on leukocytes is crucial for a successful immune response and the platelet integrin αIIbβ3 initiates the process of blood clotting through binding fibrinogen1-3. Integrins on circulating cells bind poorly to their ligands but become active after ‘inside-out’ signaling through other membrane receptors4,5. Subjects with leukocyte adhesion deficiency-1 (LAD-I) do not express β2 integrins because of mutations in the gene specifying the β2 subunit, and they suffer recurrent bacterial infections6,7. Mutations affecting αIIbβ3 integrin cause the bleeding disorder termed Glanzmann’s thrombasthenia3. Subjects with LAD-III show symptoms of both LAD-I and Glanzmann’s thrombasthenia. Their hematopoietically-derived cells express β1, β2 and β3 integrins, but defective inside-out signaling causes immune deficiency and bleeding problems8. The LAD-III lesion has been attributed to a C→A mutation in the gene encoding calcium and diacylglycerol guanine nucleotide exchange factor (CALDAGGEF1; official symbol RASGRP2) specifying the CALDAG-GEF1 protein9, but we show that this change is not responsible for the LAD-III disorder. Instead, we identify mutations in the KINDLIN3 (official symbol FERMT3) gene specifying the KINDLIN-3 protein as the cause of LAD-III in Maltese and Turkish subjects. Two independent mutations result in decreased KINDLIN3 messenger RNA levels and loss of protein expression. Notably, transfection of the subjects’ lymphocytes with KINDLIN3 complementary DNA but not CALDAGGEF1 cDNA reverses the LAD-III defect, restoring integrin-mediated adhesion and migration.
doi:10.1038/nm.1931
PMCID: PMC2680140  PMID: 19234463
19.  R-Ras Promotes Focal Adhesion Formation through Focal Adhesion Kinase and p130Cas by a Novel Mechanism That Differs from Integrins 
Molecular and Cellular Biology  2003;23(3):933-949.
R-Ras regulates integrin function, but its effects on integrin signaling pathways have not been well described. We demonstrate that activation of R-Ras promoted focal adhesion formation and altered localization of the α2β1 integrin from cell-cell to cell-matrix adhesions in breast epithelial cells. Constitutively activated R-Ras(38V) dramatically enhanced focal adhesion kinase (FAK) and p130Cas phosphorylation upon collagen stimulation or clustering of the α2β1 integrin, even in the absence of increased ligand binding. Signaling events downstream of R-Ras differed from integrins and K-Ras, since pharmacological inhibition of Src or disruption of actin inhibited integrin-mediated FAK and p130Cas phosphorylation, focal adhesion formation, and migration in control and K-Ras(12V)-expressing cells but had minimal effect in cells expressing R-Ras(38V). Therefore, signaling from R-Ras to FAK and p130Cas has a component that is Src independent and not through classic integrin signaling pathways and a component that is Src dependent. R-Ras effector domain mutants and pharmacological inhibition suggest a partial role for phosphatidylinositol 3-kinase (PI3K), but not Raf, in R-Ras signaling to FAK and p130Cas. However, PI3K cannot account for the Src-independent pathway, since simultaneous inhibition of both PI3K and Src did not completely block effects of R-Ras on FAK phosphorylation. Our results suggest that R-Ras promotes focal adhesion formation by signaling to FAK and p130Cas through a novel mechanism that differs from but synergizes with the α2β1 integrin.
doi:10.1128/MCB.23.3.933-949.2003
PMCID: PMC140691  PMID: 12529399
20.  General In Vivo Assay for the Study of Integrin Cell Membrane Receptor Microclustering 
Analytical chemistry  2007;79(8):3142-3147.
