The Talin Dimer Structure Orientation Is Mechanically Regulated

Formation of a stable cell-substrate contact can be regulated by mechanical force, especially at the focal adhesion. Individual proteins that make up the focal adhesions, such as talin, can exhibit mechanosensing. We previously described one mode of talin mechanosensing in which the vinculin-binding site of talin is exposed after force-induced stretch of a single talin rod domain. Here, we describe a second mode of talin mechanosensing in which the talin dimer itself can adopt different orientations in response to mechanical stimulation. Using molecular dynamics models, we demonstrate that the C-terminus region of the talin dimer is flexible mainly at the linker between the dimerization helices and the nearby actin-binding helical bundle. Our molecular dynamics simulations reveal two possible orientations of the talin dimer at its C-terminus. The extracellular matrix (ECM)-bound integrins cross-linked by talin can be forced apart leading to an elongated orientation of the talin dimer, and the ECM-bound integrins can be forced together by the ECM producing a collapsed orientation of the talin dimer. Formation of the elongated orientation is shown to be more favorable. Switching between the two talin dimer orientations constitutes a mode of mechanosensing.     

Distinct ECM mechanosensing pathways regulate microtubule dynamics to control endothelial cell branching morphogenesis

During angiogenesis, cytoskeletal dynamics that mediate endothelial cell branching morphogenesis during vascular guidance are thought to be regulated by physical attributes of the extracellular matrix (ECM) in a process termed mechanosensing. Here, we tested the involvement of microtubules in linking mechanosensing to endothelial cell branching morphogenesis. We used a recently developed microtubule plus end-tracking program to show that specific parameters of microtubule assembly dynamics, growth speed and growth persistence, are globally and regionally modified by, and contribute to, ECM mechanosensing. We demonstrated that engagement of compliant two-dimensional or three-dimensional ECMs induces local differences in microtubule growth speed that require myosin II contractility. Finally, we found that microtubule growth persistence is modulated by myosin II-mediated compliance mechanosensing when cells are cultured on two-dimensional ECMs, whereas three-dimensional ECM engagement makes microtubule growth persistence insensitive to changes in ECM compliance. Thus, compliance and dimensionality ECM mechanosensing pathways independently regulate specific and distinct microtubule dynamics parameters in endothelial cells to guide branching morphogenesis in physically complex ECMs.     

Predicting YAP/TAZ nuclear translocation in response to ECM mechanosensing

Cells translate mechanical cues from the extracellular matrix (ECM) into signaling that can affect the nucleus. One pathway by which such nuclear mechanotransduction occurs is a signaling axis that begins with integrin-ECM bonds and continues through a cascade of chemical reactions and structural changes that lead to nuclear translocation of YAP/TAZ. This signaling axis is self-reinforcing, with stiff ECM promoting integrin binding and thus facilitating polymerization and tension in the cytoskeletal contractile apparatus, which can compress nuclei, open nuclear pore channels, and enhance nuclear accumulation of YAP/TAZ. We previously developed a computational model of this mechanosensing axis for the linear elastic ECM by assuming that there is a linear relationship between the nucleocytoplasmic ratio of YAP/TAZ and nuclear flattening. Here, we extended our previous model to more general ECM behaviors (e.g., viscosity, viscoelasticity, and viscoplasticity) and included detailed YAP/TAZ translocation dynamics based on nuclear deformation. This model was predictive of diverse mechanosensing responses in a broad range of cells. Results support the hypothesis that diverse mechanosensing phenomena across many cell types arise from a simple, unified set of mechanosensing pathways.     

Talin tension sensor reveals novel features of focal adhesion force transmission and mechanosensitivity

Integrin-dependent adhesions are mechanosensitive structures in which talin mediates a linkage to actin filaments either directly or indirectly by recruiting vinculin. Here, we report the development and validation of a talin tension sensor. We find that talin in focal adhesions is under tension, which is higher in peripheral than central adhesions. Tension on talin is increased by vinculin and depends mainly on actin-binding site 2 (ABS2) within the middle of the rod domain, rather than ABS3 at the far C terminus. Unlike vinculin, talin is under lower tension on soft substrates. The difference between central and peripheral adhesions requires ABS3 but not vinculin or ABS2. However, differential stiffness sensing by talin requires ABS2 but not vinculin or ABS3. These results indicate that central versus peripheral adhesions must be organized and regulated differently, and that ABS2 and ABS3 have distinct functions in spatial variations and stiffness sensing. Overall, these results shed new light on talin function and constrain models for cellular mechanosensing.     

