A Computational Study of Stress Fiber-Focal Adhesion Dynamics Governing Cell Contractility

We apply a recently developed model of cytoskeletal force generation to study a cell’s intrinsic contractility, as well as its response to external loading. The model is based on a nonequilibrium thermodynamic treatment of the mechanochemistry governing force in the stress fiber-focal adhesion system. Our computational study suggests that the mechanical coupling between the stress fibers and focal adhesions leads to a complex, dynamic, mechanochemical response. We collect the results in response maps whose regimes are distinguished by the initial geometry of the stress fiber-focal adhesion system, and by the external load on the cell. The results from our model connect qualitatively with recent studies on the force response of smooth muscle cells on arrays of polymeric microposts.     

Prestress and Adhesion Site Dynamics Control Cell Sensitivity to Extracellular Stiffness

This study aims at improving the understanding of mechanisms responsible for cell sensitivity to extracellular environment. We explain how substrate mechanical properties can modulate the force regulation of cell sensitive elements primarily adhesion sites. We present a theoretical and experimental comparison between two radically different approaches of the force regulation of adhesion sites that depends on their either stationary or dynamic behavior. The most classical stationary model fails to predict cell sensitivity to substrate stiffness whereas the dynamic model predicts extracellular stiffness dependence. This is due to a time dependent reaction force in response to actomyosin traction force exerted on cell sensitive elements. We purposely used two cellular models, i.e., alveolar epithelial cells and alveolar macrophages exhibiting respectively stationary and dynamic adhesion sites, and compared their sensitivity to theoretical predictions. Mechanical and structural results show that alveolar epithelial cells exhibit significant prestress supported by evident stress fibers and lacks sensitivity to substrate stiffness. On the other hand, alveolar macrophages exhibit low prestress and exhibit sensitivity to substrate stiffness. Altogether, theory and experiments consistently show that adhesion site dynamics and cytoskeleton prestress control cell sensitivity to extracellular environment with an optimal sensitivity expected in the intermediate range.     

Model of coupled transient changes of Rac, Rho, adhesions and stress fibers alignment in endothelial cells responding to shear stress

Interactions of cell adhesions, Rho GTPases and actin in the endothelial cells' response to external forces are complex and not fully understood, but a qualitative understanding of the mechanosensory response begins to emerge. Here, we formulate a mathematical model of the coupled dynamics of cell adhesions, small GTPases Rac and Rho and actin stress fibers guiding a directional reorganization of the actin cytoskeleton. The model is based on the assumptions that the interconnected cytoskeleton transfers the shear force to the adhesion sites, which in turn transduce the force into a chemical signal that activates integrins at the basal surface of the cell. Subsequently, activated and ligated integrins signal and transiently de-activate Rho, causing the disassembly of actin stress fibers and inhibiting the maturation of focal complexes into focal contacts. Focal complexes and ligated integrins activate Rac, which in turn enhances focal complex assembly. When Rho activity recovers, stress fibers re-assemble and promote the maturation of focal complexes into focal contacts. Merging stress fibers self-align, while the elevated level of Rac activity at the downstream edge of the cell is translated into an alignment of the cells and the newly forming stress fibers in the flow direction. Numerical solutions of the model equations predict transient changes in Rac and Rho that compare well with published experimental results. We report quantitative data on early alignment of the stress fibers and its dependence on cell shape that agrees with the model.     

Stability of adhesion clusters and cell reorientation under lateral cyclic tension

This work is motivated by experimental observations that cells on stretched substrate exhibit different responses to static and dynamic loads. A model of focal adhesion that can consider the mechanics of stress fiber, adhesion bonds, and substrate was developed at the molecular level by treating the focal adhesion as an adhesion cluster. The stability of the cluster under dynamic load was studied by applying cyclic external strain on the substrate. We show that a threshold value of external strain amplitude exists beyond which the adhesion cluster disrupts quickly. In addition, our results show that the adhesion cluster is prone to losing stability under high-frequency loading, because the receptors and ligands cannot get enough contact time to form bonds due to the high-speed deformation of the substrate. At the same time, the viscoelastic stress fiber becomes rigid at high frequency, which leads to significant deformation of the bonds. Furthermore, we find that the stiffness and relaxation time of stress fibers play important roles in the stability of the adhesion cluster. The essence of this work is to connect the dynamics of the adhesion bonds (molecular level) with the cell's behavior during reorientation (cell level) through the mechanics of stress fiber. The predictions of the cluster model are consistent with experimental observations.     

