Catch Bonds Research Articles

Non-channel mechanosensors working at focal adhesion-stress fiber complex.

Mechanosensitive ion channels (MSCs) have long been the only established molecular class of cell mechanosensors; however, in the last decade, a variety of non-channel type mechanosensor molecules have been identified. Many of them are focal adhesion-associated proteins that include integrin, talin, and actin. Mechanosensors must be non-soluble molecules firmly interacting with relatively rigid cellular structures such as membranes (in terms of lateral stiffness), cytoskeletons, and adhesion structures. The partner of MSCs is the membrane in which MSC proteins efficiently transduce changes in the membrane tension into conformational changes that lead to channel opening. By contrast, the integrin, talin, and actin filament form a linear complex of which both ends are typically anchored to the extracellular matrices via integrins. Upon cell deformation by forces, this structure turns out to be a portion that efficiently transduces the generated stress into conformational changes of composite molecules, leading to the activation of integrin (catch bond with extracellular matrices) and talin (unfolding to induce vinculin bindings). Importantly, this structure also serves as an "active" mechanosensor to detect substrate rigidity by pulling the substrate with contraction of actin stress fibers (SFs), which may induce talin unfolding and an activation of MSCs in the vicinity of integrins. A recent study demonstrates that the actin filament acts as a mechanosensor with unique characteristics; the filament behaves as a negative tension sensor in which increased torsional fluctuations by tension decrease accelerate ADF/cofilin binding, leading to filament disruption. Here, we review the latest progress in the study of those non-channel mechanosensors and discuss their activation mechanisms and physiological roles.

Regulation of shear stress on rolling behaviors of HL-60 cells on P-selectin

Circulating leukocytes in trafficking to the inflammatory sites, will be first tether to, and then roll on the vascular surface. This event is mediated through specific interaction of P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1), and regulated by hemodynamics. Poor data were reported in understanding P-selectin-mediated rolling. With the flow chamber technique, we herein observed HL-60 cell rolling on P-selectin with or without 3% Ficoll at various wall shear stresses from 0.05 to 0.4 dyn/cm2. The results demonstrated that force rather than transport regulated the rolling, similar to rolling on L- and E-selectin. The rolling was accelerated quickly by an increasing force below the optimal shear threshold of 0.15 dyn/cm2 first and then followed by a slowly decelerating phase starting at the optimum, showing a catch-slip transition and serving as a mechanism for the rolling. The catch-slip transition was completely reflected to the tether lifetime and other rolling parameters, such as the mean and fractional stop time. The narrow catch bond regime stabilized the rolling quickly, through steeply increasing fractional stop time to a plateau of about 0.85. Data presented here suggest that the low shear stress threshold serves as a mechanism for most cell rolling events through P-selectin.

Plasticity of hydrogen bond networks regulates mechanochemistry of cell adhesion complexes.

Mechanical forces acting on cell adhesion receptor proteins regulate a range of cellular functions by formation and rupture of noncovalent interactions with ligands. Typically, force decreases the lifetimes of intact complexes ("slip bonds"), making the discovery that these lifetimes can also be prolonged ("catch bonds") a surprise. We created a microscopic analytic theory by incorporating the structures of selectin and integrin receptors into a conceptual framework based on the theory of stochastic equations, which quantitatively explains a wide range of experimental data (including catch bonds at low forces and slip bonds at high forces). Catch bonds arise due to force-induced remodeling of hydrogen bond networks, a finding that also accounts for unbinding in structurally unrelated integrin-fibronectin and actomyosin complexes. For the selectin family, remodeling of hydrogen bond networks drives an allosteric transition resulting in the formation of the maximum number of hydrogen bonds determined only by the structure of the receptor and independent of the ligand. A similar transition allows us to predict the increase in the number of hydrogen bonds in a particular allosteric state of α5β1 integrin-fibronectin complex, a conformation which is yet to be crystallized. We also make a testable prediction that a single point mutation (Tyr51Phe) in the ligand associated with selectin should dramatically alter the nature of the catch bond compared with the wild type. Our work suggests that nature uses a ductile network of hydrogen bonds to engineer function over a broad range of forces.

Resolving the molecular mechanism of cadherin catch bond formation.

