Intracellular stress transmission through actin stress fiber network in adherent vascular cells

Intracellular stress transmission through subcellular structural components has been proposed to affect activation of localized mechano-sensing sites such as focal adhesions in adherent cells. Previous studies reported that physiological extracellular forces produced heterogeneous spatial distributions of cytoplasmic strain. However, mechanical signaling pathway involved in intracellular force transmission through basal actin stress fibers (SFs), a mechano-responsive cytoskeletal structure, remains elusive. In the present study, we investigated force balance within the basal SFs of cultured smooth muscle cells and endothelial cells by (i) removing the cell membrane and cytoplasmic constituents except for materials physically attaching to the substrate (i.e., SF-focal adhesion complexities) or (ii) dislodging either mechanically or chemically the cell processes of the cells expressing fluorescent proteins-labeled actin and focal adhesions in order, to examine stress-release-induced deformation of the basal SFs. The result showed that a removal of mechanical restrictions for SFs resulted in a decrease in the length of the remaining SFs, which means SFs bear tension. In addition, a release of the preexisting tension in a single SF was transmitted to another SF physically linked to the former, but not transmitted to the other ones physically independent of the former, suggesting that the prestress is balanced in tensed SF networks. These results support a hypothesis regarding cell structural architecture that physiological extracellular forces can produce in the basal SF network a directional intracellular stress or strain distribution. Therefore, consideration of the coexistence of the directional stretching strain along the axial direction of SFs and the heterogeneous strain in the other cytoplasmic region will be essential for understanding intracellular stress transmission in the adherent cells.     

Integrins and extracellular matrix in mechanotransduction

Integrins bind extracellular matrix fibrils and associate with intracellular actin filaments through a variety of cytoskeletal linker proteins to mechanically connect intracellular and extracellular structures. Each component of the linkage from the cytoskeleton through the integrin-mediated adhesions to the extracellular matrix therefore transmits forces that may derive from both intracellular, myosin-generated contractile forces and forces from outside the cell. These forces activate a wide range of signaling pathways and genetic programs to control cell survival, fate, and behavior. Additionally, cells sense the physical properties of their surrounding environment through forces exerted on integrin-mediated adhesions. This article first summarizes current knowledge about regulation of cell function by mechanical forces acting through integrin-mediated adhesions and then discusses models for mechanotransduction and sensing of environmental forces.     

Crosstalk between Cell Adhesion Complexes in Regulation of Mechanotransduction

Physical forces regulate numerous biological processes during development, physiology, and pathology. Forces between the external environment and intracellular actin cytoskeleton are primarily transmitted through integrin-containing focal adhesions and cadherin-containing adherens junctions. Crosstalk between these complexes is well established and modulates the mechanical landscape of the cell. However, integrins and cadherins constitute large families of adhesion receptors and form multiple complexes by interacting with different ligands, adaptor proteins, and cytoskeletal filaments. Recent findings indicate that integrin-containing hemidesmosomes oppose force transduction and traction force generation by focal adhesions. The cytolinker plectin mediates this crosstalk by coupling intermediate filaments to the actin cytoskeleton. Similarly, cadherins in desmosomes might modulate force generation by adherens junctions. Moreover, mechanotransduction can be influenced by podosomes, clathrin lattices, and tetraspanin-enriched microdomains. This review discusses mechanotransduction by multiple integrin- and cadherin-based cell adhesion complexes, which together with the associated cytoskeleton form an integrated network that allows cells to sense, process, and respond to their physical environment.     

The consequence of substrates of large-scale rigidity on actin network tension in adherent cells

Abstract

There is compelling evidence that substrate stiffness affects cell adhesion as well as cytoskeleton organization and contractile activity. This work was designed to study the cytoskeletal contractile activity of single cells plated on micropost substrates of different stiffness using a numerical model simulating the intracellular tension of individual cells. We allowed cells to adhere onto micropost substrates of various rigidities and used experimental traction force data to infer cell contractility using a numerical model. The model shows that higher substrate stiffness leads to an increase in intracellular tension. The strength of this model is its ability to calculate the mechanical state of each cell in accordance to its individual cytoskeletal structure. This is achieved by regenerating a numerical cytoskeleton based on microscope images of the actin network of each cell. The resulting numerical structure consequently represents pulling characteristics on its environment similar to those generated by the cell in-vivo. From actin imaging we can calculate and better understand how forces are transmitted throughout the cell.     