Modeling and simulation of chemomechanics at the cell-matrix interface

Chemomechanical characteristics of the extracellular materials with which cells interact can have a profound impact on cell adhesion and migration. To understand and modulate such complex multiscale processes, a detailed understanding of the feedback between a cell and the adjacent microenvironment is crucial. Here, we use computational modeling and simulation to examine the cell-matrix interaction at both the molecular and continuum lengthscales. Using steered molecular dynamics, we consider how extracellular matrix (ECM) stiffness and extracellular pH influence the interaction between cell surface adhesion receptors and extracellular matrix ligands, and we predict potential consequences for focal adhesion formation and dissolution. Using continuum level finite element simulations and analytical methods to model cell-induced ECM deformation as a function of ECM stiffness and thickness, we consider the implications toward design of synthetic substrata for cell biology experiments that intend to decouple chemical and mechanical cues.Key words: cell adhesion, focal adhesion, steered molecular dynamics, finite element, chemomechanics, multiscale modeling, elasticity theory     

The structure of the talin/integrin complex at a lipid bilayer: an NMR and MD simulation study

Integrins are cell surface receptors crucial for cell migration and adhesion. They are activated by interactions of the talin head domain with the membrane surface and the integrin β cytoplasmic tail. Here, we use coarse-grained molecular dynamic simulations and nuclear magnetic resonance spectroscopy to elucidate the membrane-binding surfaces of the talin head (F2-F3) domain. In particular, we show that mutations in the four basic residues (K258E, K274E, R276E, and K280E) in the F2 binding surface reduce the affinity of the F2-F3 for the membrane and modify its orientation relative to the bilayer. Our results highlight the key role of anionic lipids in talin/membrane interactions. Simulation of the F2-F3 in complex with the α/β transmembrane dimer reveals information for its orientation relative to the membrane. Our studies suggest that the perturbed orientation of talin relative to the membrane in the F2 mutant would be expected to in turn perturb talin/integrin interactions.     

Talin contains a C-terminal calpain2 cleavage site important in focal adhesion dynamics

Talin is a large (～2540 residues) dimeric adaptor protein that associates with the integrin family of cell adhesion molecules in cell-extracellular matrix junctions (focal adhesions; FAs), where it both activates integrins and couples them to the actin cytoskeleton. Calpain2-mediated cleavage of talin between the head and rod domains has previously been shown to be important in FA turnover. Here we identify an additional calpain2-cleavage site that removes the dimerisation domain from the C-terminus of the talin rod, and show that an E2492G mutation inhibits calpain cleavage at this site in vitro, and increases the steady state levels of talin1 in vivo. Expression of a GFP-tagged talin1 E2492G mutant in CHO.K1 cells inhibited FA turnover and the persistence of cell protrusion just as effectively as a L432G mutation that inhibits calpain cleavage between the talin head and rod domains. Moreover, incorporation of both mutations into a single talin molecule had an additive effect clearly demonstrating that calpain cleavage at both the N- and C-terminal regions of talin contribute to the regulation of FA dynamics. However, the N-terminal site was more sensitive to calpain cleavage suggesting that lower levels of calpain are required to liberate the talin head and rod fragments than are needed to clip off the C-terminal dimerisation domain. The talin head and rod liberated by calpain2 cleavage have recently been shown to play roles in an integrin activation cycle important in FA turnover and in FAK-dependent cell cycle progression respectively. The half-life of the talin head is tightly regulated by ubiquitination and we suggest that removal of the C-terminal dimerisation domain from the talin rod may provide a mechanism both for terminating the signalling function of the talin rod and indeed for inactivating full-length talin thereby promoting FA turnover at the rear of the cell.     