Classical cadherin Ca(2+)-dependent cell-cell adhesion proteins play key roles in embryogenesis and in maintaining tissue integrity. Cadherins mediate robust adhesion by binding in multiple conformations. One of these adhesive states, called an X-dimer, forms catch bonds that strengthen and become longer lived in the presence of mechanical force. Here we use single-molecule force-clamp spectroscopy with an atomic force microscope along with molecular dynamics and steered molecular dynamics simulations to resolve the molecular mechanisms underlying catch bond formation and the role of Ca(2+) ions in this process. Our data suggest that tensile force bends the cadherin extracellular region such that they form long-lived, force-induced hydrogen bonds that lock X-dimers into tighter contact. When Ca(2+) concentration is decreased, fewer de novo hydrogen bonds are formed and catch bond formation is eliminated.

Structural mechanism of TCR-pMHC catch bond revealed by molecular dynamics, mutagenesis, and single-molecule kinetics analyses (IRM10P.752)

Abstract Mechanical force is increasingly recognized as an important axis in regulation of T cell function. Recently, we showed that force elicits agonist-specific TCR-pMHC catch bond (where force counterintuitively prolongs bond lifetime), which provides a biophysical mechanism of force-triggered T cell Ca2+ flux (Liu et al., Cell, in press). Here, we performed steered molecular dynamics (SMD) simulation and single-bond lifetime experiments to investigate the molecular mechanism of TCR-pMHC catch bond. SMD simulation of 2C TCR-agonist dissociation showed that pulling TCR-pMHC bond leads to characteristic rotation of MHCI α1/α2 domain relative to TCR variable region, yielding multiple new atomic interactions (e.g., hydrogen bond) that are absent in crystal structure. Force-regulated bond lifetime measured on naïve 2C T cells validated two major predictions of such catch bond mechanism. (1) Pulling with a geometry that favors the relative domain rotation (by pulling pMHC β chain rather than c-terminal of heavy chain) produced a more pronounced catch bond; (2) a single point mutation on pMHC that abolishes new hydrogen bond formation eliminated catch bond and lowered EC50 (proliferation) by more than three orders of magnitude with only two fold decrease in affinity and a longer zero force lifetime. These data highlight a dynamic perspective of TCR-pMHC structure that reveals novel insights on TCR-pMHC interaction and T cell function.

Accumulation of Dynamic Catch Bonds between TCR and Agonist Peptide-MHC Triggers T Cell Signaling

TCR-pMHC interactions initiate adaptive immune responses, but the mechanism of how such interactions under force induce T cell signaling is unclear. We show that force prolongs lifetimes of single TCR-pMHC bonds for agonists (catch bonds) but shortens those for antagonists (slip bonds). Both magnitude and duration of force are important, as the highest Ca(2+) responses were induced by 10 pN via both pMHC catch bonds whose lifetime peaks at this force and anti-TCR slip bonds whose maximum lifetime occurs at 0 pN. High Ca(2+) levels require early and rapid accumulation of bond lifetimes, whereas short-lived bonds that slow early accumulation of lifetimes correspond to low Ca(2+) responses. Our data support a model in which force on the TCR induces signaling events depending on its magnitude, duration, frequency, and timing, such that agonists form catch bonds that trigger the T cell digitally, whereas antagonists form slip bonds that fail to activate.

Role of Force-Sensitive Amyloid-Like Interactions in Fungal Catch Bonding and Biofilms

The Candida albicans Als adhesin Als5p has an amyloid-forming sequence that is required for aggregation and formation of model biofilms on polystyrene. Because amyloid formation can be triggered by force, we investigated whether laminar flow could activate amyloid formation and increase binding to surfaces. Shearing Saccharomyces cerevisiae cells expressing Als5p or C. albicans at 0.8 dyne/cm(2) increased the quantity and strength of cell-to-surface and cell-to-cell binding compared to that at 0.02 dyne/cm(2). Thioflavin T fluorescence showed that the laminar flow also induced adhesin aggregation into surface amyloid nanodomains in Als5p-expressing cells. Inhibitory concentrations of the amyloid dyes thioflavin S and Congo red or a sequence-specific anti-amyloid peptide decreased binding and biofilm formation under flow. Shear-induced binding also led to formation of robust biofilms. There was less shear-activated increase in adhesion, thioflavin fluorescence, and biofilm formation in cells expressing the amyloid-impaired V326N-substituted Als5p. Similarly, S. cerevisiae cells expressing Flo1p or Flo11p flocculins also showed shear-dependent binding, amyloid formation, biofilm formation, and inhibition by anti-amyloid compounds. Together, these results show that laminar flow activated amyloid formation and led to enhanced adhesion of yeast cells to surfaces and to biofilm formation.

