CD98hc (SLC3A2) Participates in Fibronectin Matrix Assembly by Mediating Integrin Signaling

The Journal of Cell Biology. Aug, 2007  |  Pubmed ID: 17682053

Integrin-dependent assembly of the fibronectin (Fn) matrix plays a central role in vertebrate development. We identify CD98hc, a membrane protein, as an important component of the matrix assembly machinery both in vitro and in vivo. CD98hc was not required for biosynthesis of cellular Fn or the maintenance of the repertoire or affinity of cellular Fn binding integrins, which are important contributors to Fn assembly. Instead, CD98hc was involved in the cell's ability to exert force on the matrix and did so by dint of its capacity to interact with integrins to support downstream signals that lead to activation of RhoA small GTPase. Thus, we identify CD98hc as a membrane protein that enables matrix assembly and establish that it functions by interacting with integrins to support RhoA-driven contractility. CD98hc expression can vary widely; our data show that these variations in CD98hc expression can control the capacity of cells to assemble an Fn matrix, a process important in development, wound healing, and tumorigenesis.

High Resolution Traction Force Microscopy Based on Experimental and Computational Advances

Biophysical Journal. Jan, 2008  |  Pubmed ID: 17827246

Cell adhesion and migration crucially depend on the transmission of actomyosin-generated forces through sites of focal adhesion to the extracellular matrix. Here we report experimental and computational advances in improving the resolution and reliability of traction force microscopy. First, we introduce the use of two differently colored nanobeads as fiducial markers in polyacrylamide gels and explain how the displacement field can be computationally extracted from the fluorescence data. Second, we present different improvements regarding standard methods for force reconstruction from the displacement field, which are the boundary element method, Fourier-transform traction cytometry, and traction reconstruction with point forces. Using extensive data simulation, we show that the spatial resolution of the boundary element method can be improved considerably by splitting the elastic field into near, intermediate, and far field. Fourier-transform traction cytometry requires considerably less computer time, but can achieve a comparable resolution only when combined with Wiener filtering or appropriate regularization schemes. Both methods tend to underestimate forces, especially at small adhesion sites. Traction reconstruction with point forces does not suffer from this limitation, but is only applicable with stationary and well-developed adhesion sites. Third, we combine these advances and for the first time reconstruct fibroblast traction with a spatial resolution of approximately 1 microm.

Biophysics. Clutch Dynamics

Science (New York, N.Y.). Dec, 2008  |  Pubmed ID: 19074337

Traction Stress in Focal Adhesions Correlates Biphasically with Actin Retrograde Flow Speed

The Journal of Cell Biology. Dec, 2008  |  Pubmed ID: 19075110

How focal adhesions (FAs) convert retrograde filamentous actin (F-actin) flow into traction stress on the extracellular matrix to drive cell migration is unknown. Using combined traction force and fluorescent speckle microscopy, we observed a robust biphasic relationship between F-actin speed and traction force. F-actin speed is inversely related to traction stress near the cell edge where FAs are formed and F-actin motion is rapid. In contrast, larger FAs where the F-actin speed is low are marked by a direct relationship between F-actin speed and traction stress. We found that the biphasic switch is determined by a threshold F-actin speed of 8-10 nm/s, independent of changes in FA protein density, age, stress magnitude, assembly/disassembly status, or subcellular position induced by pleiotropic perturbations to Rho family guanosine triphosphatase signaling and myosin II activity. Thus, F-actin speed is a fundamental regulator of traction force at FAs during cell migration.

