Effects of Mutations in the Cytoplasmic Domain of Integrin β1 to Talin Binding and Cell Spreading

Integrins are transmembrane proteins linking the extracellular matrix or certain cell–cell contacts to the cytoskeleton. To study integrin–cytoskeleton interactions we wanted to relate talin–integrin interaction to integrin function in cell spreading and formation of focal adhesions. For talin-binding studies we used fusion proteins of glutathione S-transferase and the cytoplasmic domain of integrin β₁ (GST-cytoβ₁) expressed in bacteria. For functional studies chimeric integrins containing the extracellular and transmembrane parts of β₃ linked to the cytoplasmic domain of β₁ were expressed in CHO cells as a dimer with the α_IIb subunit. Point mutations in the amino acid sequence N⁷⁸⁵PIY⁷⁸⁸ of β₁ disrupted both the integrin–talin interaction and the ability of the integrin to mediate cell spreading. COOH-terminal truncation of β₁ at the amino acid position 797 disrupted its ability to mediate cell spreading, whereas the disruption of talin binding required deletion of five more amino acids (truncation at position 792). A synthetic peptide from this region of β₁ (W⁷⁸⁰DTGENPIYKSAV⁷⁹²) bound to purified talin and inhibited talin binding to GST-cytoβ₁. The ability of the mutants to mediate focal adhesion formation or to codistribute to focal adhesions formed by other integrins correlated with their ability to mediate cell spreading. These results confirm the previous finding that a talin-binding site in the integrin β₁ tail resides at or close to the central NPXY motif and suggest that the integrin–talin interaction is necessary but not sufficient for integrin-mediated cell spreading.     

Quantitative characterization of single‐cell adhesion properties by atomic force microscopy using protein‐functionalized microbeads

A method was developed to characterize the adhesion properties of single cells by using protein‐functionalized atomic force microscopy (AFM) probes. The quantification by force spectroscopy of the mean detachment force between cells and a gelatin‐functionalized colloidal tip reveals differences in cell adhesion properties that are not within reach of a traditional bulk technique, the washing assay. In this latter method, experiments yield semiquantitative and average adhesion properties of a large population of cells. They are also limited to stringent conditions and cannot highlight disparities in adhesion in the subset of adherent cells. In contrast, this AFM‐based method allows for a reproducible and quantitative investigation of the adhesive properties of individual cells in common cell culture conditions and allows for the detection of adhesive subpopulations of cells. These characteristics meet the critical requirements of many fields, such as the study of cancer cell migratory abilities.     

Mg2+ modulates integrin–extracellular matrix interaction in vascular smooth muscle cells studied by atomic force microscopy

Atomic force microscopy (AFM) was used to investigate the interaction between α5β1 integrin and fibronectin (FN) in the presence of divalent cations. AFM probes were labeled with FN and used to measure binding strength between α5β1 integrin and FN by quantifying the force required to break single FN–integrin bonds on a physiological range of loading rates (100–10 000 pN/s). The force necessary to rupture single α5β1–FN bond increased twofold over the regime of loading rates investigated. Changes in Mg²⁺ and Ca²⁺ concentration affected the thermodynamical parameters of the interaction and modulated the binding energy. These data indicate that the external ionic environment in which vascular smooth muscle cells reside, influences the mechanical parameters that define the interaction between the extracellular matrix and integrins. Thus, in a dynamic mechanical environment such as the vascular wall, thermodynamic binding properties between FN and α5β1 integrin vary in relation to locally applied loads and divalent cations concentrations. These changes can be recorded as direct measurements on live smooth muscle cells by using AFM. Copyright © 2009 John Wiley & Sons, Ltd.     