Loop 2 of myosin is a force-dependent inhibitor of the rigor bond

Myosin's actin-binding loop (loop 2) carries a charge opposite to that of its binding site on actin and is thought to play an important role in ionic interactions between the two molecules during the initial binding step. However, no subsequent role has been identified for loop 2 in actin-myosin binding. We used an optical trap to measure bond formation and bond rupture between actin and rigor heavy meromyosin when loaded perpendicular to the filament axis. We studied HMM with intact or proteolytically cleaved loop 2 at low and physiologic ionic strength. Here we show that the presence of intact loop 2 allows actomyosin bonds to form quickly and that they do so in a short-lived bound state. Increasing tensile load causes the transition to a long-lived state-the distinguishing behavior of a catch bond. When loop 2 was cleaved catch bond behavior was abrogated leaving only a long-lived state. These data suggest that in addition to its role in locating binding sites on actin, loop 2 is also a force-dependent inhibitor of the long-lived actomyosin complex. This may be important for reducing the duty ratio and increasing the shortening velocity of actomyosin at low forces.

Catching up on Slip: Focal Adhesion Composition and Mechanosensing

Unlike slip bonds, catch bonds experience reinforcement under tension. Cell adheres to the surface, using integrins forming both catch- and slip- bonds with the surface receptors. How will the catch and slip bonds interact with each other on a single adhesion scale? How does the intracellular structure vary depending on the extracellular matrix stiffness? I discuss the implications of single catch-bond characteristics for the behavior of a load-sharing cluster of such bonds: these are shown to possess a regime of strengthening with increasing applied force, similar to the manner in which focal adhesions become selectively reinforced. In addition, I present numerical simulations of mixtures of catch and slip bonds within single focal adhesion, and propose a model of how they can influence cytoskeletal reorganization, force generation and adhesion growth, interacting indirectly through applied force. Our results may shed new light on the fundamental processes that allow cells to sense the mechanical properties of their environment and in particular show how single focal adhesions may act, autonomously, as local rigidity sensors.

Resolving the Molecular Determinants of Cadherin Catch Bond Formation

Classical cadherin Ca2+-dependent cell-cell adhesion proteins play key roles in embryogenesis and in maintaining tissue integrity. Cadherins mediate robust adhesion by binding in multiple conformations. We recently showed that one of these conformations, called an X-dimer, forms catch bonds that strengthen and become longer lived in the presence of mechanical stress. Here we use single molecule force clamp spectroscopy with an Atomic Force Microscope along with Molecular Dynamics and Steered Molecular Dynamics simulations to identify key interactions that mediate X-dimer catch bond formation and to resolve the role of Ca2+ ions in this process. We show that tensile force bends the cadherin extracellular region such that they form force-induced hydrogen bonds that lock X-dimers into tighter contact. When Ca2+ concentration is decreased, these hydrogen bonds are eliminated and catch bond formation is abolished. Based on these results, we formulate a simple ‘flex and lock’ kinetic scheme that quantitatively describes X-dimer catch bonds.

Single Molecule Force Spectroscopy Reveals Force-Enhanced Binding of Calcium Ions by Gelsolin

