Single Stress Fibers Research Articles

An Inverse Modeling Approach to Estimate Three-Dimensional Aortic Valve Interstitial Cell Stress Fiber Force Levels.

Within the aortic valve (AV) leaflet exists a population of interstitial cells (AVICs) that maintain the constituent tissues by extracellular matrix (ECM) secretion, degradation, and remodeling. AVICs can transition from a quiescent, fibroblast-like phenotype to an activated, myofibroblast phenotype in response to growth or disease. AVIC dysfunction has been implicated in AV disease processes, yet our understanding of AVIC function remains quite limited. A major characteristic of the AVIC phenotype is its contractile state, driven by contractile forces generated by the underlying stress fibers (SF). However, direct assessment of the AVIC SF contractile state and structure within physiologically mimicking three-dimensional environments remains technically challenging, as the size of single SFs are below the resolution of light microscopy. Therefore, in the present study, we developed a three-dimensional (3D) computational approach of AVICs embedded in 3D hydrogels to estimate their SF local orientations and contractile forces. One challenge with this approach is that AVICs will remodel the hydrogel, so that the gel moduli will vary spatially. We thus utilized our previous approach (Khang et al. 2023, "Estimation of Aortic Valve Interstitial Cell-Induced 3D Remodeling of Poly (Ethylene Glycol) Hydrogel Environments Using an Inverse Finite Element Approach," Acta Biomater., 160, pp. 123-133) to define local hydrogel mechanical properties. The AVIC SF model incorporated known cytosol and nucleus mechanical behaviors, with the cell membrane assumed to be perfectly bonded to the surrounding hydrogel. The AVIC SFs were first modeled as locally unidirectional hyperelastic fibers with a contractile force component. An adjoint-based inverse modeling approach was developed to estimate local SF orientation and contractile force. Substantial heterogeneity in SF force and orientations were observed, with the greatest levels of SF alignment and contractile forces occurring in AVIC protrusions. The addition of a dispersed SF orientation to the modeling approach did not substantially alter these findings. To the best of our knowledge, we report the first fully 3D computational contractile cell models which can predict locally varying stress fiber orientation and contractile force levels.

Measuring viscoelastic properties of single stress fibers during 3D confined migration

Actomyosin stress fibers (SFs) allow cells to exert tensile forces on their surrounding microenvironment, which in turn underlies cell structure, mechanics, and migration. While the tensile properties and functional contributions of SFs have been extensively explored in 2D geometries, much remains unknown about how SFs function in 3D, particularly in confined geometries relevant to invasion and metastasis. Here we integrate 3D printing with single-SF femtosecond laser ablation to measure SF viscoelastic properties in well-defined 3D confined geometries.

Mechanical Control of Cell Migration by the Metastasis Suppressor Tetraspanin CD82/KAI1.

The plasma membrane is a key actor of cell migration. For instance, its tension controls persistent cell migration and cell surface caveolae integrity. Then, caveolae constituents such as caveolin-1 can initiate a mechanotransduction loop that involves actin- and focal adhesion-dependent control of the mechanosensor YAP to finely tune cell migration. Tetraspanin CD82 (also named KAI-1) is an integral membrane protein and a metastasis suppressor. Its expression is lost in many cancers including breast cancer. It is a strong inhibitor of cell migration by a little-known mechanism. We demonstrated here that CD82 controls persistent 2D migration of EGF-induced single cells, stress fibers and focal adhesion sizes and dynamics. Mechanistically, we found that CD82 regulates membrane tension, cell surface caveolae abundance and YAP nuclear translocation in a caveolin-1-dependent manner. Altogether, our data show that CD82 controls 2D cell migration using membrane-driven mechanics involving caveolin and the YAP pathway.