Retrograde fluxes of focal adhesion proteins in response to cell migration and mechanical signals

Recent studies suggest that mechanical signals mediated by the extracellular matrix play an essential role in various physiological and pathological processes; yet, how cells respond to mechanical stimuli remains elusive. Using live cell fluorescence imaging, we found that actin filaments, in association with a number of focal adhesion proteins, including zyxin and vasodilator-stimulated phosphoprotein, undergo retrograde fluxes at focal adhesions in the lamella region. This flux is inversely related to cell migration, such that it is amplified in fibroblasts immobilized on micropatterned islands. In addition, the flux is regulated by mechanical signals, including stretching forces applied to flexible substrates and substrate stiffness. Conditions favoring the flux share the common feature of causing large retrograde displacements of the interior actin cytoskeleton relative to the substrate anchorage site, which may function as a switch translating mechanical input into chemical signals, such as tyrosine phosphorylation. In turn, the stimulation of actin flux at focal adhesions may function as part of a feedback mechanism, regulating structural assembly and force production in relation to cell migration and mechanical load. The retrograde transport of associated focal adhesion proteins may play additional roles in delivering signals from focal adhesions to the interior of the cell.     

Marching at the front and dragging behind: differential alphaVbeta3-integrin turnover regulates focal adhesion behavior.

Integrins are cell-substrate adhesion molecules that provide the essential link between the actin cytoskeleton and the extracellular matrix during cell migration. We have analyzed alphaVbeta3-integrin dynamics in migrating cells using a green fluorescent protein-tagged beta3-integrin chain. At the cell front, adhesion sites containing alphaVbeta3-integrin remain stationary, whereas at the rear of the cell they slide inward. The integrin fluorescence intensity within these different focal adhesions, and hence the relative integrin density, is directly related to their mobility. Integrin density is as much as threefold higher in sliding compared with stationary focal adhesions. High intracellular tension under the control of RhoA induced the formation of high-density contacts. Low-density adhesion sites were induced by Rac1 and low intracellular tension. Photobleaching experiments demonstrated a slow turnover of beta3-integrins in low-density contacts, which may account for their stationary nature. In contrast, the fast beta3-integrin turnover observed in high-density contacts suggests that their apparent sliding may be caused by a polarized renewal of focal contacts. Therefore, differential acto-myosin-dependent integrin turnover and focal adhesion densities may explain the mechanical and behavioral differences between cell adhesion sites formed at the front, and those that move in the retracting rear of migrating cells.     

Geometry Regulates Traction Stresses in Adherent Cells

Cells generate mechanical stresses via the action of myosin motors on the actin cytoskeleton. Although the molecular origin of force generation is well understood, we currently lack an understanding of the regulation of force transmission at cellular length scales. Here, using 3T3 fibroblasts, we experimentally decouple the effects of substrate stiffness, focal adhesion density, and cell morphology to show that the total amount of work a cell does against the substrate to which it is adhered is regulated by the cell spread area alone. Surprisingly, the number of focal adhesions and the substrate stiffness have little effect on regulating the work done on the substrate by the cell. For a given spread area, the local curvature along the cell edge regulates the distribution and magnitude of traction stresses to maintain a constant strain energy. A physical model of the adherent cell as a contractile gel under a uniform boundary tension and mechanically coupled to an elastic substrate quantitatively captures the spatial distribution and magnitude of traction stresses. With a single choice of parameters, this model accurately predicts the cell’s mechanical output over a wide range of cell geometries.     

Geometry Regulates Traction Stresses in Adherent Cells

Cells generate mechanical stresses via the action of myosin motors on the actin cytoskeleton. Although the molecular origin of force generation is well understood, we currently lack an understanding of the regulation of force transmission at cellular length scales. Here, using 3T3 fibroblasts, we experimentally decouple the effects of substrate stiffness, focal adhesion density, and cell morphology to show that the total amount of work a cell does against the substrate to which it is adhered is regulated by the cell spread area alone. Surprisingly, the number of focal adhesions and the substrate stiffness have little effect on regulating the work done on the substrate by the cell. For a given spread area, the local curvature along the cell edge regulates the distribution and magnitude of traction stresses to maintain a constant strain energy. A physical model of the adherent cell as a contractile gel under a uniform boundary tension and mechanically coupled to an elastic substrate quantitatively captures the spatial distribution and magnitude of traction stresses. With a single choice of parameters, this model accurately predicts the cell’s mechanical output over a wide range of cell geometries.     

