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1.  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.
PMCID: PMC2782178  PMID: 20011123
2.  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.
PMCID: PMC3281566  PMID: 21937733
3.  The Talin Head Domain Reinforces Integrin-Mediated Adhesion by Promoting Adhesion Complex Stability and Clustering 
PLoS Genetics  2014;10(11):e1004756.
Talin serves an essential function during integrin-mediated adhesion in linking integrins to actin via the intracellular adhesion complex. In addition, the N-terminal head domain of talin regulates the affinity of integrins for their ECM-ligands, a process known as inside-out activation. We previously showed that in Drosophila, mutating the integrin binding site in the talin head domain resulted in weakened adhesion to the ECM. Intriguingly, subsequent studies showed that canonical inside-out activation of integrin might not take place in flies. Consistent with this, a mutation in talin that specifically blocks its ability to activate mammalian integrins does not significantly impinge on talin function during fly development. Here, we describe results suggesting that the talin head domain reinforces and stabilizes the integrin adhesion complex by promoting integrin clustering distinct from its ability to support inside-out activation. Specifically, we show that an allele of talin containing a mutation that disrupts intramolecular interactions within the talin head attenuates the assembly and reinforcement of the integrin adhesion complex. Importantly, we provide evidence that this mutation blocks integrin clustering in vivo. We propose that the talin head domain is essential for regulating integrin avidity in Drosophila and that this is crucial for integrin-mediated adhesion during animal development.
Author Summary
Cells are the building blocks of our bodies. How do cells rearrange to form three-dimensional body plans and maintain specific tissue structures? Specialized adhesion molecules on the cell surface mediate attachment between cells and their surrounding environment to hold tissues together. Our work uses the developing fruit fly embryo to demonstrate how such connections are regulated during tissue growth. Since the genes and molecules involved in this process are highly similar between flies and humans, we can also apply our findings to our understanding of how human tissues form and are maintained. We observe that, in late developing muscles, clusters of cell adhesion molecules concentrate together to create stronger attachments between muscle cells and tendon cells. This strengthening mechanism allows the fruit fly to accommodate increasing amounts of force imposed by larger, more active muscles. We identify specific genetic mutations that disrupt these strengthening mechanisms and lead to severe developmental defects during fly development. Our results illustrate how subtle fine-tuning of the connections between cells and their surrounding environment is important to form and maintain normal tissue structure across the animal kingdom.
PMCID: PMC4230843  PMID: 25393120
4.  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.
PMCID: PMC2588347  PMID: 18363565
cell adhesion; integrin; Rap1; signal transduction; talin; thrombosis
5.  Neuropilin-1/GIPC1 Signaling Regulates α5β1 Integrin Traffic and Function in Endothelial Cells 
PLoS Biology  2009;7(1):e1000025.
Neuropilin 1 (Nrp1) is a coreceptor for vascular endothelial growth factor A165 (VEGF-A165, VEGF-A164 in mice) and semaphorin 3A (SEMA3A). Nevertheless, Nrp1 null embryos display vascular defects that differ from those of mice lacking either VEGF-A164 or Sema3A proteins. Furthermore, it has been recently reported that Nrp1 is required for endothelial cell (EC) response to both VEGF-A165 and VEGF-A121 isoforms, the latter being incapable of binding Nrp1 on the EC surface. Taken together, these data suggest that the vascular phenotype caused by the loss of Nrp1 could be due to a VEGF-A164/SEMA3A-independent function of Nrp1 in ECs, such as adhesion to the extracellular matrix. By using RNA interference and rescue with wild-type and mutant constructs, we show here that Nrp1 through its cytoplasmic SEA motif and independently of VEGF-A165 and SEMA3A specifically promotes α5β1-integrin-mediated EC adhesion to fibronectin that is crucial for vascular development. We provide evidence that Nrp1, while not directly mediating cell spreading on fibronectin, interacts with α5β1 at adhesion sites. Binding of the homomultimeric endocytic adaptor GAIP interacting protein C terminus, member 1 (GIPC1), to the SEA motif of Nrp1 selectively stimulates the internalization of active α5β1 in Rab5-positive early endosomes. Accordingly, GIPC1, which also interacts with α5β1, and the associated motor myosin VI (Myo6) support active α5β1 endocytosis and EC adhesion to fibronectin. In conclusion, we propose that Nrp1, in addition to and independently of its role as coreceptor for VEGF-A165 and SEMA3A, stimulates through its cytoplasmic domain the spreading of ECs on fibronectin by increasing the Rab5/GIPC1/Myo6-dependent internalization of active α5β1. Nrp1 modulation of α5β1 integrin function can play a causal role in the generation of angiogenesis defects observed in Nrp1 null mice.