Chapter 19: Mechanical Response of Cytoskeletal Networks

Methods in Cell Biology. 2008  |  Pubmed ID: 19118688

The cellular cytoskeleton is a dynamic network of filamentous proteins, consisting of filamentous actin (F-actin), microtubules, and intermediate filaments. However, these networks are not simple linear, elastic solids; they can exhibit highly nonlinear elasticity and a thermal dynamics driven by ATP-dependent processes. To build quantitative mechanical models describing complex cellular behaviors, it is necessary to understand the underlying physical principles that regulate force transmission and dynamics within these networks. In this chapter, we review our current understanding of the physics of networks of cytoskeletal proteins formed in vitro. We introduce rheology, the technique used to measure mechanical response. We discuss our current understanding of the mechanical response of F-actin networks, and how the biophysical properties of F-actin and actin cross-linking proteins can dramatically impact the network mechanical response. We discuss how incorporating dynamic and rigid microtubules into F-actin networks can affect the contours of growing microtubules and composite network rigidity. Finally, we discuss the mechanical behaviors of intermediate filaments.

Identification and Characterization of a Small Molecule Inhibitor of Formin-mediated Actin Assembly

Chemistry & Biology. Nov, 2009  |  Pubmed ID: 19942139

Formins stimulate actin filament assembly for fundamental cellular processes including division, adhesion, establishing polarity, and motility. A formin inhibitor would be useful because most cells express multiple formins whose functions are not known and because metastatic tumor formation depends on the deregulation of formin-dependent processes. We identified a general small molecule inhibitor of formin homology 2 domains (SMIFH2) by screening compounds for the ability to prevent formin-mediated actin assembly in vitro. SMIFH2 targets formins from evolutionarily diverse organisms including yeast, nematode worm, and mice, with a half-maximal inhibitor concentration of approximately 5 to 15 microM. SMIFH2 prevents both formin nucleation and processive barbed end elongation and decreases formin's affinity for the barbed end. Furthermore, low micromolar concentrations of SMIFH2 disrupt formin-dependent, but not Arp2/3 complex-dependent, actin cytoskeletal structures in fission yeast and mammalian NIH 3T3 fibroblasts.

Optimization of Traction Force Microscopy for Micron-sized Focal Adhesions

Journal of Physics. Condensed Matter : an Institute of Physics Journal. May, 2010  |  Pubmed ID: 20523913

To understand how adherent cells regulate traction forces on their surrounding extracellular matrix (ECM), quantitative techniques are needed to measure forces at the cell-ECM interface. Microcontact printing is used to create a substrate of 1 μm diameter circles of ECM ligand to experimentally study the reconstruction of traction stresses at constrained, point-like focal adhesions. Traction reconstruction with point forces (TRPF) and Fourier transform traction cytometry (FTTC) are used to calculate the traction forces and stress field, respectively, at isolated adhesions. We find that the stress field calculated with FTTC peaks near the center of individual adhesions but propagates several microns beyond the adhesion location. We find the optimal set of FTTC parameters that yield the highest stress magnitude, minimizing information lost from over-smoothing and sampling of the displacement or stress field. A positive correlation between the TRPF and FTTC measurements exists, but integrating the FTTC stress field over the adhesion area yields only a small fraction of the force calculated by TRPF. An effective area similar to that defined by the width of the stress distribution measured with FTTC is required to reconcile these measurements. These measurements set bounds on the spatial resolution and precision of FTTC measurements on micron-sized adhesions.

Transient Frictional Slip Between Integrin and the ECM in Focal Adhesions Under Myosin II Tension

Current Biology : CB. Jul, 2010  |  Pubmed ID: 20541412

The spatiotemporal regulation of adhesion to the extracellular matrix is important in metazoan cell migration and mechanosensation. Although adhesion assembly depends on intracellular and extracellular tension, the biophysical regulation of force transmission between the actin cytoskeleton and extracellular matrix during this process remains largely unknown.