The measurement of Bacillus mycoides spore adhesion using atomic force microscopy,simple counting methods,and a spinning disk technique

An atomic force microscope has been used to study the adhesion of Bacillus mycoides spores to a hydrophilic glass surface and a hydrophobic-coated glass surface. AFM images of spores attached to the hydrophobic-coated mica surface allowed the measurement of spore dimensions in an aqueous environment without desiccation. The spore exosporium was observed to be flexible and to promote the adhesion of the spore by increasing the area of spore contact with the surface. Results from counting procedures using light microscopy matched the density of spores observed on the hydrophobic-coated glass surface with AFM. However, no spores were observed on the hydrophilic glass surface with AFM, a consequence of the weaker adhesion of the spores at this surface. AFM was also used to quantify directly the interactions of B. mycoides spores at the two surfaces in an aqueous environment. The measurements used "spore probes" constructed by immobilizing a single spore at the apex of a tipless AFM cantilever. The data showed that stretching and sequential bond breaking occurred as the spores were retracted from the hydrophilic glass surface. The greatest spore adhesion was measured at the hydrophobic-coated glass surface. An attractive force on the spores was measured as the spores approached the hydrophobic-coated surface. At the hydrophilic glass surface, only repulsive forces were measured during the approach of the spores. The AFM force measurements were in qualitative agreement with the results of a hydrodynamic shear adhesion assay that used a spinning disk technique. Quantitatively, AFM measurements of adhesive force were up to 4 x 10(3) times larger than the estimates made using the spinning disk data. This is a consequence of the different types of forces applied to the spore in the different adhesion assays. AFM has provided some unique insights into the interactions of spores with surfaces. No other instrument can make such direct measurements for single microbiological cells.     

A primer to traction force microscopy

Traction force microscopy (TFM) has emerged as a versatile technique for the measurement of single-cell-generated forces. TFM has gained wide use among mechanobiology laboratories, and several variants of the original methodology have been proposed. However, issues related to the experimental setup and, most importantly, data analysis of cell traction datasets may restrain the adoption of TFM by a wider community. In this review, we summarize the state of the art in TFM-related research, with a focus on the analytical methods underlying data analysis. We aim to provide the reader with a friendly compendium underlying the potential of TFM and emphasizing the methodological framework required for a thorough understanding of experimental data. We also compile a list of data analytics tools freely available to the scientific community for the furtherance of knowledge on this powerful technique.     

Probing elasticity and adhesion of live cells by atomic force microscopy indentation

Atomic force microscopy (AFM) indentation has become an important technique for quantifying the mechanical properties of live cells at nanoscale. However, determination of cell elasticity modulus from the force–displacement curves measured in the AFM indentations is not a trivial task. The present work shows that these force–displacement curves are affected by indenter-cell adhesion force, while the use of an appropriate indentation model may provide information on the cell elasticity and the work of adhesion of the cell membrane to the surface of the AFM probes. A recently proposed indentation model (Sirghi, Rossi in Appl Phys Lett 89:243118, 2006), which accounts for the effect of the adhesion force in nanoscale indentation, is applied to the AFM indentation experiments performed on live cells with pyramidal indenters. The model considers that the indentation force equilibrates the elastic force of the cell cytoskeleton and the adhesion force of the cell membrane. It is assumed that the indenter-cell contact area and the adhesion force decrease continuously during the unloading part of the indentation (peeling model). Force–displacement curves measured in indentation experiments performed with silicon nitride AFM probes with pyramidal tips on live cells (mouse fibroblast Balb/c3T3 clone A31-1-1) in physiological medium at 37°C agree well with the theoretical prediction and are used to determine the cell elasticity modulus and indenter-cell work of adhesion. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Anchorage‐dependent binding of integrin I‐domain to adhesion ligands

The dynamic interactions between leukocyte integrin receptors and ligands in the vascular endothelium, extracellular matrix, or invading pathogens result in leukocyte adhesion, extravasation, and phagocytosis. This work examined the mechanical strength of the connection between iC3b, a complement component that stimulates phagocytosis, and the ligand‐binding domain, the I‐domain, of integrin α_Mβ₂. Single‐molecule force measurements of α_M I‐domain–iC3b complexes were conducted by atomic force microscope. Strikingly, depending on loading rates, immobilization of the I‐domain via its C‐terminus resulted in a 1.3‐fold to 1.5‐fold increase in unbinding force compared with I‐domains immobilized via the N‐terminus. The force spectra (unbinding force versus loading rate) of the I‐domain–iC3b complexes revealed that the enhanced mechanical strength is due to a 2.4‐fold increase in the lifetime of the I‐domain–iC3b bond. Given the structural and functional similarity of all integrin I‐domains, our result supports the existing allosteric regulatory model by which the ligand binding strength of integrin can be increased rapidly when a force is allowed to stretch the C‐terminus of the I‐domain. This type of mechanism may account for the rapid ligand affinity adjustment during leukocyte migration. Copyright © 2015 John Wiley & Sons, Ltd.     