Inhibition of Intercellular Adhesion in Herpex Simplex Virus Infection by Glycyrrhizin

Herpes simplex virus (HSV) is one of the most common viruses infecting humans and animals. Cellular adhesion is increased in HSV and plays a role in pathogenesis of inflammatory response during this viral infection. In our study, we studied a potential role of glycyrrhizin in disrupting cellular adhesion in HSV. We isolated rat cerebral capillary vessel endothelial cells (CCECs) and polymorphonuclear leukocytes (PMN) and evaluated intercellular adhesion between these cells by micropipette aspiration technique. The adhesion force and stress between CCEC and PMN were significantly (P < 0.01) increased in HSV infection. Glycyrrhizin perfusion significantly (P < 0.01) reduced adhesion force and stress between CCEC and PMN. In conclusion, glycyrrhizin may attenuate inflammatory responses in HSV by inhibition of adhesion between CCEC and PMN.     

Direct detection of cellular adaptation to local cyclic stretching at the single cell level by atomic force microscopy

The cellular response to external mechanical forces has important effects on numerous biological phenomena. The sequences of molecular events that underlie the observed changes in cellular properties have yet to be elucidated in detail. Here we have detected the responses of a cultured cell against locally applied cyclic stretching and compressive forces, after creating an artificial focal adhesion under a glass bead attached to the cantilever of an atomic force microscope. The cell tension initially increased in response to the tensile stress and then decreased within ∼1 min as a result of viscoelastic properties of the cell. This relaxation was followed by a gradual increase in tension extending over several minutes. The slow recovery of tension ceased after several cycles of force application. This tension-recovering activity was inhibited when cells were treated with cytochalasin D, an inhibitor of actin polymerization, or with (−)-blebbistatin, an inhibitor of myosin II ATPase activity, suggesting that the activity was driven by actin-myosin interaction. To our knowledge, this is the first quantitative analysis of cellular mechanical properties during the process of adaptation to locally applied cyclic external force.     

A theoretical model study of the influence of fluid stresses on a cell adhering to a microchannel wall.

We predict the amplification of mechanical stress, force, and torque on an adherent cell due to flow within a narrow microchannel. We model this system as a semicircular bulge on a microchannel wall, with pressure-driven flow. This two-dimensional model is solved computationally by the boundary element method. Algebraic expressions are developed by using forms suggested by lubrication theory that can be used simply and accurately to predict the fluid stress, force, and torque based upon the fluid viscosity, muoffhannel height, H, cell size, R, and flow rate per unit width, Q2-d. This study shows that even for the smallest cells (gamma = R/H << 1), the stress, force, and torque can be significantly greater than that predicted based on flow in a cell-free system. Increased flow resistance and fluid stress amplification occur with bigger cells (gamma > 0.25), because of constraints by the channel wall. In these cases we find that the shear stress amplification is proportional to Q2-d(1-gamma)-2, and the force and torque are proportional to Q2-d(1-gamma2)-5/2. Finally, we predict the fluid mechanical influence on three-dimensional immersed objects. These algebraic expressions have an accuracy of approximately 10% for flow in channels and thus are useful for the analysis of cells in flow chambers. For cell adhesion in tubes, the approximations are accurate to approximately 25% when gamma > 0.5. These calculations may thus be used to simply predict fluid mechanical interactions with cells in these constrained settings. Furthermore, the modeling approach may be useful in understanding more complex systems that include cell deformability and cell-cell interactions.     