Force is increasingly recognized as an important element in controlling biological processes. Forces are able to deform native protein conformations leading to protein-specific effects. Although in a few cases, the exposure of novel protein binding sites by force has been reported, mechanical stress generally leads to the decrease of protein-protein binding affinities. In this paper, we demonstrate that the calcium-binding affinity of the actin-binding protein gelsolin domain G6 is enhanced, rather than decreased, by mechanical force. Using a recently developed single molecule-binding assay based on atomic force microscopy, we establish that the calcium-binding affinity of G6 increases exponentially with the applied force, up to the point of G6 unfolding. This implies that gelsolin will be activated at lower calcium ion levels when subjected to tensile forces and suggests a basis for enhanced cooperativity during multi-cation induced activation. This is, to our knowledge, the first experimental verification of the force-amplified ligand binding concept at the molecular level. Such force-amplified ligand binding is fundamentally different from the widely accepted ideas of "catch bond" and force activated binding. The demonstration that cation-protein binding affinities can be force-dependent provides a new paradigm in understanding the complex interactions of cation-regulated proteins in stressful cellular environments, such as those found in the cytoskeleton-rich leading edge and at cell adhesions.

Cyclic Mechanical Reinforcement of Actin Depolymerization and Polarity of Interaction

Biomolecular bonds in focal adhesion and the actin cytoskeleton experience dynamic forces over time and the history of force loading could serve as mechanical preconditioning. Force-dependence of mean lifetime was observed in G-actin/G-actin (GG) and G-actin/F-actin (GF) dissociation, which was a biphasic transition from a catch bond to a slip bond. In the interaction of integrin/fibronectin, application of cyclic forces significantly prolonged bond lifetime. Therefore, we investigated actin depolymerization under a range of dynamic force environments.A custom-made atomic force microscopy and force-clamping procedures were used. To mimic dynamic force, cyclic forces were applied with peak forces of 10-30 pN, and minimum forces of 5-8 pN. 1.5, 2.5 and 3.5-cycles with a 10-12 pN peak force were also applied. To investigate polarity of interactions, barbed and pointed ends were distinguished by adding capping proteins, CapZ to block the barbed end and Tmod3 for the pointed end.After applying cyclic forces, the post-conditioning average lifetimes of GG interaction were significantly prolonged (3-18 fold) at low force range (5-10 pN), instead of reverting instantly to a low affinity state. The lifetime distributions switched from a one-state to a two-state dissociation. In the GF interactions, at the pointed end, the bond lifetimes were significantly prolonged (6-30 fold) and exhibited two-state dissociation kinetics similar to the GG interactions. On the other hand, the effect of cyclic mechanical reinforcement (CMR) was significantly suppressed at the barbed end.Our study demonstrated that actin depolymerization kinetics are force-history dependent and that mechanical preconditioning by cyclic force loading significantly enhances bond strength in actin/actin interactions. We hypothesize that this CMR is an important regulatory mechanism underlying cytoskeletal dynamics by affecting the actin depolymerization kinetics and more effective than the traditional catch bond.

DNA-Based “Digital” Tension Probes with Pn Sensitivity Reveal Early Cell Adhesion Mechanics at the Single Molecule Level

Mechanical forces are involved in important processes such as cell division, migration and gene expression, signifying their critical importance to living systems. However, the lack of methods to visualize cellular forces at the molecular scale has hampered the study of mechanotransduction. To address this need, we developed a new class of DNA-based molecular tension fluorescence microscopy (MTFM) probes that function as a reversible digital switch, and are ideally suited to investigate the pN-range forces applied by individual integrins. We show that focal adhesion maturation involves an increase in tension per molecule coupled with recruitment of a greater density of integrins. By engaging cells to sensor chips presenting mixtures of spectrally-encoded probes with different mechanical responses, we find that integrins display both chemical and mechanical specificity at the single-molecule level. Integrins show mechanical preference for more rigid ligands within nascent adhesions, thus suggesting focal adhesions function as rigidity sensors at the single integrin level. Moreover, this observation may be related to the catch bond model, where the integrin-ligand bond lifetime increases under a mechanical load.