Modeling cells spreading, motility, and receptors dynamics: a general framework

The response of cells during spreading and motility is dictated by several multi-physics events, which are triggered by extracellular cues and occur at different time-scales. For this sake, it is not completely appropriate to provide a cell with classical notions of the mechanics of materials, as for “rheology” or “mechanical response”. Rather, a cell is an alive system with constituents that show a reproducible response, as for the contractility for single stress fibers or for the mechanical response of a biopolymer actin network, but that reorganize in response to external cues in a non-exactly-predictable and reproducible way. Aware of such complexity, in this note we aim at formulating a multi-physics framework for modeling cells spreading and motility, accounting for the relocation of proteins on advecting lipid membranes.Graphic We study the mechanical response under compression/extension of an assembly composed of 8 helical rods, pin-jointed and arranged in pairs with opposite chirality. In compression we find that, whereas a single rod buckles (a), the rods of the assembly deform as stable helical shapes (b). We investigate the effect of different boundary conditions and elastic properties on the mechanical response, and find that the deformed geometries exhibit a common central region where rods remain circular helices. Our findings highlight the key role of mutual interactions in the ensemble response and shed some light on the reasons why tubular helical assemblies are so common and persistent.

Cofilin is required for polarization of tension in stress fiber networks during migration.

Cell migration is associated with the establishment of defined leading and trailing edges, which in turn requires polarization of contractile forces. While the actomyosin stress fiber (SF) network plays a critical role in enforcing this polarity, precisely how this asymmetry is established remains unclear. Here, we provide evidence for a model in which the actin-severing protein cofilin (specifically cofilin-1) participates in symmetry breakage by removing low-tension actomyosin filaments during transverse arc assembly. Cofilin knockdown (KD) produces a non-polarized SF architecture that cannot be rescued with chemokines or asymmetric matrix patterns. Whereas cofilin KD increases whole-cell prestress, it decreases prestress within single SFs, implying an accumulation of low-tension SFs. This notion is supported by time-lapse imaging, which reveals weakly contractile and incompletely fused transverse arcs. Confocal and super-resolution imaging further associate this failed fusion with the presence of crosslinker-rich, tropomyosin-devoid nodes at the junctions of multiple transverse arc fragments and dorsal SFs. These results support a model in which cofilin facilitates the formation of high-tension transverse arcs, thereby promoting mechanical asymmetry.

Retardation Can Quantify Tension in Single Stress Fibers?

Tension in stress fibers plays an important role in cell functions such as cell migration, division, and contraction. We devised a method to evaluate a cell contraction force from retardation using a photo-elasticity technique (S. Sugita et al., Scientific Reports 9: 3690, 2019) and confirmed that the method can differentiate the phenotypes of vascular smooth muscle cells: contractile and synthetic types. It, however, remains unclear whether this method quantifies the “force” itself and why the retardation can sense the changes in force. Because the retardation is determined not only by the force but also by the density and alignment degree of birefringent material of stress fibers, cell activities might have changed such density or alignment. In this study, we isolated a single stress fiber from a cell to avoid cell responses and investigated the effect of force on the retardation. The vascular smooth muscle cells were isolated from porcine thoracic aortas, incubated on a dish, and treated with an osmotic shock solution to leave only stress fibers on the dish. The stress fibers were observed under a birefringent imaging system, and their retardation was measured. Application of ATP solutions to increase tension in the stress fibers led to an increase in the retardation, and retardation correlated significantly (P< 0.05) with the concentration of the ATP solutions. To elucidate the mechanism of retardation changes, the width of the stress fibers was evaluated from shadow images of the stress fibers under a phase-contrast microscope. When ATP solutions were applied, the width of the stress fiber decreased while its length was kept constant. These results demonstrated that the elevation of the retardation was attributed to the condensation of stress fibers induced by the tension.