The mechanochemistry of cytoskeletal force generation

In this communication, we propose a model to study the non-equilibrium process by which actin stress fibers develop force in contractile cells. The emphasis here is on the non-equilibrium thermodynamics, which is necessary to address the mechanics as well as the chemistry of dynamic cell contractility. In this setting, we are able to develop a framework that relates (a) the dynamics of force generation within the cell and (b) the cell’s response to external stimuli to the chemical processes occurring within the cell, as well as to the mechanics of linkage between the stress fibers, focal adhesions and extracellular matrix.     

A Mechanochemical Model of Cell Reorientation on Substrates under Cyclic Stretch

We report a theoretical study on the cyclic stretch-induced reorientation of spindle-shaped cells. Specifically, by taking into account the evolution of sub-cellular structures like the contractile stress fibers and adhesive receptor-ligand clusters, we develop a mechanochemical model to describe the dynamics of cell realignment in response to cyclically stretched substrates. Our main hypothesis is that cells tend to orient in the direction where the formation of stress fibers is energetically most favorable. We show that, when subjected to cyclic stretch, the final alignment of cells reflects the competition between the elevated force within stress fibers that accelerates their disassembly and the disruption of cell-substrate adhesion as well, and an effectively increased substrate rigidity that promotes more stable focal adhesions. Our model predictions are consistent with various observations like the substrate rigidity dependent formation of stable adhesions and the stretching frequency, as well as stretching amplitude, dependence of cell realignment. This theory also provides a simple explanation on the regulation of protein Rho in the formation of stretch-induced stress fibers in cells.     

Characterizing Cell Adhesion by Using Micropipette Aspiration

We have developed a technique to directly quantify cell-substrate adhesion force using micropipette aspiration. The micropipette is positioned perpendicular to the surface of an adherent cell and a constant-rate aspiration pressure is applied. Since the micropipette diameter and the aspiration pressure are our control parameters, we have direct knowledge of the aspiration force, whereas the cell behavior is monitored either in brightfield or interference reflection microscopy. This setup thus allows us to explore a range of geometric parameters, such as projected cell area, adhesion area, or pipette size, as well as dynamical parameters such as the loading rate. We find that cell detachment is a well-defined event occurring at a critical aspiration pressure, and that the detachment force scales with the cell adhesion area (for a given micropipette diameter and loading rate), which defines a critical stress. Taking into account the cell adhesion area, intrinsic parameters of the adhesion bonds, and the loading rate, a minimal model provides an expression for the critical stress that helps rationalize our experimental results.     

拉伸作用对成骨细胞粘附、铺展、粘弹性的影响

采用四点弯曲梁实验装置（自行研制）对离体培养的大鼠成骨细胞,施以拉伸应变影响,通过微管吸吮系统、显微摄录、计算机图像系统了解细胞的粘附、铺展行为和细胞的粘弹性变化,认识细胞形态、粘附力及变形性对机械刺激的响应。发现：（1）机械拉伸2h成骨细胞与基底粘附力以及细胞单位面积粘附力较对照组明显升高,但加载后期与对照无明显;（2）成骨细胞粘弹性较对照组略低;（3）加载24h(500με）实验组细胞增殖比对照组快。机械拉伸有成骨细胞生长,并可通过粘附、铺展调整、削减应变影响。     

Induction Kinetics of a Conditional pH Stress Response System in Escherichia coli

The analysis of stress response systems in microorganisms can reveal molecular strategies for regulatory control and adaptation. In this study, we focused on the Cad module, a subsystem of Escherichia coli’s response to acidic stress that is conditionally activated at low pH only when lysine is available. When expressed, the Cad system counteracts the elevated H⁺ concentration by converting lysine to cadaverine under the consumption of H⁺ and exporting cadaverine in exchange for external lysine. Surprisingly, the cad operon displays a transient response, even when the conditions for its induction persist. To quantitatively characterize the regulation of the Cad module, we experimentally recorded and theoretically modeled the dynamics of important system variables. We established a quantitative model that adequately describes and predicts the transient expression behavior for various initial conditions. Our quantitative analysis of the Cad system supports negative feedback by external cadaverine as the origin of the transient response. Furthermore, the analysis puts causal constraints on the precise mechanism of signal transduction via the regulatory protein CadC.     