Dynamic and structural signatures of lamellar actomyosin force generation

The regulation of cellular traction forces on the extracellular matrix is critical to cell adhesion, migration, proliferation, and differentiation. Diverse lamellar actin organizations ranging from contractile lamellar networks to stress fibers are observed in adherent cells. Although lamellar organization is thought to reflect the extent of cellular force generation, understanding of the physical behaviors of the lamellar actin cytoskeleton is lacking. To elucidate these properties, we visualized the actomyosin dynamics and organization in U2OS cells over a broad range of forces. At low forces, contractile lamellar networks predominate and force generation is strongly correlated to actomyosin retrograde flow dynamics with nominal change in organization. Lamellar networks build ～60% of cellular tension over rapid time scales. At high forces, reorganization of the lamellar network into stress fibers results in moderate changes in cellular tension over slower time scales. As stress fibers build and tension increases, myosin band spacing decreases and α-actinin bands form. On soft matrices, force generation by lamellar networks is unaffected, whereas tension-dependent stress fiber assembly is abrogated. These data elucidate the dynamic and structural signatures of the actomyosin cytoskeleton at different levels of tension and set a foundation for quantitative models of cell and tissue mechanics.     

Cytoskeletal regulation of pulmonary vascular permeability.

The endothelial cell (EC) lining of the pulmonary vasculature forms a semipermeable barrier between the blood and the interstitium of the lung. Disruption of this barrier occurs during inflammatory disease states such as acute lung injury and acute respiratory distress syndrome and results in the movement of fluid and macromolecules into the interstitium and pulmonary air spaces. These processes significantly contribute to the high morbidity and mortality of patients afflicted with acute lung injury. The critical importance of pulmonary vascular barrier function is shown by the balance between competing EC contractile forces, which generate centripetal tension, and adhesive cell-cell and cell-matrix tethering forces, which regulate cell shape. Both competing forces in this model are intimately linked through the endothelial cytoskeleton, a complex network of actin microfilaments, microtubules, and intermediate filaments, which combine to regulate shape change and transduce signals within and between EC. A key EC contractile event in several models of agonist-induced barrier dysfunction is the phosphorylation of regulatory myosin light chains catalyzed by Ca(2+)/calmodulin-dependent myosin light chain kinase and/or through the activity of the Rho/Rho kinase pathway. Intercellular contacts along the endothelial monolayer consist primarily of two types of complexes (adherens junctions and tight junctions), which link to the actin cytoskeleton to provide both mechanical stability and transduction of extracellular signals into the cell. Focal adhesions provide additional adhesive forces in barrier regulation by forming a critical bridge for bidirectional signal transduction between the actin cytoskeleton and the cell-matrix interface. Increasingly, the effects of mechanical forces such as shear stress and ventilator-induced stretch on EC barrier function are being recognized. The critical role of the endothelial cytoskeleton in integrating these multiple aspects of pulmonary vascular permeability provides a fertile area for the development of clinically important barrier-modulating therapies.     

Mechanotransduction and focal adhesions

Cellular FAs (focal adhesions) respond to internal and external mechanical stresses which make them prime candidates for mechanotransduction. Recent observations showed that the FA proteins including vinculin, FAK (FA kinase) and p130Cas are crucial for the ability of cells to transmit forces and to generate cytoskeletal tension. When mechanically stimulated, cells respond by modulating the spreading area, remodel the actin cytoskeleton, activate actomyosin interactions, recruit integrins and reinforce FAs and cytoskeletal structures. These complex cellular responses are orchestrated such that mechanical stresses within the FA complex remained within a narrow range.     

Protein Kinase C,Focal Adhesions and the Regulation of Cell Migration

Cell adhesion to extracellular matrix is a complex process involving protrusive activity driven by the actin cytoskeleton, engagement of specific receptors, followed by signaling and cytoskeletal organization. Thereafter, contractile and endocytic/recycling activities may facilitate migration and adhesion turnover. Focal adhesions, or focal contacts, are widespread organelles at the cell-matrix interface. They arise as a result of receptor interactions with matrix ligands, together with clustering. Recent analysis shows that focal adhesions contain a very large number of protein components in their intracellular compartment. Among these are tyrosine kinases, which have received a great deal of attention, whereas the serine/threonine kinase protein kinase C has received much less. Here the status of protein kinase C in focal adhesions and cell migration is reviewed, together with discussion of its roles and potential substrates.     