Platelet Cytoskeletal Force Transmission Through The GPIb-IX-V Complex

Platelets are the primary cellular components of the hemostatic plug that forms during primary hemostasis. The first step in this process is platelet adhesion from the flowing blood to a surface, carried out by the platelet glycoprotein (GP) Ib-IX-V binding to immobilized von Willebrand factor (VWF). Adhesion is followed by activation of integrin αIIbβ3, which mediates the attachment of platelets to each other by binding multivalent ligands such as VWF or fibrinogen. To stabilize the hemostatic plug and strengthen its attachment to the wound site, platelets must transmit contractile forces from actin and myosin proteins in their cytoskeleton to extracellular matrix proteins within the vessel wall or to the adhesive proteins between adjacent platelets. Integrin αIIbβ3 is one of the membrane proteins capable of transmitting these forces, having a direct link to the platelet cytoskeleton through talin and other focal adhesion related proteins. In the current study, we investigated whether the GPIb-IX-V complex is also capable of force transmission after binding ligand.The GPIb-IX-V complex contains 4 polypeptides, GPIbα, GPIbβ, GPIX and GPV. Only GPIbα binds VWF, which it does through VWF's A1 domain. GPIbα also attaches the complex to the actin and membrane skeletons through its cytoplasmic domain, with the large skeletal protein filamin functioning as the intermediary. There is strong evidence that the GPIbα-A1 bond is force sensitive, becoming stronger as force is applied to it, a property that defines it as a “catch bond”. For this reason, we investigated the role of GPIbα in transmitting platelet forces using a new tool that we have developed to measure contractile forces generated by platelets. This tool, composed of arrays of nanoposts separated by 2 μm (Figure 1), was fabricated using e-beam lithography. VWF was adsorbed to the tips of the nanoposts and platelets were allowed to adhere, spread, and contract. To assess the contribution of αIIbβ3 and GPIbα to force generation, we blocked these receptors with the antibodies 7E3 and AK2, respectively. Treatment with 7E3 significantly lowered the force generated, but did not eliminate it completely (57% reduction). AK2 had a smaller effect (20% reduction), and the combination of the two usually abolished force generation. We observed a similar force reduction (30%) as AK2 treatment when we blocked the VWF A1 domain with recombinant GPIbα N-terminus. Because VWF contains binding sites for more than one platelet receptor, and although purified, could have trace amounts of other plasma proteins, we also evaluated force generation on nanoposts coated with recombinant VWF A1 domain, which should only bind GPIbα. In this case, the platelets generated forces similar to those observed when αIIbβ3 was blocked by 7E3, providing further evidence that GPIbα can transmit forces by binding the A1 domain. [Display omitted] As a final test of the ability of GPIbα to support force generation, we examined whether Chinese hamster ovary (CHO) cells expressing the GPIb-IX complex (CHOαβIX, fully functional but lacking GPV) could generate force on VWF or A1 domain (Figure 2). CHOαβIX cells adhered, spread and generated forces of similar magnitude on microposts (larger because of the larger cell size) coated with either substrate. CHOβIX cells, lacking GPIbα, did not adhere to either substrate. [Display omitted] To investigate the requirement for cytoskeletal attachment of the complex in force generation, we studied a CHOαβIX line containing GPIbα truncated after residue 518 and therefore lacking almost the entire cytoplasmic domain. These cells adhered and spread on VWF-coated microposts, but generated minimal contractile force.Together, these results indicate that the GPIb-IX-V complex is able to transmit cytoskeletal contractile forces to its ligand, VWF, in a process requiring the cytoplasmic domain of GPIbα. This is the first example of a non-integrin transmitting force to an external substrate. Disclosures:Sniadecki:Stasys Medical Corporation: Equity Ownership, Founder Other.

Stochastic dynamics of small ensembles of non-processive molecular motors: The parallel cluster model

Non-processive molecular motors have to work together in ensembles in order to generate appreciable levels of force or movement. In skeletal muscle, for example, hundreds of myosin II molecules cooperate in thick filaments. In non-muscle cells, by contrast, small groups with few tens of non-muscle myosin II motors contribute to essential cellular processes such as transport, shape changes, or mechanosensing. Here we introduce a detailed and analytically tractable model for this important situation. Using a three-state crossbridge model for the myosin II motor cycle and exploiting the assumptions of fast power stroke kinetics and equal load sharing between motors in equivalent states, we reduce the stochastic reaction network to a one-step master equation for the binding and unbinding dynamics (parallel cluster model) and derive the rules for ensemble movement. We find that for constant external load, ensemble dynamics is strongly shaped by the catch bond character of myosin II, which leads to an increase of the fraction of bound motors under load and thus to firm attachment even for small ensembles. This adaptation to load results in a concave force-velocity relation described by a Hill relation. For external load provided by a linear spring, myosin II ensembles dynamically adjust themselves towards an isometric state with constant average position and load. The dynamics of the ensembles is now determined mainly by the distribution of motors over the different kinds of bound states. For increasing stiffness of the external spring, there is a sharp transition beyond which myosin II can no longer perform the power stroke. Slow unbinding from the pre-power-stroke state protects the ensembles against detachment.