The Non-Equilibrium Thermodynamics and Kinetics of Focal Adhesion Dynamics

Background

We consider a focal adhesion to be made up of molecular complexes, each consisting of a ligand, an integrin molecule, and associated plaque proteins. Free energy changes drive the binding and unbinding of these complexes and thereby controls the focal adhesion''s dynamic modes of growth, treadmilling and resorption.

Principal Findings

We have identified a competition among four thermodynamic driving forces for focal adhesion dynamics: (i) the work done during the addition of a single molecular complex of a certain size, (ii) the chemical free energy change associated with the addition of a molecular complex, (iii) the elastic free energy change associated with deformation of focal adhesions and the cell membrane, and (iv) the work done on a molecular conformational change. We have developed a theoretical treatment of focal adhesion dynamics as a nonlinear rate process governed by a classical kinetic model. We also express the rates as being driven by out-of-equilibrium thermodynamic driving forces, and modulated by kinetics. The mechanisms governed by the above four effects allow focal adhesions to exhibit a rich variety of behavior without the need to introduce special constitutive assumptions for their response. For the reaction-limited case growth, treadmilling and resorption are all predicted by a very simple chemo-mechanical model. Treadmilling requires symmetry breaking between the ends of the focal adhesion, and is achieved by driving force (i) above. In contrast, depending on its numerical value (ii) causes symmetric growth, resorption or is neutral, (iii) causes symmetric resorption, and (iv) causes symmetric growth. These findings hold for a range of conditions: temporally-constant force or stress, and for spatially-uniform and non-uniform stress distribution over the FA. The symmetric growth mode dominates for temporally-constant stress, with a reduced treadmilling regime.

Significance

In addition to explaining focal adhesion dynamics, this treatment can be coupled with models of cytoskeleton dynamics and contribute to the understanding of cell motility.     

Kinetic and thermodynamic analyses of adhesion of a peptide,Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm), and human formyl peptide receptor (hFPR)

The kinetic and thermodynamic properties of a peptide–receptor interaction was investigated by measuring the adhesion force in the reaction via atomic force microscopy (AFM). Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm), considered as a model system in the present study, is a potent neutrophil chemo-attractant. Since being identified as an agonist for formyl peptide receptor (FPR), WKYMVm’s high affinity to FPR has been verified through investigation of its kinetic and physiological behaviors via conventional methods. However, there have been no reports on the adhesion force of WKYMVm-FPR. In this research, we measured the adhesion force of WKYMVm-FPR using AFM. Kinetic parameters obtained from the relationship between the adhesion force and loading rate were used to characterize the thermodynamic properties of WKYMVm-hFPR binding.     

Mechanical Principle of Enhancing Cell-Substrate Adhesion via Pre-Tension in the Cytoskeleton

Motivated by our earlier study on the effect of pre-tension in gecko adhesion, here we investigate whether and how pre-tension in cytoskeleton influences cell adhesion by developing a stochastic-elasticity model of a stress fiber attached on a rigid substrate via molecular bonds. By comparing the variations in adhesion lifetime and observing the sequences of bond breaking with and without pre-tension in the stress fiber under the same applied force, we demonstrate that the effect of pre-tension is to shift the interfacial failure mode from cracklike propagation toward uniform bond failure within the contact region, thereby greatly increasing the adhesion lifetime. Since stress fibers are the primary load-bearing components of cells, as well as the basic functional units of cytoskeleton that facilitate cell adhesion, this study suggests a feasible mechanism by which cell adhesion could be actively controlled via cytoskeletal contractility and proposes that pre-tension may be a general principle in biological adhesion.     