Direct,dynamic assessment of cell-matrix interactions inside fibrillar collagen lattices

Cell mechanical behavior has traditionally been studied using 2-D planar elastic substrates. The goal of this study was to directly assess cell-matrix mechanical interactions inside more physiologic 3-D collagen matrices. Rabbit corneal fibroblasts transfected to express GFP-zyxin were plated at low density inside 100 micro m-thick type I collagen matrices. 3-D datasets of isolated cells were acquired at 1-3-min intervals for up to 5 h using fluorescent and Nomarski DIC imaging. Unlike cells on 2-D substrates, cells inside the collagen matrices had a bipolar morphology with thin pseudopodial processes, and without lamellipodia. The organization of the collagen fibrils surrounding each cell was clearly visualized using DIC. Using time-lapse color overlays of GFP and DIC images, displacement and/or realignment of collagen fibrils by focal adhesions could be directly visualized. During pseudopodial extension, new focal adhesions often formed in a line along collagen fibrils in front of the cell, while existing adhesions moved backward. This process generated tractional forces as indicated by the pulling in of collagen fibrils in front of the cell. Meanwhile, adhesions on both the dorsal and ventral surface of the cell body generally moved forward, resulting in contractile shortening along the pseudopodia and localized extracellular matrix (ECM) compression. Cytochalasin D induced rapid disassembly of focal adhesions, cell elongation, and ECM relaxation. This experimental model allows direct, dynamic assessment of cell-matrix interactions inside a 3-D fibrillar ECM. The data suggest that adhesions organize along actin-based contractile elements that are much less complex than the network of actin filaments that mechanically links lamellar adhesions on 2-D substrates.     

The novel actin/focal adhesion-associated protein MISP is involved in mitotic spindle positioning in human cells

Accurate mitotic spindle positioning is essential for the regulation of cell fate choices, cell size and cell position within tissues. The most prominent model of spindle positioning involves a cortical pulling mechanism, where the minus end-directed microtubule motor protein dynein is attached to the cell cortex and exerts pulling forces on the plus ends of astral microtubules that reach the cortex. In nonpolarized cultured cells integrin-dependent, retraction fiber-mediated cell adhesion is involved in spindle orientation. Proteins serving as intermediaries between cortical actin or retraction fibers and astral microtubules remain largely unknown. In a recent genome-wide RNAi screen we identified a previously uncharacterized protein, MISP (C19ORF21) as being involved in centrosome clustering, a process leading to the clustering of supernumerary centrosomes in cancer cells into a bipolar mitotic spindle array by microtubule tension. Here, we show that MISP is associated with the actin cytoskeleton and focal adhesions and is expressed only in adherent cell types. During mitosis MISP is phosphorylated by Cdk1 and localizes to retraction fibers. MISP interacts with the +TIP EB1 and p150^glued, a subunit of the dynein/dynactin complex. Depletion of MISP causes mitotic arrest with reduced tension across sister kinetochores, chromosome misalignment and spindle multipolarity in cancer cells with supernumerary centrosomes. Analysis of spindle orientation revealed that MISP depletion causes randomization of mitotic spindle positioning relative to cell axes and cell center. Together, we propose that MISP links microtubules to the actin cytoskeleton and focal adhesions in order to properly position the mitotic spindle.     

A helping hand: How vinculin contributes to cell-matrix and cell-cell force transfer

Vinculin helps cells regulate and respond to mechanical forces. It is a scaffolding protein that tightly regulates its interactions with potential binding partners within adhesive structures—including focal adhesions that link the cell to the extracellular matrix and adherens junctions that link cells to each other—that physically connect the force-generating actin cytoskeleton (CSK) with the extracellular environment. This tight control of binding partner interaction—mediated by vinculin's autoinhibitory head–tail interaction—allows vinculin to rapidly interact and detach in response to changes in the dynamic forces applied through the cell. In doing so, vinculin modulates the structural composition of focal adhesions and the cell's ability to generate traction forces and adhesion strength. Recent evidence suggests that vinculin plays a similar role in regulating the fate and function of cell–cell junctions, further underscoring the importance of this protein. Using our lab's recent work as a starting point, this commentary explores several outstanding questions regarding the nature of vinculin activation and its function within focal adhesions and adherens junctions.     

Stretch-activated force shedding, force recovery, and cytoskeletal remodeling in contractile fibroblasts

The stress fiber network within contractile fibroblasts structurally reinforces and provides tension, or "tone", to tissues such as those found in healing wounds. Stress fibers have previously been observed to polymerize in response to mechanical forces. We observed that, when stretched sufficiently, contractile fibroblasts diminished the mechanical tractions they exert on their environment through depolymerization of actin filaments then restored tissue tension and rebuilt actin stress fibers through staged Ca(++)-dependent processes. These staged Ca(++)-modulated contractions consisted of a rapid phase that ended less than a minute after stretching, a plateau of inactivity, and a final gradual phase that required several minutes to complete. Active contractile forces during recovery scaled with the degree of rebuilding of the actin cytoskeleton. This complementary action demonstrates a programmed regulatory mechanism that protects cells from excessive stretch through choreographed active mechanical and biochemical healing responses.     