Apoptotic effect of cleaved high molecular weight kininogen is regulated by extracellular matrix proteins

We previously reported that cleaved high molecular weight kininogen (HKa) and its domain 5 (D5) inhibit critical steps required for angiogenesis and in vivo neovascularization (Colman et al. 2000: Blood 95:543-550). We have further shown that D5 is able to induce apoptosis of endothelial cells, which may represent a critical part of the anti-angiogenic activity of HKa and D5 (Guo et al. 2001: Arterioscler Thromb Vasc Biol 21:1427-1433). In this study, we demonstrate that HKa- and D5-induced apoptosis is closely correlated with their anti-adhesive effect. An important new finding is that the apoptotic activity of HKa and D5 is highly regulated by their interactions with different extracellular matrix (ECM) proteins. HKa inhibited cell adhesion to vitronectin (Vn, 90%) and gelatin (Gel) (40%), but it had no apparent effect on cell adhesion to fibronectin (Fn). D5 showed a similar pattern on cell adhesion but was less potent than HKa. HKa induced apoptosis of endothelial cells grown on Vn and Gel but not cells grown on Fn which closely parallels with its anti-adhesive potency. Further results revealed that the anti-adhesive effect and the apoptotic effect of HKa are associated with its ability to inhibit phosphorylation of focal adhesion kinase (FAK) and paxillin, two important signal molecules required for cell adhesion and cell viability. We conclude that the anti-adhesive activity of HKa and D5 is responsible for their apoptotic effect and that Vn is likely an ECM component that mediates the effect of HKa and D5.     

Prolin-rich tyrosine kinase 2 (PYK2) expression and localization in mouse testis

Prolin-rich kinase 2 (PYK2) is a nonreceptor tyrosine kinase related to the focal adhesion kinase (FAK) p125(FAK). PYK2 is rapidly phosphorylated on tyrosine residues in response to various stimuli, such as tumor necrosis factor-alpha (TNF-alpha), changes in osmolarity, elevation in intracellular calcium concentration, angiotensin, and UV irradiation. PYK2 has ligand sequences for Src homology 2 and 3 (SH-2 and SH-3), and has binding sites for paxillin and p130(cas). Activation of PYK2 leads to modulation of ion channel function, phosphorylation of tyrosine residues, and activation of the MAP kinase signaling pathways. Immunocytochemistry shows that PYK2 is present in mouse germinal and Sertoli cells (ser). Northern blot and immunoprecipitation analysis demonstrate that, among germinal cells, PYK2 is more abundant in spermatocytes (spc) and spermatids (spt); in addition, immunofluorescence analysis clearly shows that the diffuse cytoplasmic localization of PYK2 changes in a specific cellular compartment in spt and spermatozoa.     

The translocation of focal adhesion kinase in brain synaptosomes is regulated by phosphorylation and actin assembly

Focal adhesion kinase (FAK) and the related proline-rich tyrosine kinase 2 (PYK2) are non-receptor protein tyrosine kinases that transduce extracellular signals through the activation of Src family kinases and are highly enriched in neurones. To further elucidate the regulation of FAK and PYK2 in nervous tissue, we investigated their distribution in brain subcellular fractions and analysed their translocation between membrane and cytosolic compartments. We have found that FAK and PYK2 are present in a small membrane-associated pool and a larger cytosolic pool in various neuronal compartments including nerve terminals. In intact nerve terminals, inhibition of Src kinases inhibited the membrane association of FAK, but not of PYK2, whereas tyrosine phosphatase inhibition sharply increased the membrane association of both FAK and PYK2. Disruption of the actin cytoskeleton was followed by a decrease in the membrane-associated pool of FAK, but not of PYK2. For both kinases, a significant correlation was found between autophosphorylation and membrane association. The data indicate that FAK and PYK2 are present in nerve terminals and that the membrane association of FAK is regulated by both phosphorylation and actin assembly, whereas that of PKY2 is primarily dependent on its phosphorylation state.     