Author Summary
The vascular system is a hierarchical network of blood vessels lined by endothelial cells that, by means of the transmembrane integrin proteins, bind to the surrounding proteinaceous extracellular matrix (ECM). Integrins are required for proper cardiovascular development and exist in bent (inactive) and extended (active) shapes that are correspondingly unable and able to attach to the ECM. Extracellular guidance cues, such as vascular endothelial growth factor and semaphorins, bind the transmembrane protein neuropilin-1 (Nrp1) and then activate biochemical signals that, respectively, activate or inactivate endothelial integrins. Here, we show that Nrp1, via its short cytoplasmic domain and independently of vascular endothelial growth factor and semaphorins, specifically promotes endothelial cell attachment to the ECM protein fibronectin, which is known to be crucial for vascular development. Notably, Nrp1 favors cell adhesion by associating with fibronectin-binding integrins and promoting the fast vesicular traffic of their extended form back and forth from the endothelial cell-to-ECM contacts. Binding of the Nrp1 cytoplasmic domain with the adaptor protein GIPC1, which in turn associates with proteins required for integrin internalization and vesicle motility, is required as well. It is likely that such an integrin treadmill could act as a major regulator of cell adhesion in general.
The transmembrane protein neuropilin-1 promotes endothelial cell attachment to the extracellular matrix by enhancing active integrin treadmilling at cell-adhesion sites.
PMCID: PMC2631072  PMID: 19175293
6.  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.
PMCID: PMC2812850  PMID: 20048261
7.  Induction of β3-Integrin Gene Expression by Sustained Activation of the Ras-Regulated Raf–MEK–Extracellular Signal-Regulated Kinase Signaling Pathway 
Molecular and Cellular Biology  2001;21(9):3192-3205.
Alterations in the expression of integrin receptors for extracellular matrix (ECM) proteins are strongly associated with the acquisition of invasive and/or metastatic properties by human cancer cells. Despite this, comparatively little is known of the biochemical mechanisms that regulate the expression of integrin genes in cells. Here we demonstrate that the Ras-activated Raf–MEK–extracellular signal-regulated kinase (ERK) signaling pathway can specifically control the expression of individual integrin subunits in a variety of human and mouse cell lines. Pharmacological inhibition of MEK1 in a number of human melanoma and pancreatic carcinoma cell lines led to reduced cell surface expression of α6- and β3-integrin. Consistent with this, conditional activation of the Raf-MEK-ERK pathway in NIH 3T3 cells led to a 5 to 20-fold induction of cell surface α6- and β3-integrin expression. Induced β3-integrin was expressed on the cell surface as a heterodimer with αv-integrin; however, the overall level of αv-integrin expression was not altered by Ras or Raf. Raf-induced β3-integrin was observed in primary and established mouse fibroblast lines and in mouse and human endothelial cells. Consistent with previous reports of the ability of the Raf-MEK-ERK signaling pathway to induce β3-integrin gene transcription in human K-562 erythroleukemia cells, Raf activation in NIH 3T3 cells led to elevated β3-integrin mRNA. However, unlike immediate-early Raf targets such as heparin binding epidermal growth factor and Mdm2, β3-integrin mRNA was induced by Raf in a manner that was cycloheximide sensitive. Surprisingly, activation of the Raf-MEK-ERK signaling pathway by growth factors and mitogens had little or no effect on β3-integrin expression, suggesting that the expression of this gene requires sustained activation of this signaling pathway. In addition, despite the robust induction of cell surface αvβ3-integrin expression by Raf in NIH 3T3 cells, such cells display decreased spreading and adhesion, with a loss of focal adhesions and actin stress fibers. These data suggest that oncogene-induced alterations in integrin gene expression may participate in the changes in cell adhesion and migration that accompany the process of oncogenic transformation.
PMCID: PMC86954  PMID: 11287623
8.  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.
PMCID: PMC3197140  PMID: 22043318
9.  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.
10.  β1 Integrin Signaling Maintains Human Epithelial Progenitor Cell Survival In Situ and Controls Proliferation, Apoptosis and Migration of Their Progeny 
PLoS ONE  2013;8(12):e84356.