Integrating actin dynamics,mechanotransduction and integrin activation: The multiple functions of actin binding proteins in focal adhesions

Focal adhesions are clusters of integrin transmembrane receptors that mechanically couple the extracellular matrix to the actin cytoskeleton during cell migration. Focal adhesions sense and respond to variations in force transmission along a chain of protein-protein interactions linking successively actin filaments, actin binding proteins, integrins and the extracellular matrix to adapt cell-matrix adhesion to the composition and mechanical properties of the extracellular matrix. This review focuses on the molecular mechanisms by which actin binding proteins integrate actin dynamics, mechanotransduction and integrin activation to control force transmission in focal adhesions.     

Mapping the dynamics of shear stress-induced structural changes in endothelial cells

Hemodynamic shear stress regulates endothelial cell biochemical processes that govern cytoskeletal contractility, focal adhesion dynamics, and extracellular matrix (ECM) assembly. Since shear stress causes rapid strain focusing at discrete locations in the cytoskeleton, we hypothesized that shear stress coordinately alters structural dynamics in the cytoskeleton, focal adhesion sites, and ECM on a time scale of minutes. Using multiwavelength four-dimensional fluorescence microscopy, we measured the displacement of rhodamine-fibronectin and green fluorescent protein-labeled actin, vimentin, paxillin, and/or vinculin in aortic endothelial cells before and after onset of steady unidirectional shear stress. In the cytoskeleton, the onset of shear stress increased actin polymerization into lamellipodia, altered the angle of lateral displacement of actin stress fibers and vimentin filaments, and decreased centripetal remodeling of actin stress fibers in subconfluent and confluent cell layers. Shear stress induced the formation of new focal complexes and reduced the centripetal remodeling of focal adhesions in regions of new actin polymerization. The structural dynamics of focal adhesions and the fibronectin matrix varied with cell density. In subconfluent cell layers, shear stress onset decreased the displacement of focal adhesions and fibronectin fibrils. In confluent monolayers, the direction of fibronectin and focal adhesion displacement shifted significantly toward the downstream direction within 1 min after onset of shear stress. These spatially coordinated rapid changes in the structural dynamics of cytoskeleton, focal adhesions, and ECM are consistent with focusing of mechanical stress and/or strain near major sites of shear stress-mediated mechanotransduction.     

A computational model of ameboid deformation and locomotion

Traditional continuum models of ameboid deformation and locomotion are limited by the computational difficulties intrinsic in free boundary conditions. A new model using the immersed boundary method overcomes these difficulties by representing the cell as a force field immersed in fluid domain. The forces can be derived from a direct mechanical interpretation of such cell components as the cell membrane, the actin cortex, and the transmembrane adhesions between the cytoskeleton and the substratum. The numerical cytoskeleton, modeled as a dynamic network of immersed springs, is able to qualitatively model the passive mechanical behavior of a shear-thinning viscoelastic fluid (Bottino 1997). The same network is used to generate active protrusive and contractile forces. When coordinated with the attachment-detachment cycle of the cell's adhesions to the substratum, these forces produce directed locomotion of the model ameba. With this model it is possible to study the effects of altering the numerical parameters upon the motility of the model cell in a manner suggestive of genetic deletion experiments. In the context of this ameboid cell model and its numerical implementation, simulations involving multicellular interaction, detailed internal signaling, and complex substrate geometries are tractable. Received: 5 January 1998 / Revised version: 23 March 1998 / Accepted: 26 March 1998     

Mechanical model of cytoskeleton structuration during cell adhesion and spreading

The biomechanical behavior of an adherent cell is intimately dependent on its cytoskeleton structure. Several models have been proposed to study this structure taking into account its existing internal forces. However, the structural and geometrical complexities of the cytoskeleton's filamentous networks lead to difficulties for determining a biologically realistic architecture. The objective of this paper is to present a mechanical model, combined with a numerical method, devoted to the form-finding of the cytoskeleton structure (shape and internal forces) when a cell adheres on a substrate. The cell is modeled as a granular medium, using rigid spheres (grains) corresponding to intracellular cross-linking proteins and distant mechanical interactions to reproduce the cytoskeleton filament internal forces. At the initial state (i.e., before adhesion), these interactions are tacit. The adhesion phenomenon is then simulated by considering microtubules growing from the centrosome towards transmembrane integrin-like receptors. The simulated cell shape changes in this process and results in a mechanically equilibrated structure with traction and compression forces, in interaction with the substrate reactions. This leads to a compressive microtubule network and a corresponding tensile actin-filament network. The results provide coherent shape and forces information for developing a mechanical model of the cytoskeleton structure, which can be exploitable in future biomechanical studies of adherent cells.