Regulation of Cell Motile Behavior by Crosstalk Between Cadherin- and Integrin-mediated Adhesions

Proceedings of the National Academy of Sciences of the United States of America. Jul, 2010  |  Pubmed ID: 20566866

During normal development and in disease, cohesive tissues undergo rearrangements that require integration of signals from cell adhesions to neighboring cells and to the extracellular matrix (ECM). How a range of cell behaviors is coordinated by these different adhesion complexes is unknown. To analyze epithelial cell motile behavior in response to combinations of cell-ECM and cell-cell adhesion cues, we took a reductionist approach at the single-cell scale by using unique, functionalized micropatterned surfaces comprising alternating stripes of ECM (collagenIV) and adjustable amounts of E-cadherin-Fc (EcadFc). On these surfaces, individual cells spatially segregated integrin- and cadherin-based complexes between collagenIV and EcadFc surfaces, respectively. Cell migration required collagenIV and did not occur on surfaces functionalized with only EcadFc. However, E-cadherin adhesion dampened lamellipodia activity on both collagenIV and EcadFc surfaces and biased the direction of cell migration without affecting the migration rate, all in an EcadFc concentration-dependent manner. Traction force microscopy showed that spatial confinement of integrin-based adhesions to collagenIV stripes induced anisotropic cell traction on collagenIV and migration directional bias. Selective depletion of different pools of alphaE-catenin, an E-cadherin and actin binding protein, identified a membrane-associated pool required for E-cadherin-mediated adhesion and down-regulation of lamellipodia activity and a cytosolic pool that down-regulated the migration rate in an E-cadherin adhesion-independent manner. These results demonstrate that there is crosstalk between E-cadherin- and integrin-based adhesion complexes and that E-cadherin regulates lamellipodia activity and cell migration directionality, but not cell migration rate.

Conserved F-actin Dynamics and Force Transmission at Cell Adhesions

Current Opinion in Cell Biology. Oct, 2010  |  Pubmed ID: 20728328

Adhesions are a central mechanism by which cells mechanically interact with the surrounding extracellular matrix (ECM) and neighboring cells. In both cell-ECM and cell-cell adhesions, forces generated within the actin cytoskeleton are transmitted to the surrounding environment and are essential for numerous morphogenic processes. Despite differences in many molecular components that regulate cell-cell and cell-ECM adhesions, the roles of F-actin dynamics and mechanical forces in adhesion regulation are surprisingly similar. Moreover, force transmission at adhesions occurs concomitantly with dynamic F-actin; proteins comprising the adhesion of F-actin to the plasma membrane must accommodate this movement while still facilitating force transmission. Thus, despite different molecular architectures, integrin and cadherin-mediated adhesions operate with common biophysical characteristics to transmit and respond to mechanical forces in multicellular tissue.

A Zyxin-mediated Mechanism for Actin Stress Fiber Maintenance and Repair

Developmental Cell. Sep, 2010  |  Pubmed ID: 20833360

To maintain mechanical homeostasis, cells must recognize and respond to changes in cytoskeletal integrity. By imaging live cells expressing fluorescently tagged cytoskeletal proteins, we observed that actin stress fibers undergo local, acute, force-induced elongation and thinning events that compromise their stress transmission function, followed by stress fiber repair that restores this capability. The LIM protein zyxin rapidly accumulates at sites of strain-induced stress fiber damage and is essential for stress fiber repair and generation of traction force. Zyxin promotes recruitment of the actin regulatory proteins α-actinin and VASP to compromised stress fiber zones. α-Actinin plays a critical role in restoration of actin integrity at sites of local stress fiber damage, whereas both α-actinin and VASP independently contribute to limiting stress fiber elongation at strain sites, thus promoting stabilization of the stress fiber. Our findings demonstrate a mechanism for rapid repair and maintenance of the structural integrity of the actin cytoskeleton.

Mechanical Integration of Actin and Adhesion Dynamics in Cell Migration

Annual Review of Cell and Developmental Biology. Nov, 2010  |  Pubmed ID: 19575647

Directed cell migration is a physical process that requires dramatic changes in cell shape and adhesion to the extracellular matrix. For efficient movement, these processes must be spatiotemporally coordinated. To a large degree, the morphological changes and physical forces that occur during migration are generated by a dynamic filamentous actin (F-actin) cytoskeleton. Adhesion is regulated by dynamic assemblies of structural and signaling proteins that couple the F-actin cytoskeleton to the extracellular matrix. Here, we review current knowledge of the dynamic organization of the F-actin cytoskeleton in cell migration and the regulation of focal adhesion assembly and disassembly with an emphasis on how mechanical and biochemical signaling between these two systems regulate the coordination of physical processes in cell migration.