β1 integrin regulates multiple epithelial cell functions by connecting cells with the extracellular matrix (ECM). While β1 integrin-mediated signaling in murine epithelial stem cells is well-studied, its role in human adult epithelial progenitor cells (ePCs) in situ remains to be defined. Using microdissected, organ-cultured human scalp hair follicles (HFs) as a clinically relevant model for studying human ePCs within their natural topobiological habitat, β1 integrin-mediated signaling in ePC biology was explored by β1 integrin siRNA silencing, specific β1 integrin-binding antibodies and pharmacological inhibition of integrin-linked kinase (ILK), a key component of the integrin-induced signaling cascade. β1 integrin knock down reduced keratin 15 (K15) expression as well as the proliferation of outer root sheath keratinocytes (ORSKs). Embedding of HF epithelium into an ECM rich in β1 integrin ligands that mimic the HF mesenchyme significantly enhanced proliferation and migration of ORSKs, while K15 and CD200 gene and protein expression were inhibited. Employing ECM-embedded β1 integrin-activating or -inhibiting antibodies allowed to identify functionally distinct human ePC subpopulations in different compartments of the HF epithelium. The β1 integrin-inhibitory antibody reduced β1 integrin expression in situ and selectively enhanced proliferation of bulge ePCs, while the β1 integrin-stimulating antibody decreased hair matrix keratinocyte apoptosis and enhanced transferrin receptor (CD71) immunoreactivity, a marker of transit amplifying cells, but did not affect bulge ePC proliferation. That the putative ILK inhibitor QLT0267 significantly reduced ORSK migration and proliferation and induced massive ORSK apoptosis suggests a key role for ILK in mediating the ß1 integrin effects. Taken together, these findings demonstrate that ePCs in human HFs require β1 integrin-mediated signaling for survival, adhesion, and migration, and that different human HF ePC subpopulations differ in their response to β1 integrin signaling. These insights may be exploited for cell-based regenerative medicine strategies that employ human HF-derived ePCs.
PMCID: PMC3874009  PMID: 24386370
11.  Coordinate role for cell surface chondroitin sulfate proteoglycan and alpha 4 beta 1 integrin in mediating melanoma cell adhesion to fibronectin 
The Journal of Cell Biology  1992;118(2):431-444.
Cellular recognition and adhesion to the extracellular matrix (ECM) has a complex molecular basis, involving both integrins and cell surface proteoglycans (PG). The current studies have used specific inhibitors of chondroitin sulfate proteoglycan (CSPG) synthesis along with anti- alpha 4 integrin subunit monoclonal antibodies to demonstrate that human melanoma cell adhesion to an A-chain derived, 33-kD carboxyl- terminal heparin binding fragment of human plasma fibronectin (FN) involves both cell surface CSPG and alpha 4 beta 1 integrin. A direct role for cell surface CSPG in mediating melanoma cell adhesion to this FN fragment was demonstrated by the identification of a cationic synthetic peptide, termed FN-C/H-III, within the fragment. FN-C/H-III is located close to the amino terminal end of the fragment, representing residues #1721-1736 of intact FN. FN-C/H-III binds CSPG directly, can inhibit CSPG binding to the fragment, and promotes melanoma cell adhesion by a CSPG-dependent, alpha 4 beta 1 integrin- independent mechanism. A scrambled version of FN-C/H-III does not inhibit CSPG binding or cell adhesion to the fragment or to FN-C/H-III, indicating that the primary sequence of FN-C/H-III is important for its biological properties. Previous studies have identified three other synthetic peptides from within this 33-kD FN fragment that promote cell adhesion by an arginyl-glycyl-aspartic acid (RGD) independent mechanism. Two of these synthetic peptides (FN-C/H-I and FN-C/H-II) bind heparin and promote cell adhesion, implicating cell surface PG in mediating cellular recognition of these two peptides. Additionally, a third synthetic peptide, CS1, is located in close proximity to FN-C/H-I and FN-C/H-II and it promotes cell adhesion by an alpha 4 beta 1 integrin-dependent mechanism. In contrast to FN-C/H-III, cellular recognition of these three peptides involved contributions from both CSPG and alpha 4 integrin subunits. Of particular importance are observations demonstrating that CS1-mediated melanoma cell adhesion could be inhibited by interfering with CSPG synthesis or expression. Since CS1 does not bind CSPG, the results suggest that CSPG may modify the function and/or activity of alpha 4 beta 1 integrin on the surface of human melanoma cells. Together, these results support a model in which the PG and integrin binding sites within the 33-kD fragment may act in concert to focus these two cell adhesion receptors into close proximity on the cell surface, thereby influencing initial cellular recognition events that contribute to melanoma cell adhesion on this fragment.