A method for measuring the microclustering of a class of cell surface receptors called integrins is reported. Integrins are proteins involved in bi-directional signaling across the cell membrane, and are important in cell adhesion, growth and survival. Their activity is regulated by changes in protein conformation and protein clustering. The developed in vivo clustering assay uses fluorescence resonance energy transfer (FRET), and has the benefit of requiring a single cloning step to generate FRET donors and acceptors that can be used to measure the clustering of a series of integrin mutants. The FRET reporters contain extracellular donor or acceptor fluorescent protein attached to native integrin cytoplasmic and transmembrane domains, and these are expressed along with wild-type or mutant integrins. Expression of the FRET reporters has no affect on the ligand binding properties of co-expressed integrins. FRET values are calculated for cell lines spreading on ligand coated surfaces, and these values are independent of fluorescent protein expression. No FRET is observed in cell lines expressing the reporters in the absence of integrins. Integrin dependent FRET values increase approximately 2- to 3-fold when the integrins contain mutations that result in increased ligand binding affinities.
doi:10.1021/ac062008i
PMCID: PMC2538944  PMID: 17346031
21.  Critical roles for the COOH-terminal NITY and RGT sequences of the integrin β3 cytoplasmic domain in inside-out and outside-in signaling 
The Journal of Cell Biology  2003;162(2):329-339.
Bidirectional signaling of integrin αIIbβ3 requires the β3 cytoplasmic domain. To determine the sequence in the β3 cytoplasmic domain that is critical to integrin signaling, cell lines were established that coexpress the platelet receptor for von Willebrand factor (vWF), glycoprotein Ib-IX, integrin αIIb, and mutants of β3 with truncations at sites COOH terminal to T741, Y747, F754, and Y759. Truncation at Y759 did not affect integrin activation, as indicated by vWF-induced fibrinogen binding, but affected cell spreading and stable adhesion. Thus, the COOH-terminal RGT sequence of β3 is important for outside-in signaling but not inside-out signaling. In contrast, truncation at F754, Y747, or T741 completely abolished integrin activation. A point mutation replacing Y759 with alanine also abolished integrin activation. Thus, the T755NITY759 sequence of β3, containing an NXXY motif, is critical to inside-out signaling, whereas the intact COOH terminus is important for outside-in signaling. In addition, we found that the calcium-dependent protease calpain preferentially cleaves at Y759 in a population of β3 during platelet aggregation and adhesion, suggesting that calpain may selectively regulate integrin outside-in signaling.
doi:10.1083/jcb.200303120
PMCID: PMC2172800  PMID: 12860973
integrin; platelet; calpain; signaling; platelet activation
22.  DPP in the matrix mediates cell adhesion but is not restricted to stickiness 
Cell Adhesion & Migration  2012;6(4):307-311.
Cell adhesion to DPP substrate is an integrin-mediated event and involves integrin binding, clustering, assembly of focal adhesion complexes and cytoskeletal organization. Cells perceive the DPP substrate through the integrin receptor αvβ1 and bind the actin cytoskeleton to the membrane via focal adhesion sites. The cells respond to this proteinaceous rigid substrate by activating the mechano-chemical signaling events leading to cell spreading and formation of focal adhesions. Focal adhesions, which are sites of integrin binding to the extracellular matrix, form in the leading edge during cell migration. These sites are dynamic and the supramolecular assemblies contain structural and signaling components regulating cell functions. In our study, we present a scenario that integrins utilize the actin network to permit activation of the mitogen-activated kinase modules to transduce signals through the cytoplasm to the nucleus in the presence of DPP. We specifically demonstrate that ERK-mediated transcriptional events impinge on activation of transcription factors leading to cell differentiation.
doi:10.4161/cam.20627
PMCID: PMC3478251  PMID: 22588498
DPP; integrin; FAK; extracellular matrix; adhesion; MAP kinase
23.  Nanolithographic control of the spatial organization of cellular adhesion receptors at the single-molecule level 
Nano letters  2011;11(3):1306-1312.