The N-terminal Flanking Region of the A1 Domain Regulates the Force-dependent Binding of von Willebrand Factor to Platelet Glycoprotein Ibα

Binding of platelet glycoprotein Ibα (GPIbα) to von Willebrand factor (VWF) initiates platelet adhesion to disrupted vascular surface under arterial blood flow. Flow exerts forces on the platelet that are transmitted to VWF-GPIbα bonds, which regulate their dissociation. Mutations in VWF and/or GPIbα may alter the mechanical regulation of platelet adhesion to cause hemostatic defects as found in patients with von Willebrand disease (VWD). Using a biomembrane force probe, we observed biphasic force-decelerated (catch) and force-accelerated (slip) dissociation of GPIbα from VWF. The VWF A1 domain that contains the N-terminal flanking sequence Gln(1238)-Glu(1260) (1238-A1) formed triphasic slip-catch-slip bonds with GPIbα. By comparison, using a short form of A1 that deletes this sequence (1261-A1) abolished the catch bond, destabilizing its binding to GPIbα at high forces. Importantly, shear-dependent platelet rolling velocities on these VWF ligands in a flow chamber system mirrored the force-dependent single-bond lifetimes. Adding the Gln(1238)-Glu(1260) peptide, which interacted with GPIbα and 1261-A1 but not 1238-A1, to whole blood decreased platelet attachment under shear stress. Soluble Gln(1238)-Glu(1260) reduced the lifetimes of GPIbα bonds with VWF and 1238-A1 but rescued the catch bond of GPIbα with 1261-A1. A type 2B VWD 1238-A1 mutation eliminated the catch bond by prolonging lifetimes at low forces, a type 2M VWD 1238-A1 mutation shifted the respective slip-catch and catch-slip transition points to higher forces, whereas a platelet type VWD GPIbα mutation enhanced the bond lifetime in the entire force regime. These data reveal the structural determinants of VWF activation by hemodynamic force of the circulation.

Cytoskeletal function in the immune system

Cytoskeletal function in the immune system

Macrophages lift off surface-bound bacteria using a filopodium-lamellipodium hook-and-shovel mechanism

To clear pathogens from host tissues or biomaterial surfaces, phagocytes have to break the adhesive bacteria-substrate interactions. Here we analysed the mechanobiological process that enables macrophages to lift-off and phagocytose surface-bound Escherichia coli (E. coli). In this opsonin-independent process, macrophage filopodia hold on to the E. coli fimbriae long enough to induce a local protrusion of a lamellipodium. Specific contacts between the macrophage and E. coli are formed via the glycoprotein CD48 on filopodia and the adhesin FimH on type 1 fimbriae (hook). We show that bacterial detachment from surfaces occurrs after a lamellipodium has protruded underneath the bacterium (shovel), thereby breaking the multiple bacterium-surface interactions. After lift-off, the bacterium is engulfed by a phagocytic cup. Force activated catch bonds enable the long-term survival of the filopodium-fimbrium interactions while soluble mannose inhibitors and CD48 antibodies suppress the contact formation and thereby inhibit subsequent E. coli phagocytosis.

Mechanochemitry: A Molecular Biomechanics View of Mechanosensing

Molecular biomechanics includes two themes: the study of mechanical aspects of biomolecules and the study of molecular biology of the cell using mechanical tools. The two themes are interconnected for obvious reasons. The present review focuses on one of the interconnected areas-the mechanical regulation of molecular interaction and conformational change. Recent conceptual developments are summarized, including catch bonds, regulation of molecular interaction by the history of force application, and cyclic mechanical reinforcement. These studies elucidate the mechanochemistry of some of the candidate mechanosensing molecules, thereby providing a natural connection to mechanobiology.

Catch Me Because You Can: A Mathematical Model for Mechanosensing

Catch Me Because You Can: A Mathematical Model for Mechanosensing