Measurement of microscopic deformation in a single stress fiber using FRET-based tension sensor:

Measurement of microscopic deformation in a single stress fiber using FRET-based tension sensor:

Mechanical Control of Cell Migration by the Metastasis Suppressor Tetraspanin CD82/KAI1

The plasma membrane is a key actor of cell migration. For instance, its tension controls persistent cell migration and cell surface caveolae integrity. Then, caveolae constituents like caveolin-1 can initiate a mechano-tranduction loop that involves actin-and focal adhesions- dependent control of the mechanosensor YAP to finely tune cell migration. Tetraspanin CD82 (also named KAI-1) is an integral membrane protein and a metastasis suppressor. Its expression is lost in many cancers including breast cancer. It is a strong inhibitor of cell migration by a not well-known mechanism. We demonstrated here that CD82 controls persistent 2D migration of EGF-induced single cell, stress fibers and focal adhesion sizes and dynamics. In addition, CD82 regulates cell mechanics e.g. membrane tension, cell surface caveolae abundance and YAP nuclear translocation in a caveolin-1 dependent manner. Altogether, our data strongly suggest that CD82 controls 2D cell migration using a membrane-driven mechanical loop involving caveolin and the YAP pathway.

A novel FRET analysis method for tension dynamics in a single actin stress fiber: Application to MC3T3-E1 cells during movement on a substrate

A novel FRET analysis method for tension dynamics in a single actin stress fiber: Application to MC3T3-E1 cells during movement on a substrate

Anisotropic Mechanical Properties of Living Cells Revealed by Integrated Spinning Disk Confocal and Atomic Force Microscopy

Recent developments in fluorescent live-cell imaging and biophysical methods have significantly advanced our understanding of the dynamic biochemical and mechanical processes underlying cellular functions such as cell migration. One of most frequently used techniques for assessment of cell mechanical properties is the indentation experiments conducted with the atomic force microscope (AFM), due primarily to AFM's relative ease of operation, its high precision of force measurement, and high spatial resolution. We used the AFM in conjunction with a spinning disk confocal (SDC) microscope to directly visualize AFM indentation of living cells with high spatial and temporal resolution. With novel live cell imaging probes to fluorescently label F-actin, microtubules, and membrane, we were able to directly observe structural changes during the indentation process of a living cell (NIH 3T3 fibroblasts) with a spherical indenter. The most notable observation was a deformation of single apical stress fibers (ASF) under the action of the probe. The presence of ASF (also known as perinuclear actin cap) correlated with low cell height and high stiffness. Moreover, the presence of ASF caused an anisotropic indentation geometry as observed from surface displacement profiles calculated from the SDC images. Isotropic indentation profile was found in cells without ASF: cancerous cells MDA-MB-231 which are naturally lacking actin cap and NIH 3T3 cells in which ASF were disrupted by latrunculin A. Anisotropic indentation behavior suggests an anisotropic deformability and stiffness of the cell. We performed finite element simulations to extract anisotropic properties from the surface displacement data. Simulations suggest a very strong level of anisotropy in cells which role should be further elucidated.

Rock and MlCK Tune Regional Stress Fiber Mechanics via Preferential Myosin Light Chain Phosphorylation

Non-muscle myosin II (NMMII) is the key force-generating component of actomyosin stress fibers (SFs), which transmit tension throughout the cytoskeleton and to the extracellular matrix. NMMII motor activity is strongly promoted by phosphorylation of its regulatory light chains (RLCs) at Ser19 (p-RLC) and additionally enhanced by further phosphorylation at Thr18 (pp-RLC). The main RLC regulatory kinases are myosin RLC kinase (MLCK) and Rho-associated kinase (ROCK). While prior work suggests that MLCK and ROCK respectively regulate SFs located in the cellular center and periphery, it is unclear how these kinases differ in their effects on RLC phosphorylation state and how each phosphospecies influences the mechanics of central and peripheral SFs. Here we address these questions by combining graded induction of ROCK or MLCK expression with phospho-RLC immuno-analysis and mechanical measurement of single SFs in living cells. Immunoblots and quantitative immunofluorescence imaging reveal that graded MLCK overexpression preferentially produces graded increases in p-RLC, which localizes to peripheral SFs, while graded ROCK overexpression increases pp-RLC, which localizes to central SFs. Single-SF laser ablation studies revealed that MLCK increases the effective viscosity and stored elastic energy in peripheral SFs but not central SFs, while ROCK preferentially increases these parameters in central SFs. Graded variations in kinase activities revealed surprising and previously unappreciated nonlinearities between phosphorylation levels and SF viscoelasticity. To establish causal relationships between RLC phosphospecies and SF mechanics, we overexpressed RLC mono- and di-phosphomimetic mutants. We found that phosphomimetic p-RLC localized to and selectively influenced the mechanics of peripheral SFs while di-phosphomimetic pp-RLC localized to and selectively influenced the mechanics of central SFs. This work supports a model in which MLCK regulates peripheral SF viscoelasticity via p-RLC while ROCK regulates central SF viscoelasticity via pp-MLC.