The C terminus of talin links integrins to cell cycle progression

Integrins are cell adhesion receptors that sense the extracellular matrix (ECM) environment. One of their functions is to regulate cell fate decisions, although the question of how integrins initiate intracellular signaling is not fully resolved. In this paper, we examine the role of talin, an adapter protein at cell-matrix attachment sites, in outside-in signaling. We used lentiviral small hairpin ribonucleic acid to deplete talin in mammary epithelial cells. These cells still attached to the ECM in an integrin-dependent manner and spread. They had a normal actin cytoskeleton, but vinculin, paxillin, focal adhesion kinase (FAK), and integrin-linked kinase were not recruited to adhesion sites. Talin-deficient cells showed proliferation defects, and reexpressing a tail portion of the talin rod, but not its head domain, restored integrin-mediated FAK phosphorylation, suppressed p21 expression, and rescued cell cycle. Thus, talin recruits and activates focal adhesion proteins required for proliferation via the C terminus of its rod domain. Our study reveals a new function for talin, which is to link integrin adhesions with cell cycle progression.     

Modeling and simulation of chemomechanics at the cell-matrix interface

Chemomechanical characteristics of the extracellular materials with which cells interact can have a profound impact on cell adhesion and migration. To understand and modulate such complex multiscale processes, a detailed understanding of the feedback between a cell and the adjacent microenvironment is crucial. Here, we use computational modeling and simulation to examine the cell-matrix interaction at both the molecular and continuum lengthscales. Using steered molecular dynamics, we consider how extracellular matrix (ECM) stiffness and extracellular pH influence the interaction between cell surface adhesion receptors and extracellular matrix ligands, and we predict potential consequences for focal adhesion formation and dissolution. Using continuum-level finite element simulations and analytical methods to model cell-induced ECM deformation as a function of ECM stiffness and thickness, we consider the implications toward design of synthetic substrata for cell biology experiments that intend to decouple chemical and mechanical cues.     

The conformational states of talin autoinhibition complex and its activation under forces

Talin is an integrin-binding protein located at focal adhesion site and serves as both an adapter and a force transmitter. Its integrin binding activity is regulated by the intramolecular autoinhibition interaction between its F3 and RS domains. Here, we used atomic force microscopy to measure the strength of talin autoinhibition complex. Our results suggest that the lifetime of talin autoinhibition complex shows weak catch bond behavior and does not change significantly at smaller forces, while it drops rapidly at larger forces (>10 pN). Moreover, besides the complex conformation revealed by crystal structure, our molecular dynamics (MD) simulations indicate the possible existence of another stable conformation. Further analysis indicates that forces may regulate the equilibrium of the two stable binding states and result in the non-exponential force dependence of the binding lifetime. Our findings reveal a negative regulation mechanism on talin activation and provide a new point of view on the function of talin in focal adhesion.     

Transitions in microtubule C-termini conformations as a possible dendritic signaling phenomenon

We model the dynamical states of the C-termini of tubulin dimers that comprise neuronal microtubules. We use molecular dynamics and other computational tools to explore the time-dependent behavior of conformational states of a C-terminus of tubulin within a microtubule and assume that each C-terminus interacts via screened Coulomb forces with the surface of a tubulin dimer, with neighboring C-termini and also with any adjacent microtubule-associated protein 2 (MAP2). Each C-terminus can either bind to the tubulin surface via one of the several positively charged regions or can be allowed to explore the space available in the solution surrounding the dimer. We find that the preferential orientation of each C-terminus is away from the tubulin surface but binding to the surface may also take place, albeit at a lower probability. The results of our model suggest that perturbations generated by the C-termini interactions with counterions surrounding a MAP2 may propagate over distances greater than those between adjacent microtubules. Thus, the MAP2 structure is able to act as a kind of biological wire (or a cable) transmitting local electrostatic perturbations resulting in ionic concentration gradients from one microtubule to another. We briefly discuss the implications the current dynamic modeling may have on synaptic activation and potentiation.     