Force-induced adsorption and anisotropic growth of focal adhesions

Focal adhesions are micrometer-sized protein aggregates that connect actin stress fibers to the extracellular matrix, a network of macromolecules surrounding tissue cells. The actin fibers are under tension due to actin-myosin contractility. Recent measurements have shown that as the actin force is increased, these adhesions grow in size and in the direction of the force. This is in contrast to the growth of condensed domains of surface-adsorbed molecules in which the dynamics are isotropic. We predict these force-sensitive, anisotropic dynamics of focal adhesions from a model for the adsorption of proteins from the cytoplasm to the adhesion site. Our theory couples the mechanical forces and elasticity to the adsorption dynamics via force-induced conformational changes of molecular-sized mechanosensors located in the focal adhesion. We predict the velocity of both the front and back of the adhesion as a function of the applied force. In addition, our results show that the relative motion of the front and back of the adhesion is asymmetric and in different ranges of forces, the adhesion can either shrink or grow in the direction of the force.     

机械应力对巨噬细胞的功能调节作用及机制

巨噬细胞作为先天免疫系统识别外界环境刺激的先锋,能够响应环境细微变化对自身功能实现适应性调节,在维持体内稳态和抵抗感染方面起关键作用。生理或病理组织微环境存在多种机械应力刺激,对巨噬细胞的免疫功能具有重要影响。本文围绕机械应力对巨噬细胞的功能调节,从影响细胞行为和极化的角度综述,总结巨噬细胞机械应力感知及转导的分子机制,展望巨噬细胞力学生物学研究在组织工程、再生医学以及肿瘤免疫治疗领域的应用前景,为深入理解巨噬细胞功能可塑性提供有力支持。     

Three-dimensional model of the human craniofacial skeleton: method and preliminary results using finite element analysis

The purpose of this study was to develop a three-dimensional finite element model of the craniofacial skeleton using a dry human skull. The model consisted of 2918 nodes and 1776 solid elements, and was used to investigate the biomechanical effect of a distally directed orthopaedic force on the craniofacial complex. The force was applied at the level of the maxillary first molar. The results indicated that in response to the force system applied: the nasomaxillary complex displaces in a backward and downward direction and rotates in clockwise sense; the nasomaxillary complex, including the zygomatic bone, experiences high stress levels in comparison with those at the remaining bones; the stress distribution in the maxillary basal bone area is relatively uniform; and the stress distribution across the opposing surface of the bony margins of the sutures is non-uniform.     

Binding Kinetics of Bisintercalator Triostin A with Optical Tweezers Force Mechanics

The binding kinetics of the intercalative binding of Triostin A to λ-DNA was investigated by measuring the force extension response of the DNA-ligand complexes with an optical tweezers system. These force response curves, containing the information about different binding properties, were analyzed based on a recent method (put forth by another research group) for monointercalators that was extended to bisintercalators. Our binding analysis reveals an exponential dependence of the association constant on the applied external force as well as a decreasing binding site size. In general, our results are in agreement with those for the monointercalator ethidium. However, to explain the high-force binding site size, a new model for bisintercalation of Triostin A at high forces is proposed.     

FAP52 regulates actin organization via binding to filamin.

FAP52, a focal adhesion-associated phosphoprotein, is a member of a FAP52/PACSIN/syndapin family of proteins. They share a multidomain structure and are implicated in actin-based and endocytotic functions. We show, by using both native and recombinant proteins, that FAP52 selectively binds to the actin cross-linking protein filamin (ABP-280). This was based on an affinity purification followed by a sequence determination by mass spectrometry, co-immunoprecipitation, overlay binding, and surface plasmon resonance analysis. Binding studies with deletion mutants showed that the sites of the interaction map to the highly alpha-helical N-terminal part of FAP52 and to the C-terminal region of filamin, which also contains binding sites to some transmembrane signaling proteins. In immunofluorescence and immunoelectron microscopy of cultured fibroblasts, a different overall subcellular distribution was seen for filamin and FAP52 except for a stress fiber-focal adhesion junction where they showed a notable overlap. Overexpression of the full-length and mutant forms of FAP52 led to an extensive reorganization of actin and filamin in cultured fibroblasts. Thus, the results show that FAP52 interacts with filamin, and we propose that this interaction is important in linking and coordinating the events between focal adhesions and the actin cytoskeleton.