Tensile test of isolated single stress fiber expressing FRET-based tension sensor

Tensile test of isolated single stress fiber expressing FRET-based tension sensor

Activation of ROCK and MLCK tunes regional stress fiber formation and mechanics via preferential myosin light chain phosphorylation.

The assembly and mechanics of actomyosin stress fibers (SFs) depend on myosin regulatory light chain (RLC) phosphorylation, which is driven by myosin light chain kinase (MLCK) and Rho-associated kinase (ROCK). Although previous work suggests that MLCK and ROCK control distinct pools of cellular SFs, it remains unclear how these kinases differ in their regulation of RLC phosphorylation or how phosphorylation influences individual SF mechanics. Here, we combine genetic approaches with biophysical tools to explore relationships between kinase activity, RLC phosphorylation, SF localization, and SF mechanics. We show that graded MLCK overexpression increases RLC monophosphorylation (p-RLC) in a graded manner and that this p-RLC localizes to peripheral SFs. Conversely, graded ROCK overexpression preferentially increases RLC diphosphorylation (pp-RLC), with pp-RLC localizing to central SFs. Interrogation of single SFs with subcellular laser ablation reveals that MLCK and ROCK quantitatively regulate the viscoelastic properties of peripheral and central SFs, respectively. The effects of MLCK and ROCK on single-SF mechanics may be correspondingly phenocopied by overexpression of mono- and diphosphomimetic RLC mutants. Our results point to a model in which MLCK and ROCK regulate peripheral and central SF viscoelastic properties through mono- and diphosphorylation of RLC, offering new quantitative connections between kinase activity, RLC phosphorylation, and SF viscoelasticity.

Geometry and network connectivity govern the mechanics of stress fibers

Actomyosin stress fibers (SFs) play key roles in driving polarized motility and generating traction forces, yet little is known about how tension borne by an individual SF is governed by SF geometry and its connectivity to other cytoskeletal elements. We now address this question by combining single-cell micropatterning with subcellular laser ablation to probe the mechanics of single, geometrically defined SFs. The retraction length of geometrically isolated SFs after cutting depends strongly on SF length, demonstrating that longer SFs dissipate more energy upon incision. Furthermore, when cell geometry and adhesive spacing are fixed, cell-to-cell heterogeneities in SF dissipated elastic energy can be predicted from varying degrees of physical integration with the surrounding network. We apply genetic, pharmacological, and computational approaches to demonstrate a causal and quantitative relationship between SF connectivity and mechanics for patterned cells and show that similar relationships hold for nonpatterned cells allowed to form cell-cell contacts in monolayer culture. Remarkably, dissipation of a single SF within a monolayer induces cytoskeletal rearrangements in cells long distances away. Finally, stimulation of cell migration leads to characteristic changes in network connectivity that promote SF bundling at the cell rear. Our findings demonstrate that SFs influence and are influenced by the networks in which they reside. Such higher order network interactions contribute in unexpected ways to cell mechanics and motility.