Structural basis for phosphatidylinositol phosphate kinase type Igamma binding to talin at focal adhesions

The cytoskeletal protein talin binds to a short C-terminal sequence in phosphatidylinositol phosphate kinase type Igamma (PIPKIgamma), activating the enzyme and promoting the local production of phosphatidylinositol 4,5 bisphosphate, which regulates focal adhesion dynamics as well as clathrin-mediated endocytosis in neuronal cells. Here we show by crystallographic, NMR, and calorimetric analysis that the phosphotyrosine binding (PTB)-like domain of talin engages the PIPKIgamma C terminus in a mode very similar to that of integrin binding. However, PIPKIgamma binds in the canonical PTB-peptide mode with an SPLH motif replacing the classic NPXY motif. The tighter packing of the SPLH motif against the hydrophobic core of talin may explain the stronger binding of PIPKIgamma. Two tyrosine residues flanking the SPLH motif (Tyr-644 and Tyr-649) have been implicated in the regulation of talin binding. We show that phosphorylation at Tyr-644, a Src phosphorylation site in vivo, has little effect on the binding mode or strength, which is consistent with modeling studies in which the phosphotyrosine makes surface-exposed salt bridges, and we suggest that its strong activating effect arises from the release of autoinhibitory restraints in the full-length PIPKIgamma. Modeling studies suggest that phosphorylation of Tyr-649 will likewise have little effect on talin binding, whereas phosphorylation of the SPLH serine is predicted to be strongly disruptive. Our data are consistent with the proposal that Src activity promotes a switch from integrin binding to PIPKIgamma binding that regulates focal adhesion turnover.     

Properties of talin from chicken gizzard smooth muscle

This paper describes the structural and biochemical characterization of talin, a protein localized to various cellular sites where bundles of actin filaments attach to the plasma membrane. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein has a molecular mass of 225,000 +/- 5,000 daltons. Hydrodynamic measurements at protein concentrations less than 0.72 mg/ml indicate a monomeric protein with a native molecular mass of 213,000 +/- 15,000 daltons. Sedimentation equilibrium experiments indicate self-association at protein concentrations of 0.72 mg/ml and higher. The data suggest that this self-association is a simple monomer:dimer equilibrium over the range of concentrations observed. At low protein concentrations where talin is a monomer, the Stokes radius and sedimentation coefficient vary with ionic strength. Under low ionic strength conditions (5-20 mM NaCl), talin has a Stokes radius of 6.5 nm and a sedimentation value of 9.4, suggesting an asymmetric globular molecule; whereas under high ionic strength conditions (200 mM NaCl), the Stokes radius increases to 7.7 nm and the sedimentation coefficient decreases to 8.8, suggesting a more elongated protein. This conformation change is confirmed by electron microscopy which reveals a more globular protein at low ionic strength which unfolds to become an elongated flexible molecule as the ionic strength is increased to physiological and higher levels. The amino acid composition of talin indicates a low level of aromatic residues, consistent with its relatively low extinction coefficient, talin has an isoelectric point between pH 6.7 and 6.8 based on isoelectric focusing. The detailed purification of talin is described.     

A conformational switch in vinculin drives formation and dynamics of a talin-vinculin complex at focal adhesions

Dynamic interactions between the cytoskeleton and integrins control cell adhesion, but regulatory mechanisms remain largely undefined. Here, we tested the extent to which the autoinhibitory head-tail interaction (HTI) in vinculin regulates formation and lifetime of the talin-vinculin complex, a proposed mediator of integrin-cytoskeleton bonds. In an ectopic recruitment assay, mutational reduction of HTI drove assembly of talin-vinculin complexes, whereas ectopic complexes did not form between talin and wild-type vinculin. Moreover, reduction of HTI altered the dynamic assembly of vinculin and talin in focal adhesions. Using fluorescence recovery after photobleaching, we show that the focal adhesion residency time of vinculin was enhanced up to 3-fold by HTI mutations. The slow dynamics of vinculin correlated with exposure of its cryptic talin-binding site, and a talin-binding site mutation rescued the dynamics of activated vinculin. Significantly, HTI-deficient vinculin inhibited the focal adhesion dynamics of talin, but not paxillin or alpha-actinin. These data show that talin conformation in cells permits vinculin binding, whereas the autoinhibited conformation of vinculin constitutes the barrier to complex formation. Down-regulation of HTI in vinculin to Kd approximately 10(-7) is sufficient to induce talin binding, and HTI is essential to the dynamics of vinculin and talin at focal adhesions. We therefore conclude that vinculin conformation, as modulated by the strength of HTI, directly regulates the formation and lifetime of talin-vinculin complexes in cells.     

