Cell size and actin architecture determine force generation in optogenetically activated adherent cells (original) (raw)
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Biophysical Journal, 2009
We investigate the dynamic response of single cells to weak and local rigidities, applied at controlled adhesion sites. Using multiple latex beads functionalized with fibronectin, and each trapped in its own optical trap, we study the reaction in real time of single 3T3 fibroblast cells to asymmetrical tensions in the tens of pN $ mm À1 range. We show that the cell feels a rigidity gradient even at this low range of tension, and over time develops an adapted change in the force exerted on each adhesion site. The rate at which force increases is proportional to trap stiffness. Actomyosin recruitment is regulated in space and time along the rigidity gradient, resulting in a linear relationship between the amount of recruited actin and the force developed independently in trap stiffness. This time-regulated actomyosin behavior sustains a constant and rigidity-independent velocity of beads inside the traps. Our results show that the strengthening of extracellular matrix-cytoskeleton linkages along a rigidity gradient is regulated by controlling adhesion area and actomyosin recruitment, to maintain a constant deformation of the extracellular matrix.
Actin-driven protrusions generate rapid long-range membrane tension propagation in cells
Membrane tension is thought to be a long-range integrator of cell physiology. This role necessitates effective tension transmission across the cell. However, the field remains strongly divided as to whether cell membranes support or resist tension propagation, in part due to a lack of adequate tools for locally manipulating membrane tension. We overcome these limitations by leveraging optogenetics to generate localized actinbased protrusions while concurrently monitoring the propagation of membrane tension using dual-trap optical tweezers. Surprisingly, actin-driven protrusions elicit rapid global membrane tension propagation with little to no attenuation, while forces applied to the cell membrane only do not. We present a simple unifying mechanical model in which mechanical forces that act on both the membrane and actin cortex drive rapid, robust membrane tension propagation.SummaryMechanical perturbations acting on both actin cortex and plasma membrane drive global membrane tensio...
Actomyosin Cortical Mechanical Properties in Nonadherent Cells Determined by Atomic Force Microscopy
Biophysical Journal, 2016
The organization of filamentous actin and myosin II molecular motor contractility is known to modify the mechanical properties of the cell cortical actomyosin cytoskeleton. Here we describe a novel method, to our knowledge, for using force spectroscopy approach curves with tipless cantilevers to determine the actomyosin cortical tension, elastic modulus, and intracellular pressure of nonadherent cells. We validated the method by measuring the surface tension of water in oil microdrops deposited on a glass surface. We extracted an average tension of T~20.25 nN/mm, which agrees with macroscopic experimental methods. We then measured cortical mechanical properties in nonadherent human foreskin fibroblasts and THP-1 human monocytes before and after pharmacological perturbations of actomyosin activity. Our results show that myosin II activity and actin polymerization increase cortex tension and intracellular pressure, whereas branched actin networks decreased them. Interestingly, myosin II activity stiffens the cortex and branched actin networks soften it, but actin polymerization has no effect on cortex stiffness. Our method is capable of detecting changes in cell mechanical properties in response to perturbations of the cytoskeleton, allowing characterization with physically relevant parameters. Altogether, this simple method should be of broad application for deciphering the molecular regulation of cell cortical mechanical properties.
High-Resolution Probing of Cellular Force Transmission
Physical Review Letters, 2009
Cells actively probe mechanical properties of their environment by exerting internally generated forces. The response they encounter profoundly affects their behavior. Here we measure in a simple geometry the forces a cell exerts suspended by two optical traps. Our assay quantifies both the overall force and the fraction of that force transmitted to the environment. Mimicking environments of varying stiffness by adjusting the strength of the traps, we found that the force transmission is highly dependent on external compliance. This suggests a calibration mechanism for cellular mechanosensing.
Nature Materials
Actomyosin machinery endows cells with contractility at a single cell level. However, within a monolayer, cells can be contractile or extensile based on the direction of pushing or pulling forces exerted by their neighbours or on the substrate. It has been shown that a monolayer of fibroblasts behaves as a contractile system while epithelial or neural progentior monolayers behave as an extensile system. Through a combination of cell culture experiments and in silico modeling, we reveal the mechanism behind this switch in extensile to contractile as the weakening of intercellular contacts. This switch promotes the buildup of tension at the cell-substrate interface through an increase in actin stress fibers and traction forces. This is accompanied by mechanotransductive changes in vinculin and YAP activation. We further show that contractile and extensile differences in cell activity sort cells in mixtures, uncovering a generic mechanism for pattern formation during cell competition, and morphogenesis. Main text The ability of cell monolayers to self-organize, migrate and evolve depends crucially on the interplay between cell-matrix and cell-cell interactions [1-4] which controls various phenomena including tissue morphogenesis [5, 6], epithelial-mesenchymal transition [1], wound healing and tumor progression [7]. Cells are active systems, engines that operate away from thermal equilibrium, transducing chemical energy into motion. Single isolated cells generate contractile force dipoles: the resultant of the forces due to actomyosin contraction, pulling on focal adhesion sites on the substrate, is typically a pair of approximately equal and opposite forces acting inwards along the cellular long axis [8] (Figure 1a). It is reasonable to expect that contractile particles also generate contractile behaviour in the monolayer [9]. However, at the collective cell level, epithelial monolayers [10, 11] and a monolayer of neural progentior cells display
Force transduction and strain dynamics in actin stress fibres in response to nanonewton forces
Journal of Cell Science, 2012
It is becoming clear that mechanical stimuli are crucial factors in regulating the biology of the cell, but the short-term structural response of a cell to mechanical forces remains relatively poorly understood. We mechanically stimulated cells transiently expressing actin-EGFP with controlled forces (0-20 nN) in order to investigate the structural response of the cell. Two clear force-dependent responses were observed: a short-term (seconds) local deformation of actin stress fibres and a long-term (minutes) force-induced remodelling of stress fibres at cell edges, far from the point of contact. By photobleaching markers along stress fibres we were also able to quantify strain dynamics occurring along the fibres throughout the cell. The results reveal that the cell exhibits complex heterogeneous negative and positive strain fluctuations along stress fibres in resting cells that indicate localized contraction and stretch dynamics. The application of mechanical force results in the activation of myosin contractile activity reflected in an ,50% increase in strain fluctuations. This approach has allowed us to directly observe the activation of myosin in response to mechanical force and the effects of cytoskeletal crosslinking on local deformation and strain dynamics. The results demonstrate that force application does not result in simplistic isotropic deformation of the cytoarchitecture, but rather a complex and localized response that is highly dependent on an intact microtubule network. Direct visualization of force-propagation and stress fibre strain dynamics have revealed several crucial phenomena that take place and ultimately govern the downstream response of a cell to a mechanical stimulus.