PMCID: PMC2290058  PMID: 1629241
12.  The Mechanism of Kindlin-mediated Activation of Integrin αIIbβ3 
Current biology : CB  2013;23(22):2288-2295.
Increased ligand binding to cellular integrins (“activation”) plays important roles in processes such as development, cell migration, extracellular matrix assembly, tumor metastasis and hemostasis and thrombosis[1-5]. Integrin activation encompasses both increased integrin monomer affinity and increased receptor clustering[6] and depends on integrin-talin interactions[5]. Loss of kindlins results in reduced activation of integrins[7-13]. Kindlins might promote talin binding to integrins through a cooperative mechanism[5, 14-16]; however, kindlins do not increase talin association with integrins[17]. Here we report that, unlike talin head domain (THD), kindlin-3 caused little effect on the affinity of purified monomeric αIIbβ3, and it didn’t enhance activation by THD. Furthermore, studies with ligands of varying valency showed that kindlins primarily increased cellular αIIbβ3 avidity rather than monomer affinity. In platelets or nucleated cells, loss of kindlins markedly reduced αIIbβ3 binding to multivalent but not monovalent ligands. Finally, silencing of kindlins reduced the clustering of ligand-occupied αIIbβ3 as revealed by total internal reflection fluorescence (TIRF) and electron microscopy. Thus, in contrast to talins, kindlins have little primary effect on integrin αIIbβ3 affinity for monovalent ligands and increase multivalent ligand binding by promoting the clustering of talin-activated integrins.
PMCID: PMC3912999  PMID: 24210614
integrin; kindlin; talin signal transduction; cell adhesion
13.  Galectin-3 Induces Clustering of CD147 and Integrin-β1 Transmembrane Glycoprotein Receptors on the RPE Cell Surface 
PLoS ONE  2013;8(7):e70011.
Proliferative vitreoretinopathy (PVR) is a blinding disease frequently occurring after retinal detachment surgery. Adhesion, migration and matrix remodeling of dedifferentiated retinal pigment epithelial (RPE) cells characterize the onset of the disease. Treatment options are still restrained and identification of factors responsible for the abnormal behavior of the RPE cells will facilitate the development of novel therapeutics. Galectin-3, a carbohydrate-binding protein, was previously found to inhibit attachment and spreading of retinal pigment epithelial cells, and thus bares the potential to counteract PVR-associated cellular events. However, the identities of the corresponding cell surface glycoprotein receptor proteins on RPE cells are not known. Here we characterize RPE-specific Gal-3 containing glycoprotein complexes using a proteomic approach. Integrin-β1, integrin-α3 and CD147/EMMPRIN, a transmembrane glycoprotein implicated in regulating matrix metalloproteinase induction, were identified as potential Gal-3 interactors on RPE cell surfaces. In reciprocal immunoprecipitation experiments we confirmed that Gal-3 associated with CD147 and integrin-β1, but not with integrin-α3. Additionally, association of Gal-3 with CD147 and integrin-β1 was observed in co-localization analyses, while integrin-α3 only partially co-localized with Gal-3. Blocking of CD147 and integrin-β1 on RPE cell surfaces inhibited binding of Gal-3, whereas blocking of integrin-α3 failed to do so, suggesting that integrin-α3 is rather an indirect interactor. Importantly, Gal-3 binding promoted pronounced clustering and co-localization of CD147 and integrin-β1, with only partial association of integrin-α3. Finally, we show that RPE derived CD147 and integrin-β1, but not integrin-α3, carry predominantly β-1,6-N-actyl-D-glucosamine-branched glycans, which are high-affinity ligands for Gal-3. We conclude from these data that extracellular Gal-3 triggers clustering of CD147 and integrin-β1 via interaction with β1,6-branched N-glycans on RPE cells and hypothesize that Gal-3 acts as a positive regulator for CD147/integrin-β1 clustering and therefore modifies RPE cell behavior contributing to the pathogenesis of PVR. Further investigations at this pathway may aid in the development of specific therapies for PVR.
PMCID: PMC3726584  PMID: 23922889
14.  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.
PMCID: PMC3747215  PMID: 23977051
15.  Localized Lipid Packing of Transmembrane Domains Impedes Integrin Clustering 
PLoS Computational Biology  2013;9(3):e1002948.