Integrin-based mechanosensing through conformational deformation

Conversion of integrins from low to high affinity states, termed activation, is important in biological processes, including immunity, hemostasis, angiogenesis, and embryonic development. Integrin activation is regulated by large-scale conformational transitions from closed, low affinity states to open, high affinity states. Although it has been suggested that substrate stiffness shifts the conformational equilibrium of integrin and governs its unbinding, here, we address the role of integrin conformational activation in cellular mechanosensing. Comparison of wild-type versus activating mutants of integrin αVβ3 show that activating mutants shift cell spreading, focal adhesion kinase activation, traction stress, and force on talin toward high stiffness values at lower stiffness. Although all activated integrin mutants showed equivalent binding affinity for soluble ligands, the β3 S243E mutant showed the strongest shift in mechanical responses. To understand this behavior, we used coarse-grained computational models derived from molecular level information. The models predicted that wild-type integrin αVβ3 displaces under force and that activating mutations shift the required force toward lower values, with S243E showing the strongest effect. Cellular stiffness sensing thus correlates with computed effects of force on integrin conformation. Together, these data identify a role for force-induced integrin conformational deformation in cellular mechanosensing.     

Integrin adhesions

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.     

Integrin adhesions: Who’s on first? What's on second?

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.     

αV-Integrins Are Required for Mechanotransduction in MDCK Epithelial Cells

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.     

Calpain-mediated proteolysis of talin regulates adhesion dynamics

Dynamic regulation of adhesion complexes is required for cell migration and has therefore emerged as a key issue in the study of cell motility. Recent progress has been made in defining some of the molecular mechanisms by which adhesion disassembly is regulated, including the contributions of adhesion adaptor proteins and tyrosine kinases. However, little is known about the potential contribution of proteolytic mechanisms to the regulation of adhesion complex dynamics. Here, we show that proteolysis of talin by the intracellular calcium-dependent protease calpain is critical for focal adhesion disassembly. We have generated a single point mutation in talin that renders it resistant to proteolysis by calpain. Quantification of adhesion assembly and disassembly rates demonstrates that calpain-mediated talin proteolysis is a rate-limiting step during adhesion turnover. Furthermore, we demonstrate that disassembly of other adhesion components, including paxillin, vinculin and zyxin, is also dependent on the ability of calpain to cleave talin, suggesting a general role for talin proteolysis in regulating adhesion turnover. Together, these findings identify calpain-mediated proteolysis of talin as a mechanism by which adhesion dynamics are regulated.     

Kindlin Is Mechanosensitive: Force-Induced Conformational Switch Mediates Cross-Talk among Integrins

Mechanical stresses directly regulate the function of several proteins of the integrin-mediated focal adhesion complex as they experience intra- and extracellular forces. Kindlin is a largely overlooked member of the focal adhesion complex whose roles in cellular mechanotransduction are only recently being identified. Recent crystallographic experiments have revealed that kindlins can form dimers that bind simultaneously to two integrins, providing a mechanistic explanation of how kindlins may promote integrin activation and clustering. In this study, using the newly identified molecular structure, we modeled the response of the kindlin2 dimer in complex with integrin β1 to mechanical cytoskeletal forces on integrins. Using molecular dynamics simulations, we show that forces on integrins are directly transmitted to the kindlin2 dimerization site, resulting in a shift in an R577-S550/E553 interaction network at this site. Under force, R577 on one protomer switches from interacting with S550 to forming new hydrogen bonds with E553 on the neighboring protomer, resulting in the strengthening of the kindlin2 dimer in complex with integrin β1. This force-induced strengthening is similar to the catch-bond mechanisms that have previously been observed in other adhesion molecules. Based on our results, we propose that the kindlin2 dimer is mechanosensitive and can strengthen integrin-mediated focal adhesions under force by shifting the interactions at its dimerization sites.