Structural basis for the phosphorylation-regulated focal adhesion targeting of type Igamma phosphatidylinositol phosphate kinase (PIPKIgamma) by talin

Phosphatidylinositol-4,5-bisphosphate (PIP2) is a key lipid messenger that regulates myriad diverse cellular signaling pathways. To ensure specificity in disparate cellular events, PIP2 must be localized to specific sub-cellular sites. At PIP2-regulated focal adhesion (FA) sites, such localization is in part mediated via the recruitment and activation of PIP2-producing enzyme, type Igamma phosphatidylinositol phosphate kinase (PIPKIgamma), by a phosphotyrosine binding (PTB) domain of talin. Transient phosphorylation of PIPKIgamma at Y644 regulates the interaction and efficient FA targeting of PIPKIgamma; however, the underlying structural basis remains elusive. We have determined the NMR structure of talin-1 PTB in complex with the Y644-phosphorylated PIPKIgamma fragment (WVpYSPLH). As compared to canonical PTB domains that typically recognize the NPXpY turn motif from a variety of signaling proteins, our structure displays an unusual non-NPXpY-based recognition mode for talin-1 PTB where K(357)RW in beta5 strand forms an antiparallel beta-sheet with the VpYS of PIPKIgamma. A specific electrostatic triad between K357/R358 of talin-1 PTB and the pY644 of PIPKIgamma was observed, which is consistent with the mutagenesis and isothermal calorimetry data. Combined with previous in vivo data, our results provide a framework for understanding how phosphorylation of Y644 in PIPKIgamma promotes its specific interaction with talin-1, leading to efficient local synthesis of PIP2 and dynamic regulation of integrin-mediated FA assembly.     

Regulation of articular chondrocyte phenotype by bone morphogenetic protein 7, interleukin 1, and cellular context is dependent on the cytoskeleton.

Bone morphogenetic proteins (BMPs) induce cartilage differentiation and morphogenesis. There are profound changes in the cytoskeletal architecture during the morphogenesis of cartilage. To investigate the possibility that morphogenetic signals such as BMPs may regulate chondrocyte phenotype by modulation of cytoskeletal protein expression, we determined whether the expression and distribution of cytoskeletal proteins in chondrocytes are regulated by bone morphogenetic protein 7 (BMP 7), interleukin 1 (IL-1), and cellular context. Addition of BMP 7, a morphogen that induces chondrogenesis, to primary cultures of bovine and murine chondrocytes induced increased expression of four cytoskeletal proteins: tensin, talin, paxillin, and focal adhesion kinase (FAK). The expression of cytoskeletal proteins is dependent on cellular context; compared to monolayer, chondrocytes in suspension exhibited increased expression of cytoskeletal components. Conversely, addition of IL-1, a catabolic cytokine, induced loss of chondrocyte phenotype and decreased the expression of these cytoskeletal components. Treatment of chondrocytes with cytochalasin D (an agent that disrupts the actin cytoskeleton) inhibited BMP 7-induced upregulation of tensin, talin, paxillin, and FAK, and blocked the effect of BMP 7 on chondrocyte phenotype. Taken together these data demonstrate that cytoskeletal components play a critical role in the response to morphogens and cytokines in the regulation of chondrocyte phenotype. (c)2001 Elsevier Science.     