Cell Stiffening in Response to External Stress is Correlated to Actin Recruitment
Biophysical Journal, 2008
We designed a micromanipulation device that allows the local application of a constant force on living cells, and the measurement of their stiffness. The force is applied through an Arg-Gly-Asp-coated bead adhering on the cell and trapped in optical tweezers controlled by a feedback loop. Epifluorescence observations of green fluorescent protein-actin in the cells are made during force application. We observe a stiffening of cells submitted to a constant force within a few minutes, coupled to actin recruitment both at the bead-cell contact and up to several micrometers from the stress application zone. Moreover, kinetics of stiffening and actin recruitment exhibit a strong correlation. This work presents the first quantification of the dynamics of cell mechanical reinforcement under stress, which is a novel insight into the elucidation of the more general phenomenon of cell adaptation to stress.
Myosin II Tension Sensors Visualize Force Generation within the Actin Cytoskeleton in Living Cells
Type II myosin motors generate cytoskeletal forces that are central to cell division, embryogenesis, muscle contraction, and many other cellular functions. However, at present there is no method that can directly measure the forces generated by myosins in living cells. Here we describe a Förster resonance energy transfer (FRET)-based tension sensor that can measure forces generated by Nonmuscle Myosin IIB (NMIIB) in living cells with piconewton (pN) sensitivity. Fluorescence lifetime imaging microscopy (FLIM)-FRET measurements indicate that the forces generated by NMIIB exhibit significant spatial and temporal heterogeneity, with inferred tensions that vary widely in different regions of the cell. This initial report highlights the potential utility of myosin-based tension sensors in elucidating the roles of cytoskeletal contractility in a wide variety of contexts.
Nature Communications
The cadherin-catenin complex at adherens junctions (AJs) is essential for the formation of cell-cell adhesion and epithelium integrity; however, studying the dynamic regulation of AJs at high spatio-temporal resolution remains challenging. Here we present an optochemical tool which allows reconstitution of AJs by chemical dimerization of the force bearing structures and their precise light-induced dissociation. For the dimerization, we reconstitute acto-myosin connection of a tailless E-cadherin by two ways: direct recruitment of α-catenin, and linking its cytosolic tail to the transmembrane domain. Our approach enables a specific ON-OFF switch for mechanical coupling between cells that can be controlled spatially on subcellular or tissue scale via photocleavage. The combination with cell migration analysis and traction force microscopy shows a wide-range of applicability and confirms the mechanical contribution of the reconstituted AJs. Remarkably, in vivo our tool is able to contr...
Biology of the Cell, 2021
Background Information: Actin cytoskeleton contractility plays a critical role in morphogenetic processes by generating forces that are then transmitted to cell-cell and cell-ECM adhesion complexes. In turn, mechanical properties of the environment are sensed and transmitted to the cytoskeleton at cell adhesion sites, influencing cellular processes such as cell migration, differentiation and survival. Anchoring of the actomyosin cytoskeleton to adhesion sites is mediated by adaptor proteins such as talin or α-catenin that link F-actin to transmembrane cell adhesion receptors, thereby allowing mechanical coupling between the intracellular and extracellular compartments. Thus, a key issue is to be able to measure the forces generated by actomyosin and transmitted to the adhesion complexes. Approaches developed in cells and those probing single molecule mechanical properties of α-catenin molecules allowed to identify α-catenin, an F-actin binding protein which binds to the cadherin complexes as a major player in cadherin-based mechanotransduction. However, it is still very difficult to bridge intercellular forces measured at cellular levels and those measured at the single-molecule level. Results: Here, we applied an intermediate approach allowing reconstruction of the actomyosin-α-catenin complex in acellular conditions to probe directly the transmitted forces. For this, we combined micropatterning of purified α-catenin and spontaneous actomyosin network assembly in the presence of G-actin and Myosin II with microforce sensor arrays used so far to measure cell-generated forces. Conclusions: Using this method, we show that self-organizing actomyosin bundles bound to micrometric α-catenin patches can apply near-nano-Newton forces. Significance: Our results pave the way for future studies on molecular/cellular mechanotransduction and mechanosensing.