Integrin clustering plays a pivotal role in a host of cell functions. Hetero-dimeric integrin adhesion receptors regulate cell migration, survival, and differentiation by communicating signals bidirectionally across the plasma membrane. Thus far, crystallographic structures of integrin components are solved only separately, and for some integrin types. Also, the sequence of interactions that leads to signal transduction remains ambiguous. Particularly, it remains controversial whether the homo-dimerization of integrin transmembrane domains occurs following the integrin activation (i.e. when integrin ectodomain is stretched out) or if it regulates integrin clustering. This study employs molecular dynamics modeling approaches to address these questions in molecular details and sheds light on the crucial effect of the plasma membrane. Conducting a normal mode analysis of the intact αllbβ3 integrin, it is demonstrated that the ectodomain and transmembrane-cytoplasmic domains are connected via a membrane-proximal hinge region, thus merely transmembrane-cytoplasmic domains are modeled. By measuring the free energy change and force required to form integrin homo-oligomers, this study suggests that the β-subunit homo-oligomerization potentially regulates integrin clustering, as opposed to α-subunit, which appears to be a poor regulator for the clustering process. If α-subunits are to regulate the clustering they should overcome a high-energy barrier formed by a stable lipid pack around them. Finally, an outside-in activation-clustering scenario is speculated, explaining how further loading the already-active integrin affects its homo-oligomerization so that focal adhesions grow in size.
Author Summary
Focal adhesions are complex, dynamic structures of multiple proteins that act as the cell's mechanical anchorage to its surrounding. Integrins are proteins linking the cell inner and outer environments, which act as a bridge that crosses the cell membrane. Integrins respond to mechanical loads exerted to them by changing their conformations. Several diseases, such as atherosclerosis and different types of cancer, are caused by altered function of integrins. Essential to the formation of focal adhesions is the process of integrin clustering. Bidirectional integrin signaling involves conformational changes in this protein, clustering, and finally the assembly of a large intracellular adhesion complex. Integrin clustering is defined as the interaction of integrins to form lateral assemblies that eventually lead to focal adhesion formation. The effect of the plasma membrane on formation of integrin clusters has been largely neglected in current literature; subsequently some apparently contradictory data has been reported by a number of researchers in the field. Using a molecular dynamics modeling approach, a computational method that simulates systems in a full-atomic scale, we probe the role of the plasma membrane in integrin clustering and hypothesize a clustering scenario that explains the relationship between integrin activation and focal adhesion growth.
PMCID: PMC3597534  PMID: 23516344
16.  α5β1 Integrin-Fibronectin Interactions Specify Liquid to Solid Phase Transition of 3D Cellular Aggregates 
PLoS ONE  2010;5(7):e11830.
Tissue organization during embryonic development and wound healing depends on the ability of cells on the one hand to exchange adhesive bonds during active rearrangement and on the other to become fixed in place as tissue homeostasis is reached. Cells achieve these contradictory tasks by regulating either cell-cell adhesive bonds, mediated by cadherins, or cell-extracellular matrix (ECM) connections, regulated by integrins. Integrin α5β1 and soluble fibronectin (sFN) are key players in cell-ECM force generation and in ECM polymerization. Here, we explore the interplay between integrin α5β1 and sFN and its influence on tissue mechanical properties and cell sorting behavior.
Methodology/Principal Findings
We generated a series of cell lines varying in α5β1 receptor density. We then systematically explored the effects of different sFN concentrations on aggregate biomechanical properties using tissue surface tensiometry. We found previously unreported complex behaviors including the observation that interactions between fibronectin and integrin α5β1 generates biphasic tissue cohesion profiles. Specifically, we show that at constant sFn concentration, aggregate cohesion increases linearly as α5β1 receptor density is increased from low to moderate levels, producing a transition from viscoelastic-liquid to pseudo viscoelastic-solid behavior. However, further increase in receptor density causes an abrupt drop in tissue cohesion and a transition back to viscoelastic-liquid properties. We propose that this may be due to depletion of sFn below a critical value in the aggregate microenvironment at high α5β1 levels. We also show that differential expression of α5β1 integrin can promote phase-separation between cells.
The interplay between α5-integrin and sFn contributes significantly to tissue cohesion and, depending on their level of expression, can mediate a shift from liquid to elastic behavior. This interplay represents a tunable level of control between integrins and the ECM that can influence tissue cohesion and other mechanical properties, which may translate to the specification of tissue structure and function. These studies provide insights into important biological processes such as embryonic development, wound healing, and for tissue engineering applications.
PMCID: PMC2912296  PMID: 20686611
17.  Clustering of Syndecan-4 and Integrin β1 by Laminin α3 Chain–derived Peptide Promotes Keratinocyte Migration 
Molecular Biology of the Cell  2009;20(13):3012-3024.
