Mechanical forces are central to developmental, physiological and pathological processes1. However, limited understanding of force transmission within sub-cellular structures is a major obstacle to unravelling molecular mechanisms. Here we describe the development of a calibrated biosensor that measures forces across specific proteins in cells with pico-Newton (pN) sensitivity, as demonstrated by single molecule fluorescence force spectroscopy2. The method is applied to vinculin, a protein that connects integrins to actin filaments and whose recruitment to focal adhesions (FAs) is force-dependent3. We show that tension across vinculin in stable FAs is ~2.5 pN and that vinculin recruitment to FAs and force transmission across vinculin are regulated separately. Highest tension across vinculin is associated with adhesion assembly and enlargement. Conversely, vinculin is under low force in disassembling or sliding FAs at the trailing edge of migrating cells. Furthermore, vinculin is required for stabilizing adhesions under force. Together, these data reveal that FA stabilization under force requires both vinculin recruitment and force transmission, and that, surprisingly, these processes can be controlled independently.

Cellular responses to mechanical forces are crucial in embryonic development and adult physiology, and are involved in numerous diseases, including atherosclerosis, hypertension, osteoporosis, muscular dystrophy, myopathies and cancer. These responses are mediated by load-bearing subcellular structures, such as the plasma membrane, cell-adhesion complexes and the cytoskeleton. Recent work has demonstrated that these structures are dynamic, undergoing assembly, disassembly and movement, even when ostensibly stable. An emerging insight is that transduction of forces into biochemical signals occurs within the context of these processes. This framework helps to explain how forces of varying strengths or dynamic characteristics regulate distinct signalling pathways.

We report the first measurements of the intrinsic strain fluctuations of living cells using a recentlydeveloped tracer correlation technique along with a theoretical framework for interpreting such data in heterogeneous media with non-thermal driving. The fluctuations' spatial and temporal correlations indicate that the cytoskeleton can be treated as a course-grained continuum with powerlaw rheology, driven by a spatially random stress tensor field. Combined with recent cell rheology results, our data imply that intracellular stress fluctuations have a nearly 1/ω 2 power spectrum, as expected for a continuum with a slowly evolving internal prestress.PACS numbers: 87.16. Ac, 87.15.Ya, 87.10.+e An accurate physical picture of the viscoelasticity and motion of the cytoskeleton is crucial for a complete understanding of processes such as intracellular transport [1], cell crawling [2], and mechano-chemical transduction [2]. Microrheology [3], based on the analysis of embedded tracer particle motion, has recently emerged as an experimental probe of cytoskeleton viscoelasticity and dynamics [4,5,6,7]. The viscoelastic properties of eucaryotic cells arise from an intricate network of protein filaments driven by specialized motor proteins and directional polymerization, that convert the chemical energy of adenosine triphosphate (ATP) to mechanical work and motion. A cell is thus a nonequilibrium soft material whose fluctuations are actively driven. Unlike the thermal fluctuations in an equilibrium material, the amplitude and spatial distribution of active fluctuations can be controlled via biochemical signaling pathways; perhaps allowing the cell to locally adjust its' mechanical properties to suit its' needs. Indeed, microscopic force generators play a central role in existing cell mechanics models such as the sol-gel [8], soft glassy rheology [4] and tensegrity [9] hypotheses.In this Letter, we extend a recently introduced method, termed two-point microrheology [10], and show that it can be used to characterize the activity of intracellular force generators by directly measuring a cell's intrinsic, random stress fluctuations. Our experimental data and theoretical framework show that a cell can be modelled as a coarse-grained viscoelastic continuum driven by a spatially random stress field having a 1/ω 2 power spectrum in our observable frequency range, 1 < ω < 60 rad/s.There are two distinct approaches to microrheology: the active approach measures the displacements of tracer particles induced by external forces and the passive approach measures fluctuations of particle positions in the absence of driving forces. The active approach provides a direct measure of the complex shear modulus µ(ω). In equilibrium systems the passive approach also measures µ(ω) because of the fluctuation-dissipation theorem (FDT) [11]. Literature results in cells using singleparticle versions of the two approaches yield shear moduli differing by orders of magnitude and exhibiting qualitatively different frequency dependencies [4,6]. These ...

Although understanding cells' responses to mechanical stimuli is seen as increasingly important for understanding cell biology, how to best measure, interpret, and model cells' mechanical properties remains unclear. We determine the frequency-dependent shear modulus of cultured mammalian cells by using four different methods, both unique and well established. This approach clarifies the effects of cytoskeletal heterogeneity, ATP-dependent processes, and cell regional variations on the interpretation of such measurements. Our results clearly indicate two qualitatively similar, but distinct, mechanical responses, corresponding to the cortical and intracellular networks, each having an unusual, weak power-law form at low frequency. The two frequency-dependent responses we observe are remarkably similar to those reported for a variety of cultured mammalian cells measured with different techniques, suggesting it is a useful consensus description. Finally, we discuss possible physical explanations for the observed mechanical response.cell mechanics ͉ cytoskeleton ͉ mechanotransduction ͉ microrheology T he important role of mechanical and physical cues in determining cell behavior is increasingly recognized. Cell shape can modulate cell differentiation (1), whereas substrate stiffness can affect tissue morphogenesis (2) and myoblast differentiation (3). The mechanisms, however, by which mechanical cues lead to molecular and biochemical responses remain largely undetermined. One approach to studying such mechano-sensing processes is to understand the mechanical properties of cells' constitutive molecules individually. Although protein conformation can respond to locally applied, small-scale mechanical signals such as molecular tension (4), how these signals may be combined to sense larger-scale mechanical properties remains unclear (5). Ultimately, an integrated physical description of cytoskeletal mechanics will be required to connect these molecular and cellular levels of description.In soft-matter and polymer physics, the mechanical response and dynamics of supramolecular assemblies are determined by using rheology, the study of the frequency-dependent elastic and viscous behavior of deformable materials. Reliably interpreting rheology measurements on living cells, however, has proven notoriously difficult. Only after decades of experimental effort have different cell measurements begun to report comparable responses, although many differences remain (6). The rheology of cells is typically inferred from either deformation in response to an applied force (termed active microrheology) or the Brownian motion of embedded or attached tracer particles (termed passive microrheology). The results of both approaches depend on theoretical models for the deformation geometry or coupling between the tracer and the cell. Passive methods will also be confounded by any non-Brownian tracer motion such as intracellular trafficking or cell crawling. The comparison of cell mechanics measurements based on different methods is currently ...

It is now widely appreciated that normal tissue morphology and function rely upon cells' ability to sense and generate forces appropriate to their correct tissue context. Although the effects of forces on cells have been studied for decades, our understanding of how those forces propagate through and act on different cell substructures remains at an early stage. The past decade has seen a resurgence of interest, with a variety of different micromechanical methods in current use that probe cells' dynamic deformation in response to a time-varying force. The ability of researchers to carefully measure the mechanical properties of cells subjected to a variety of pharmacological and genetic interventions, however, currently outstrips our ability to quantitatively interpret the data in many cases. Despite these challenges, the stage is now set for the development of detailed models for cell deformability, motility, and mechanosensing that are rooted at the molecular level.