Computational Tension Mapping of Adherent Cells Based on Actin Imaging.

Forces transiting through the cytoskeleton are known to play a role in adherent cell activity. Up to now few approaches haves been able to determine theses intracellular forces. We thus developed a computational mechanical model based on a reconstruction of the cytoskeleton of an adherent cell from fluorescence staining of the actin network and focal adhesions (FA). Our custom made algorithm converted the 2D image of an actin network into a map of contractile interactions inside a 2D node grid, each node representing a group of pixels. We assumed that actin filaments observed under fluorescence microscopy, appear brighter when thicker, we thus presumed that nodes corresponding to pixels with higher actin density were linked by stiffer interactions. This enabled us to create a system of heterogeneous interactions which represent the spatial organization of the contractile actin network. The contractility of this interaction system was then adapted to match the level of force the cell truly exerted on focal adhesions; forces on focal adhesions were estimated from their vinculin expressed size. This enabled the model to compute consistent mechanical forces transiting throughout the cell. After computation, we applied a graphical approach on the original actin image, which enabled us to calculate tension forces throughout the cell, or in a particular region or even in single stress fibers. It also enabled us to study different scenarios which may indicate the mechanical role of other cytoskeletal components such as microtubules. For instance, our results stated that the ratio between intra and extra cellular compression is inversely proportional to intracellular tension.

Dynamics of Actin Stress Fibers and Focal Adhesions during Slow Migration in Swiss 3T3 Fibroblasts: Intracellular Mechanism of Cell Turning.

To understand the mechanism regulating the spontaneous change in polarity that leads to cell turning, we quantitatively analyzed the dynamics of focal adhesions (FAs) coupling with the self-assembling actin cytoskeletal structure in Swiss 3T3 fibroblasts. Fluorescent images were acquired from cells expressing GFP-actin and RFP-zyxin by laser confocal microscopy. On the basis of the maximum area, duration, and relocation distance of FAs extracted from the RFP-zyxin images, the cells could be divided into 3 regions: the front region, intermediate lateral region, and rear region. In the intermediate lateral region, FAs appeared close to the leading edge and were stabilized gradually as its area increased. Simultaneously, bundled actin stress fibers (SFs) were observed vertically from the positions of these FAs, and they connected to the other SFs parallel to the leading edge. Finally, these connecting SFs fused to form a single SF with matured FAs at both ends. This change in SF organization with cell retraction in the first cycle of migration followed by a newly formed protrusion in the next cycle is assumed to lead to cell turning in migrating Swiss 3T3 fibroblasts.

Rheological behavior of actin stress fibers in myoblasts after nanodissection: Effectsofoxidative stress.

Cytoskeletal stress fibers (SFs) play important roles in cell rheology. Oxidative stress, as caused by excessive hydrogen peroxide (H2O2) or other reactive oxygen species, can cause cell damages via multiple pathways. Stress fiber mechanics in an oxidative environment is important for the understanding of such pathological challenges. This investigation aimed to assess the effects of oxidative stress on the mechanical conditions of single stress fibers in living cells. Utilizing a femtosecond (fs) laser to sever single SFs inside living C2C12 myoblasts, we investigated the retraction rheology of the severed single SFs to probe the mechanical conditions of the cells and the effect of H2O2 on them. The equilibration time of the retraction of the severed SFs became longer in the H2O2-treated myoblasts compared to the control. The initial gap between the two severed ends of the SF immediately after fs laser severing was larger in the H2O2-treated groups. This suggested that H2O2 exposure could promote the pre-stress in individual SFs in-situ. Oxidative stress could significantly affect the mechanical conditions of cytoskeletal SFs in myoblasts. The results were consistent with cell stiffness measured on single myoblasts under oxidative stress.