Mechanics of the F-actin Cytoskeleton

Journal of Biomechanics. Jan, 2010  |  Pubmed ID: 19913792

Dynamic regulation of the filamentous actin (F-actin) cytoskeleton is critical to numerous physical cellular processes, including cell adhesion, migration and division. Each of these processes require precise regulation of cell shape and mechanical force generation which, to a large degree, is regulated by the dynamic mechanical behaviors of a diverse assortment of F-actin networks and bundles. In this review, we review the current understanding of the mechanics of F-actin networks and identify areas of further research needed to establish physical models. We first review our understanding of the mechanical behaviors of F-actin networks reconstituted in vitro, with a focus on the nonlinear mechanical response and behavior of "active" F-actin networks. We then explore the types of mechanical response measured of cytoskeletal F-actin networks and bundles formed in living cells and identify how these measurements correspond to those performed on reconstituted F-actin networks formed in vitro. Together, these approaches identify the challenges and opportunities in the study of living cytoskeletal matter.

Dynamic and Structural Signatures of Lamellar Actomyosin Force Generation

Molecular Biology of the Cell. Apr, 2011  |  Pubmed ID: 21307339

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.

Cell-ECM Traction Force Modulates Endogenous Tension at Cell-cell Contacts

Proceedings of the National Academy of Sciences of the United States of America. Mar, 2011  |  Pubmed ID: 21383129

Cells in tissues are mechanically coupled both to the ECM and neighboring cells, but the coordination and interdependency of forces sustained at cell-ECM and cell-cell adhesions are unknown. In this paper, we demonstrate that the endogenous force sustained at the cell-cell contact between a pair of epithelial cells is approximately 100 nN, directed perpendicular to the cell-cell interface and concentrated at the contact edges. This force is stably maintained over time despite significant fluctuations in cell-cell contact length and cell morphology. A direct relationship between the total cellular traction force on the ECM and the endogenous cell-cell force exists, indicating that the cell-cell tension is a constant fraction of the cell-ECM traction. Thus, modulation of ECM properties that impact cell-ECM traction alters cell-cell tension. Finally, we show in a minimal model of a tissue that all cells experience similar forces from the surrounding microenvironment, despite differences in the extent of cell-ECM and cell-cell adhesion. This interdependence of cell-cell and cell-ECM forces has significant implications for the maintenance of the mechanical integrity of tissues, mechanotransduction, and tumor mechanobiology.

Shear Thickening of F-actin Networks Crosslinked with Non-muscle Myosin IIB

Soft Matter. Jan, 2011  |  Pubmed ID: 21552431

The material properties of cytoskeletal F-actin networks facilitate a broad range of cellular behaviors, whereby in some situations cell shape is preserved in the presence of force and, at other times, force results in irreversible shape change. These behaviors strongly suggest that F-actin networks can variably deform elastically or viscously. While a significant amount is known about the regulation of the elastic stiffness of F-actin networks, our understanding of the regulation of viscous behaviors of F-actin networks is largely lacking. Here, we study the rheological behavior of F-actin networks formed with heavy meromyosin non-muscle IIB (NMMIIB). We show that NMMIIB quenched with ADP crosslinks F-actin into networks that, for sufficient densities, display stress stiffening behavior. By performing a series of creep tests, we show that densely crosslinked actin/NMMIIB-ADP networks undergo viscous deformation over a wide range of stresses, ranging from 0.001 to 10 Pa. At high stresses, networks that stress stiffen are also observed to shear thicken, whereby the effective viscosity increases as a function of stress. Shear thickening results in a reduction in the extent of irreversible, viscous deformation in actin/NMMIIB-ADP networks at high stresses compared to that expected for a linear viscoelastic material. Thus, viscous deformation contributes less to the overall mechanical response at high levels of applied force. Our results indicate mechanisms by which the fluid-like nature of the actomyosin cytoskeleton can be reduced under high load.