Syndecans function as receptors for extracellular matrix (ECM) with integrins in cell spreading. However, the molecular mechanism of their specific involvement in cell migration or in wound healing has not been elucidated yet. Here, we report that a synthetic peptide, PEP75, which contains the syndecan-binding sequence of the laminin α3LG4 module, induces keratinocyte migration in in vitro and in vivo. Soluble PEP75 induced the clustering of syndecan-4 and conformation-modified integrin β1 colocalized with syndecan-4 in soluble PEP75-induced clusters. Treatment of cells in solution with PEP75 resulted in the exposure of the P4G11 antibody epitope of integrin β1 in immunostaining as well as in flow cytometry and augmented integrin β1–dependent cell adhesion to ECM. Pulldown assays demonstrated that PEP75 bound to syndecan-4, but not to integrin β1. A siRNA study revealed a role for syndecan-4 in PEP75-induced up-regulation of P4G11 antibody binding and migration of HaCaT cells. We conclude that binding of soluble PEP75 to syndecan-4 induces the coupling of integrin β1, which is associated with integrin β1-conformational changes and activation, and leads to keratinocyte migration. To activate integrin function through syndecans could be a novel therapeutic approach for chronic wound.
PMCID: PMC2704153  PMID: 19403697
18.  Chimeric fibronectin matrix mimetic as a functional growth- and migration-promoting adhesive substrate 
Biomaterials  2010;32(8):2077-2087.
Therapeutic protein engineering combines genetic, biochemical, and functional information to improve existing proteins or invent new protein technologies. Using these principles, we developed an approach to deliver extracellular matrix (ECM) fibronectin-specific signals to cells. Fibronectin matrix assembly is a cell-dependent process that converts the inactive, soluble form of fibronectin into biologically-active ECM fibrils. ECM fibronectin stimulates cell functions required for normal tissue regeneration, including cell growth, spreading, migration, and collagen reorganization. We have developed recombinant fibronectin fragments that mimic the effects of ECM fibronectin on cell function by coupling the cryptic heparin-binding fragment of fibronectin’s first type III repeat (FNIII1H) to the integrin-binding domain (FNIII8-10). GST/III1H,8-10 supports cell adhesion and spreading and stimulates cell proliferation to a greater extent than plasma fibronectin. Deletion and site-specific mutant constructs were generated to identify the active regions in GST/III1H,8-10 and reduce construct size. A chimeric construct in which the integrin-binding, RGDS loop was inserted into the analogous site in FNIII8 (GST/III1H,8RGD), supported cell adhesion and migration, and enhanced cell proliferation and collagen gel contraction. GST/III1H,8RGD was expressed in bacteria and purified from soluble lysate fractions by affinity chromatography. Fibronectin matrix assembly is normally up-regulated in response to tissue injury. Decreased levels of ECM fibronectin are associated with non-healing wounds. Engineering fibronectin matrix mimetics that bypass the need for cell-dependent fibronectin matrix assembly in chronic wounds is a novel approach to stimulating cellular activities critical for tissue repair.
PMCID: PMC3035968  PMID: 21185596
fibronectin; recombinant protein; extracellular matrix; cell proliferation; cell adhesion
19.  Thrombin enhances the adhesion and migration of human colon adenocarcinoma cells via increased beta 3-integrin expression on the tumour cell surface and their inhibition by the snake venom peptide, rhodostomin. 
British Journal of Cancer  1996;73(7):902-908.
The interactions between tumour cells and the microvasculature, including the adhesion of tumour cells to endothelium and extracellular matrix (ECM) as well as their migratory ability, are prerequisites for metastasis to occur. In this study we showed that thrombin is capable of enhancing in vitro tumour cell metastatic potential in terms of adhesive properties and migratory response. Following exposure to subclotting concentrations of thrombin, SW-480 human colon adenocarcinoma cells exhibited increased adhesion to both the endothelium and ECM component (i.e. fibronectin). Likewise, the pretreatment of thrombin enhanced the migratory ability of SW-480 cells. The enhanced adhesion was significantly inhibited by complexing of thrombin with its inhibitor hirudin, or by serine proteinase inhibition with 3,4-DCI, but was unaffected by pretreatment of tumour cells with actinomycin D or cycloheximide. The effect of thrombin resulted in an upregulated cell-surface expression of beta 3 integrins, a group of receptors mediating interactions between tumour cells and endothelial cells, and between tumour cells and ECM. Antibodies against beta 3 integrins effectively blocked both the enhanced adhesion and migration. This thrombin-mediated up-regulation of beta 3 integrins involved the activation of protein kinase C (PKC) as thrombin-enhanced adhesion was diminished by PKC inhibition. Rhodostomin, an Arg-Gly-Asp-containing antiplatelet snake venom peptide that antagonises the binding of ECM toward beta 3 integrins on SW-480 cells, was about 600 and 500 times, more potent that RGDS in inhibiting thrombin-enhanced adhesion and migration respectively. Our data suggest that PKC inhibitors as well as rhodostomin may serve as inhibitory agents in the prevention of thrombin-enhanced metastasis.