Spatiotemporal Tension Distribution of Individual Stress Fibers at the Cell-Matrix Interface

Actomyosin stress fibers (SFs) enable cells to exert traction on planar extracellular matrices (ECMs) by tensing focal adhesions (FAs) at the cell-ECM interface. While it is widely appreciated that the spatiotemporal distribution of these tensile forces play key roles in polarity, motility, fate choice, and other defining cell behaviors, virtually nothing is known about how an individual SF quantitatively contributes to tensile loads borne by specific molecules within associated FAs. We address this key open question by using femtosecond laser ablation to sever single SFs in cells while tracking tension across vinculin using a fluorescence resonance energy transfer (FRET) -based molecular optical sensor. We show that disruption of a single SF reduces tension across vinculin in FAs located throughout the cell, with enriched vinculin tension reduction in FAs oriented parallel to the targeted SF. Remarkably, however, some subpopulations of FAs exhibit enhanced vinculin tension upon SF irradiation and undergo dramatic, unexpected transitions between tension-enhanced and tension-reduced states. These changes depend strongly on the location of the severed SF, consistent with our earlier finding that different SF pools are regulated by distinct myosin activators. To unify these findings, we present a structural model in which central SFs are more interconnected and mutually reinforced than peripheral SFs due in part to the presence of transverse actomyosin structures that link central SFs into a cohesive network. Tension released upon compromise of a central SF is thus broadly redistributed to other stress fibers and focal adhesions, resulting in cell shape stabilization. These studies represent the most direct and high-resolution intracellular measurements of SF contributions to tension on specific FA proteins to date and offer a new paradigm for investigating regulation of adhesive complexes by cytoskeletal force. (Chang and Kumar, J Cell Sci 2013)

Vinculin tension distributions of individual stress fibers within cell-matrix adhesions

Actomyosin stress fibers (SFs) enable cells to exert traction on planar extracellular matrices (ECMs) by tensing focal adhesions (FAs) at the cell-ECM interface. Although it is widely appreciated that the spatial and temporal distribution of these tensile forces play key roles in polarity, motility, fate choice, and other defining cell behaviors, virtually nothing is known about how an individual SF quantitatively contributes to tensile loads borne by specific molecules within associated FAs. We address this key open question by using femtosecond laser ablation to sever single SFs in cells while tracking tension across vinculin using a molecular optical sensor. We show that disruption of a single SF reduces tension across vinculin in FAs located throughout the cell, with enriched vinculin tension reduction in FAs oriented parallel to the targeted SF. Remarkably, however, some subpopulations of FAs exhibit enhanced vinculin tension upon SF irradiation and undergo dramatic, unexpected transitions between tension-enhanced and tension-reduced states. These changes depend strongly on the location of the severed SF, consistent with our earlier finding that different SF pools are regulated by distinct myosin activators. We critically discuss the extent to which these measurements can be interpreted in terms of whole-FA tension and traction and propose a model that relates SF tension to adhesive loads and cell shape stability. These studies represent the most direct and high-resolution intracellular measurements of SF contributions to tension on specific FA proteins to date and offer a new paradigm for investigating regulation of adhesive complexes by cytoskeletal force.

Cyclic stretch induces cell reorientation on substrates by destabilizing catch bonds in focal adhesions.

A minimal model of cellular mechanosensing system that consists of a single stress fiber adhering on a substrate via two focal adhesions made of catch bonds is adopted to investigate the phenomena of cell reorientation on substrates induced by an applied uniaxial cyclic stretch. The model indicates that the catch bonds in the focal adhesions experience a periodically oscillating internal force with amplitude and frequency controlled by two intrinsic clocks of the stress fiber, one associated with localized activation and the other with homogeneous activation of sarcomere units along the stress fiber. It is shown that this oscillating force due to cyclic stretch tends to destabilize focal adhesions by reducing the lifetime of catch bonds. The resulting slide or relocation of focal adhesions then causes the associated stress fiber to shorten and rotate to configurations nearly perpendicular to the stretching direction. These predicted behaviors from our model are consistent with a wide range of experimental observations.