Cellular secretion studied by force microscopy

Using the optical microscope, real adventures in cellular research began in earnest in the latter half of the nineteenth century. With the development of the electron microscope, ultramicroscopy, and improved cell staining techniques, significant advances were made in defining intracellular structures at the nanometer level. The invention of force microscopy, the atomic force microscope (AFM) in the mid 1980s, and the photonic force microscope (PFM) in the mid 1990s, finally provided the opportunity to study live cellular structure-function at the nanometer level. Working with the AFM, dynamic cellular and subcellular events at the molecular level were captured in the mid 1990s, and a new cellular structure 'the porosome' in the plasma membrane of all secretory cells has been defined, where specific docking and fusion of secretory vesicles occur. The molecular mechanism of fusion of the secretory vesicle membrane at the base of the porosome membrane in cells, and the regulated release of intravesicular contents through the porosome opening to the extracellular space, has been determined. These seminal discoveries provide for the first time a molecular mechanism of cell secretion, and the possibility to ameliorate secretory defects in disease states.     

Stimulated single‐cell force spectroscopy to quantify cell adhesion receptor crosstalk

To control their attachment to substrates and other cells, cells regulate their adhesion receptors. One regulatory process is receptor crosstalk, where the binding of one type of cell adhesion molecule influences the activity of another type. To identify such crosstalk and gain insight into their mechanisms, we developed the stimulated single‐cell force spectroscopy assay. In this assay, the influence of a cells adhesion to one substrate on the strength of its adhesion to a second substrate is examined. The assay quantifies the adhesion of the cell and the contributions of specific adhesion receptors. This allows mechanisms by which the adhesion is regulated to be determined. Using the assay we identified crosstalk between collagen‐binding integrin α₁β₁ and fibronectin‐binding integrin α₅β₁ in HeLa cells. This crosstalk was unidirectional, from integrin α₁β₁ to integrin α₅β₁, and functioned by regulating the endocytosis of integrin α₅β₁. The single‐cell assay should be expandable for the screening and quantification of crosstalk between various cell adhesion molecules and other cell surface receptors.     

The beta-amyloid peptide of Alzheimer's disease decreases adhesion of vascular smooth muscle cells to the basement membrane

Cerebral amyloid angiopathy (CAA) is a major feature of Alzheimer's disease pathology. In CAA, degeneration of vascular smooth muscle cells (VSMCs) occurs close to regions of the basement membrane where the amyloid protein (Abeta) builds up. In this study, the possibility that Abeta disrupts adhesive interactions between VSMCs and the basement membrane was examined. VSMCs were cultured on a commercial basement membrane substrate (Matrigel). The presence of Abeta in the Matrigel decreased cell-substrate adhesion and cell viability. Full-length oligomeric Abeta was required for the effect, as N- and C-terminally truncated peptide analogues did not inhibit adhesion. Abeta that was fluorescently labelled at the N-terminus (fluo-Abeta) bound to Matrigel as well as to the basement membrane heparan sulfate proteoglycan (HSPG) perlecan and laminin. Adhesion of VSMCs to perlecan or laminin was decreased by Abeta. As perlecan influences VSMC viability through the extracellular signal-regulated kinase (ERK)1/2 signalling pathway, the effect of Abeta1-40 on ERK1/2 phosphorylation was examined. The level of phospho-ERK1/2 was decreased in cells following Abeta treatment. An inhibitor of ERK1/2 phosphorylation enhanced the effect of Abeta on cell adhesion. The studies suggest that Abeta can decrease VSMC viability by disrupting VSMC-extracellular matrix (ECM) adhesion.     

Identification of P2X2/P2X4/P2X6 heterotrimeric receptors using atomic force microscopy (AFM) imaging

Seven P2X purinergic receptor subunits have been identified: P2X1–P2X7. The overlapping expression of P2X2, P2X4 and P2X6 subunits has been shown in different cell types, and functional analysis of P2X receptors in Leydig cells suggests that the three subunits might interact. Here, His₆-tagged P2X2, HA-tagged P2X4 and FLAG-tagged P2X6 subunits were co-expressed in tsA 201 cells. After sequential co-immunoprecipitation using anti-HA and anti-FLAG beads, all three subunits were present, demonstrating their interaction. Atomic force microscopy (AFM) imaging revealed receptors that were specifically decorated by both an anti-His₆ antibody and an anti-HA Fab fragment, indicating the presence of a P2X2/4/6 heterotrimer. To our knowledge, this is the first report of a P2X receptor containing three different subunits.     