PMCID: PMC2074261  PMID: 8611404
20.  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.
PMCID: PMC3324407  PMID: 22509381
21.  A cell–ECM screening method to predict breast cancer metastasis 
Breast cancer preferentially spreads to the bone, brain, liver, and lung. The clinical patterns of this tissue-specific spread (tropism) cannot be explained by blood flow alone, yet our understanding of what mediates tropism to these physically and chemically diverse tissues is limited. While the microenvironment has been recognized as a critical factor in governing metastatic colonization, the role of the extracellular matrix (ECM) in mediating tropism has not been thoroughly explored. We created a simple biomaterial platform with systematic control over the ECM protein density and composition to determine if integrin binding governs how metastatic cells differentiate between secondary tissue sites. Instead of examining individual behaviors, we compiled large patterns of phenotypes associated with adhesion to and migration on these controlled ECMs. In combining this novel analysis with a simple biomaterial platform, we created an in vitro fingerprint that is predictive of in vivo metastasis. This rapid biomaterial screen also provided information on how β1, α2, and α6 integrins might mediate metastasis in patients, providing insights beyond a purely genetic analysis. We propose that this approach of screening many cell–ECM interactions, across many different heterogeneous cell lines, is predictive of in vivo behavior, and is much simpler, faster, and more economical than complex 3D environments or mouse models. We also propose that when specifically applied toward the question of tissue tropism in breast cancer, it can be used to provide insight into certain integrin subunits as therapeutic targets.
Insight, innovation, integration
We developed a high-throughput method to rapidly screen cell adhesion, motility, and growth factor responses on biomaterial surfaces. This approach is analogous to systems biology, relying on cell phenotypes in lieu of genetics. We used this technique to reveal patterns of phenotypes associated with breast cancer metastasis to possible tissue sites (bone, brain, lung). By comparing the phenotypic patterns between cell lines that metastasize to only one tissue site with heterogeneous cell lines, we provide the first method to connect in vitro phenotype to in vivo fate. This method is successful without genetic analysis, yet it also predicts outcomes related to integrin gene expression, potentially identifying new targets for tissue-specific metastasis.
PMCID: PMC4323858  PMID: 25537447
22.  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.
PMCID: PMC2173082  PMID: 12427871
MMP; adhesion; endocytosis; angiogenesis; extracellular matrix
23.  Control of Cyclin D1, p27Kip1, and Cell Cycle Progression in Human Capillary Endothelial Cells by Cell Shape and Cytoskeletal Tension 
Molecular Biology of the Cell  1998;9(11):3179-3193.
The extracellular matrix (ECM) plays an essential role in the regulation of cell proliferation during angiogenesis. Cell adhesion to ECM is mediated by binding of cell surface integrin receptors, which both activate intracellular signaling cascades and mediate tension-dependent changes in cell shape and cytoskeletal structure. Although the growth control field has focused on early integrin and growth factor signaling events, recent studies suggest that cell shape may play an equally critical role in control of cell cycle progression. Studies were carried out to determine when cell shape exerts its regulatory effects during the cell cycle and to analyze the molecular basis for shape-dependent growth control. The shape of human capillary endothelial cells was controlled by culturing cells on microfabricated substrates containing ECM-coated adhesive islands with defined shape and size on the micrometer scale or on plastic dishes coated with defined ECM molecular coating densities. Cells that were prevented from spreading in medium containing soluble growth factors exhibited normal activation of the mitogen-activated kinase (erk1/erk2) growth signaling pathway. However, in contrast to spread cells, these cells failed to progress through G1 and enter S phase. This shape-dependent block in cell cycle progression correlated with a failure to increase cyclin D1 protein levels, down-regulate the cell cycle inhibitor p27Kip1, and phosphorylate the retinoblastoma protein in late G1. A similar block in cell cycle progression was induced before this same shape-sensitive restriction point by disrupting the actin network using cytochalasin or by inhibiting cytoskeletal tension generation using an inhibitor of actomyosin interactions. In contrast, neither modifications of cell shape, cytoskeletal structure, nor mechanical tension had any effect on S phase entry when added at later times. These findings demonstrate that although early growth factor and integrin signaling events are required for growth, they alone are not sufficient. Subsequent cell cycle progression and, hence, cell proliferation are controlled by tension-dependent changes in cell shape and cytoskeletal structure that act by subjugating the molecular machinery that regulates the G1/S transition.