The ability to control the placement of individual molecules promises to enable a wide range of applications and is a key challenge in nanoscience and nanotechnology. Many biological interactions, in particular, are sensitive to the precise geometric arrangement of proteins. We have developed a technique which combines molecular-scale nanolithography with site-selective biochemistry to create biomimetic arrays of individual protein binding sites. The binding sites can be arranged in heterogeneous patterns of virtually any possible geometry with a nearly unlimited number of degrees of freedom. We have used these arrays to explore how the geometric organization of the extracellular matrix (ECM) binding ligand RGD (Arg-Gly-Asp) affects cell adhesion and spreading. Systematic variation of spacing, density and cluster size of individual integrin binding sites was used to elicit different cell behavior. Cell spreading assays on arrays of different geometric arrangements revealed a dramatic increase in spreading efficiency when at least 4 liganded sites were spaced within 60 nm or less, with no dependence on global density. This points to the existence of a minimal matrix adhesion unit for fibronectin defined in space and stoichiometry. Developing an understanding of the ECM geometries that activate specific cellular functional complexes is a critical step toward controlling cell behavior. Potential practical applications range from new therapeutic treatments to the rational design of tissue scaffolds that can optimize healing without scarring. More broadly, spatial control at the single-molecule level can elucidate factors controlling individual molecular interactions and can enable synthesis of new systems based on molecular-scale architectures.
doi:10.1021/nl104378f
PMCID: PMC3061283  PMID: 21319842
nanofabrication; nanobiology; mechanobiology; integrin clustering; cell adhesion
24.  NEDD9 Stabilizes Focal Adhesions, Increases Binding to the Extra-Cellular Matrix and Differentially Effects 2D versus 3D Cell Migration 
PLoS ONE  2012;7(4):e35058.
The speed of cell migration on 2-dimensional (2D) surfaces is determined by the rate of assembly and disassembly of clustered integrin receptors known as focal adhesions. Different modes of cell migration that have been described in 3D environments are distinguished by their dependence on integrin-mediated interactions with the extra-cellular matrix. In particular, the mesenchymal invasion mode is the most dependent on focal adhesion dynamics. The focal adhesion protein NEDD9 is a key signalling intermediary in mesenchymal cell migration, however whether NEDD9 plays a role in regulating focal adhesion dynamics has not previously been reported. As NEDD9 effects on 2D migration speed appear to depend on the cell type examined, in the present study we have used mouse embryo fibroblasts (MEFs) from mice in which the NEDD9 gene has been depleted (NEDD9 −/− MEFs). This allows comparison with effects of other focal adhesion proteins that have previously been demonstrated using MEFs. We show that focal adhesion disassembly rates are increased in the absence of NEDD9 expression and this is correlated with increased paxillin phosphorylation at focal adhesions. NEDD9−/− MEFs have increased rates of migration on 2D surfaces, but conversely, migration of these cells is significantly reduced in 3D collagen gels. Importantly we show that myosin light chain kinase is activated in 3D in the absence of NEDD9 and is conversely inhibited in 2D cultures. Measurement of adhesion strength reveals that NEDD9−/− MEFs have decreased adhesion to fibronectin, despite upregulated α5β1 fibronectin receptor expression. We find that β1 integrin activation is significantly suppressed in the NEDD9−/−, suggesting that in the absence of NEDD9 there is decreased integrin receptor activation. Collectively our data suggest that NEDD9 may promote 3D cell migration by slowing focal adhesion disassembly, promoting integrin receptor activation and increasing adhesion force to the ECM.
doi:10.1371/journal.pone.0035058
PMCID: PMC3324407  PMID: 22509381
25.  Focal Adhesion Kinase Modulates Cell Adhesion Strengthening via Integrin Activation 
Molecular Biology of the Cell  2009;20(9):2508-2519.
Focal adhesion kinase (FAK) is an essential nonreceptor tyrosine kinase regulating cell migration, adhesive signaling, and mechanosensing. Using FAK-null cells expressing FAK under an inducible promoter, we demonstrate that FAK regulates the time-dependent generation of adhesive forces. During the early stages of adhesion, FAK expression in FAK-null cells enhances integrin activation to promote integrin binding and, hence, the adhesion strengthening rate. Importantly, FAK expression regulated integrin activation, and talin was required for the FAK-dependent effects. A role for FAK in integrin activation was confirmed in human fibroblasts with knocked-down FAK expression. The FAK autophosphorylation Y397 site was required for the enhancements in adhesion strengthening and integrin-binding responses. This work demonstrates a novel role for FAK in integrin activation and the time-dependent generation of cell–ECM forces.
doi:10.1091/mbc.E08-01-0076
PMCID: PMC2675629  PMID: 19297531

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