Regulation of focal adhesion formation and filopodia extension by the cellular prion protein (PrPC)

While the prion protein (PrP) is clearly involved in neuropathology, its physiological roles remain elusive. Here, we demonstrate PrP functions in cell-substrate interaction in Drosophila S2, N2a and HeLa cells. PrP promotes cell spreading and/or filopodia formation when overexpressed, and lamellipodia when downregulated. Moreover, PrP normally accumulates in focal adhesions (FAs), and its downregulation leads to reduced FA numbers, increased FA length, along with Src and focal adhesion kinase (FAK) activation. Furthermore, its overexpression elicits the formation of novel FA-like structures, which require intact reggie/flotillin microdomains. Altogether, PrP modulates process formation and FA dynamics, possibly via signal transduction involving FAK and Src.     

Characterization of adhesion properties of the cardiomyocyte integrins and extracellular matrix proteins using atomic force microscopy

Integrins are transmembrane adhesion receptors that play important roles in the cardiovascular system by interacting with the extracellular matrix (ECM). However, direct quantitative measurements of the adhesion properties of the integrins on cardiomyocyte (CM) and their ECM ligands are lacking. In this study, we used atomic force microscopy (AFM) to quantify the adhesion force (peak force and mean force) and binding probability between CM integrins and three main heart tissue ECM proteins, ie, collagen (CN), fibronectin (FN), and laminin (LN). Functionalizing the AFM probes with ECM proteins, we found that the peak force (mean force) was 61.69 ± 5.5 pN (76.54 ± 4.0 pN), 39.26 ± 4.4 pN (59.84 ± 3.6 pN), and 108.31 ± 4.2 pN (129.63 ± 6.0 pN), respectively, for the bond of CN‐integrin, FN‐integrin, and LN‐integrin. The binding specificity between CM integrins and ECM proteins was verified by using monoclonal antibodies, where α₁₀‐ and α₁₁‐integrin bind to CN, α₃‐ and α₅‐integrin bind to FN, and α₃‐ and α₇‐integrin bind to LN. Furthermore, adhesion properties of CM integrins under physiologically high concentrations of extracellular Ca²⁺ and Mg²⁺ were tested. Additional Ca²⁺ reduced the adhesion mean force to 68.81 ± 4.0 pN, 49.84 ± 3.3 pN, and 119.21 ± 5.8 pN and binding probability to 0.31, 0.34, 0.40 for CN, FN, and LN, respectively, whereas Mg²⁺ caused very minor changes to adhesion properties of CM integrins. Thus, adhesion properties between adult murine CM integrins and its main ECM proteins were characterized, paving the way for an improved understanding of CM mechanobiology.     

Kinetics of adhesion mediated by extracellular loops of claudin-2 as revealed by single-molecule force spectroscopy

Claudins (Cldns) comprise a large family of important transmembrane proteins that localize at tight junctions where they play a central role in regulating paracellular transportation of solutes across epithelia. However, molecular interactions occurring between the extracellular domains of these proteins are poorly understood. Here, using atomic force microscopy, the adhesion strength and kinetic properties of the homophilic interactions between the two extracellular loops of Cldn2 (C2E1or C2E2) and full-length Cldn2 were characterized at the level of single molecule. Results show that while the first extracellular loop is sufficient for Cldn2/Cldn2 trans-interaction, the second extracellular loop does not interact with the full-length Cldn2, with the first extracellular loop, or with itself. Furthermore, within the range of loading rates probed (10²-10⁴ pN/s), dissociation of Cldn2/Cldn2 and C2E1/C2E1 complexes follows a two-step energy barrier model. The difference in activation energy for the inner and outer barriers of Cldn2/Cldn2 and C2E1/C2E1 dissociation was found to be 0.26 and 1.66 k_BT, respectively. Comparison of adhesion kinetics further revealed that Cldn2/Cldn2 dissociates at a much faster rate than C2E1/C2E1, indicating that the second extracellular loop probably has an antagonistic effect on the kinetic stability of Cldn2-mediated interactions. These results provide an insight into the importance of the first extracellular loop in trans-interaction of Cldn2-mediated adhesion.