PMCID: PMC25607  PMID: 9802905
24.  Osteopontin: a leading candidate adhesion molecule for implantation in pigs and sheep 
Osteopontin (OPN; also known as Secreted Phosphoprotein 1, SPP1) is a secreted extra-cellular matrix (ECM) protein that binds to a variety of cell surface integrins to stimulate cell-cell and cell-ECM adhesion and communication. It is generally accepted that OPN interacts with apically expressed integrin receptors on the uterine luminal epithelium (LE) and conceptus trophectoderm to attach the conceptus to the uterus for implantation. Research conducted with pigs and sheep has significantly advanced understanding of the role(s) of OPN during implantation through exploitation of the prolonged peri-implantation period of pregnancy when elongating conceptuses are free within the uterine lumen requiring extensive paracrine signaling between conceptus and endometrium. This is followed by a protracted and incremental attachment cascade of trophectoderm to uterine LE during implantation, and development of a true epitheliochorial or synepitheliochorial placenta exhibited by pigs and sheep, respectively. In pigs, implanting conceptuses secrete estrogens which induce the synthesis and secretion of OPN in adjacent uterine LE. OPN then binds to αvβ6 integrin receptors on trophectoderm, and the αvβ3 integrin receptors on uterine LE to bridge conceptus attachment to uterine LE for implantation. In sheep, implanting conceptuses secrete interferon tau that prolongs the lifespan of CL. Progesterone released by CL then induces OPN synthesis and secretion from the endometrial GE into the uterine lumen where OPN binds integrins expressed on trophectoderm (αvβ3) and uterine LE (identity of specific integrins unknown) to adhere the conceptus to the uterus for implantation. OPN binding to the αvβ3 integrin receptor on ovine trophectoderm cells induces in vitro focal adhesion assembly, a prerequisite for adhesion and migration of trophectoderm, through activation of: 1) P70S6K via crosstalk between FRAP1/MTOR and MAPK pathways; 2) MTOR, PI3K, MAPK3/MAPK1 (Erk1/2) and MAPK14 (p38) signaling to stimulate trohectoderm cell migration; and 3) focal adhesion assembly and myosin II motor activity to induce migration of trophectoderm cells. Further large in vivo focal adhesions assemble at the uterine-placental interface of both pigs and sheep and identify the involvement of sizable mechanical forces at this interface during discrete periods of trophoblast migration, attachment and placentation in both species.
PMCID: PMC4322467  PMID: 25671104
Implantation; Integrins; Psteoponti; Pigs; Sheep
25.  Recombinant Fibronectin Matrix Mimetics Specify Integrin Adhesion and Extracellular Matrix Assembly 
Tissue Engineering. Part A  2012;19(3-4):558-570.
Tissue engineering seeks to create functional tissues and organs by integrating natural or synthetic scaffolds with bioactive factors and cells. Creating biologically active scaffolds that support key aspects of tissue regeneration, including the re-establishment of a functional extracellular matrix (ECM), is a challenge currently facing this field. During tissue repair, fibronectin is converted from an inactive soluble form into biologically active ECM fibrils through a cell-dependent process. ECM fibronectin promotes cell processes critical to tissue regeneration and regulates the deposition and organization of other ECM proteins. We previously developed biomimetics of ECM fibronectin by directly coupling the heparin-binding fragment of the first type III repeat of fibronectin (FNIII1H) to the integrin-binding repeats (FNIII8–10). As adhesive substrates, fibronectin matrix mimetics promote cell growth, migration, and contractility through a FNIII1H-dependent mechanism. Here, we analyzed fibronectin matrix mimetic variants designed to include all or part of the integrin-binding domain for their ability to support new ECM assembly. We found that specific modifications of the integrin-binding domain produced adhesive substrates that selectively engage different integrin receptors to, in turn, regulate the amount of fibronectin and collagen deposited into the ECM. The ability of fibronectin matrix mimetics to direct cell–substrate interactions and regulate ECM assembly makes them promising candidates for use as bioactive surfaces, where precise control over integrin-binding specificity and ECM deposition are required.
PMCID: PMC3542876  PMID: 23020251

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