Reconstitution of Contractile Actomyosin Bundles

Biophysical Journal. Jun, 2011  |  Pubmed ID: 21641315

Contractile actomyosin bundles are critical for numerous aspects of muscle and nonmuscle cell physiology. Due to the varying composition and structure of actomyosin bundles in vivo, the minimal requirements for their contraction remain unclear. Here, we demonstrate that actin filaments and filaments of smooth muscle myosin motors can self-assemble into bundles with contractile elements that efficiently transmit actomyosin forces to cellular length scales. The contractile and force-generating potential of these minimal actomyosin bundles is sharply sensitive to the myosin density. Above a critical myosin density, these bundles are contractile and generate large tensile forces. Below this threshold, insufficient cross-linking of F-actin by myosin thick filaments prevents efficient force transmission and can result in rapid bundle disintegration. For contractile bundles, the rate of contraction decreases as forces build and stalls under loads of ∼0.5 nN. The dependence of contraction speed and stall force on bundle length is consistent with bundle contraction occurring by several contractile elements connected in series. Thus, contraction in reconstituted actomyosin bundles captures essential biophysical characteristics of myofibrils while lacking numerous molecular constituents and structural signatures of sarcomeres. These results provide insight into nonsarcomeric mechanisms of actomyosin contraction found in smooth muscle and nonmuscle cells.

Spatiotemporal Constraints on the Force-dependent Growth of Focal Adhesions

Biophysical Journal. Jun, 2011  |  Pubmed ID: 21689521

Focal adhesions (FAs) are the predominant mechanism by which cells mechanically couple to and exert traction forces on their extracellular matrix (ECM). It is widely presumed that FA size is modulated by force to mediate changes in adhesion strength at different levels of cellular tension. However, previous studies seeking correlations between force and FA morphology have yielded variable and often conflicting results. Here we show that a strong correlation between adhesion size and traction force exists only during the initial stages of myosin-mediated adhesion maturation and growth. For mature adhesions, no correlation between traction stress and size is observed. Rather, the tension that is sustained at mature adhesions is more strongly influenced by proximity to the cell edge, with peripheral adhesions transmitting higher tension than adhesions near the cell center. Finally, we show that mature adhesions can withstand sixfold increases in tension without changes in size. Thus, although a strong correlation between adhesion size and mechanical tension is observed during the initial stages of myosin-mediated adhesion maturation, no correlation is observed in mature, elongated adhesions. This work places spatiotemporal constraints on the force-dependent growth of adhesions and provides insight into the mechanical regulation of cell-ECM adhesion.

Tension is Required but Not Sufficient for Focal Adhesion Maturation Without a Stress Fiber Template

The Journal of Cell Biology. Feb, 2012  |  Pubmed ID: 22291038

Focal adhesion composition and size are modulated in a myosin II-dependent maturation process that controls adhesion, migration, and matrix remodeling. As myosin II activity drives stress fiber assembly and enhanced tension at adhesions simultaneously, the extent to which adhesion maturation is driven by tension or altered actin architecture is unknown. We show that perturbations to formin and α-actinin 1 activity selectively inhibited stress fiber assembly at adhesions but retained a contractile lamella that generated large tension on adhesions. Despite relatively unperturbed adhesion dynamics and force transmission, impaired stress fiber assembly impeded focal adhesion compositional maturation and fibronectin remodeling. Finally, we show that compositional maturation of focal adhesions could occur even when myosin II-dependent cellular tension was reduced by 80%. We propose that stress fiber assembly at the adhesion site serves as a structural template that facilitates adhesion maturation over a wide range of tensions. This work identifies the essential role of lamellar actin architecture in adhesion maturation.