Movement of stress fibers away from focal adhesions identifies focal adhesions as sites of stress fiber assembly in stationary cells

Force generated in contractile actin filament bundles (stress fibers-SFs) is transmitted to the extracellular matrix (ECM) via linker proteins and transmembrane integrins at focal adhesions (FAs). Though it has long been known that actin is rapidly exchanged in FAs, the connection between SFs and FAs has not been studied in detail. We introduced fiduciary marks on SFs by expressing GFP-palladin or GFP-alpha-actinin-1, which are both FA and dense body proteins, and by pattern bleaching of GFP-actin. Following fiduciary marks on SFs over time by time-lapse fluorescence microscopy, we detected assembly of SFs at FAs in stationary cells resulting in movement of SFs away from FAs with a velocity of 0.2-0.4 microm/min. Visualization of FAs in GFP-palladin/DsRed-paxillin double transfected cells showed that SF elongation was not accompanied by a change in FA length. SF elongation at FAs depended on actin polymerization and force as demonstrated by inhibitors of actin polymerization (cytochalasin D, jasplakinolide) and inhibitors of myosin-dependent contraction (blebbistatin, Y-27632), respectively. Our finding of SF assembly at FAs has important implications for SF formation, force transmission, and tension distribution within the actin cytoskeletal network of stationary cells.     

Measurements of strain on single stress fibers in living endothelial cells induced by fluid shear stress

Fluid shear stress (FSS) acting on the apical surface of endothelial cells (ECs) can be sensed by mechano-sensors in adhesive protein complexes found in focal adhesions and intercellular junctions. This sensing occurs via force transmission through cytoskeletal networks. This study quantitatively evaluated the force transmitted through cytoskeletons to the mechano-sensors by measuring the FSS-induced strain on SFs using live-cell imaging for actin stress fibers (SFs). FSS-induced bending of SFs caused the SFs to align perpendicular to the direction of the flow. In addition, the displacement vectors of the SFs were detected using image correlation and the FSS-induced axial strain of the SFs was calculated. The results indicated that FSS-induced strain on SFs spanned the range 0.01-0.1% at FSSs ranging from 2 to 10 Pa. Together with the tensile property of SFs reported in a previous study, the force exerted on SFs was estimated to range from several to several tens of pN.     

Estimation of single stress fiber stiffness in cultured aortic smooth muscle cells under relaxed and contracted states: Its relation to dynamic rearrangement of stress fibers

For a quantitative analysis of intracellular mechanotransduction, it is crucial to know the mechanical properties of actin stress fibers in situ. Here we measured tensile properties of cultured aortic smooth muscle cells (SMCs) in a quasi-in situ tensile test in relaxed and activated states to estimate stiffness of their single stress fibers (SFs). An SMC cultured on substrates was held using a pair of micropipettes and detached from the substrate while maintaining its in situ cell shape and cytoskeletal integrity. Stretching up to ～15% followed by unloading was repeated three times to stabilize their tension–strain curves in the untreated (relaxed) and 10 μM-serotonin-treated (activated) condition. Cell stiffness defined as the average slope of the loading limb of the stable loops was ～25 and ～40 nN/% in relaxed and activated states, respectively. It decreased to ～10 nN/% following SF disruption with cytochalasin D in both states. The number of SFs in each cell measured with confocal microscopy decreased significantly upon serotonin activation from 21.5±3.8 (mean±SD, n=80) to 17.5±3.9 (n=77). The dynamics of focal adhesions (FAs) were observed in adherent cells using surface reflective interference contrast microscopy. FAs aligned and elongated along the cell major axis following activation and then merged with each other, suggesting that the decrease in SFs was caused by their fusion. Average stiffness of single SFs estimated by the average decrease in whole-cell stiffness following SF disruption divided by the average number of SFs in each cell was ～0.7 and ～1.6 nN/% in the relaxed and activated states, respectively. Stiffening of single SFs following SF activation was remarkably higher than stiffening at the whole-cell level. Results indicate that SFs stiffen not only due to activation of the actomyosin interaction, but also due to their fusion, a finding which would not be obtained from analysis of isolated SFs.     

Role of marginal stress fibers formed in the rat vascular endothelial cells

Fluorescence cytochemistry using en face preparations of rat vascular endothelial cells (ECs) revealed the localization of actin, fibronectin (FN) and fibronectin receptor (FNR) along not only central stress fibers (SFs) but also the cell margins. Electron microscopy showed very close proximity between the topographical distribution of intracellular microfilament bundles and that of subendothelial FN in the EC margins. Therefore, these basal and marginal actin cables may be comparable to the well-established central SFs present in ECs. Formation of the central SFs was induced in ECs or mesothelial cells in response to tension, by which their cellular integrity seems to be effectively maintained. However, even when central SF formation was inhibited by cytochalasin D, the ECs with marginal SFs showed high resistance to mechanical tension, whereas mesenteric mesothelial cells having no such fibers easily lost their integrity. Thus, together with central SFs, the marginal SFs characteristic of rat vascular ECs may play an essential role in strengthening cell-matrix adhesion.     

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.     

Image Analysis for the Quantitative Comparison of Stress Fibers and Focal Adhesions

Actin stress fibers (SFs) detect and transmit forces to the extracellular matrix through focal adhesions (FAs), and molecules in this pathway determine cellular behavior. Here, we designed two different computational tools to quantify actin SFs and the distribution of actin cytoskeletal proteins within a normalized cellular morphology. Moreover, a systematic cell response comparison between the control cells and those with impaired actin cytoskeleton polymerization was performed to demonstrate the reliability of the tools. Indeed, a variety of proteins that were present within the string beginning at the focal adhesions (vinculin) up to the actin SFs contraction (non-muscle myosin II (NMMII)) were analyzed. Finally, the software used allows for the quantification of the SFs based on the relative positions of FAs. Therefore, it provides a better insight into the cell mechanics and broadens the knowledge of the nature of SFs.     

Tensile properties of single stress fibers isolated from cultured vascular smooth muscle cells

Stress fibers (SFs), a contractile bundle of actin filaments, play a critical role in mechanotransduction in adherent cells; yet, the mechanical properties of SFs are poorly understood. Here, we measured tensile properties of single SFs by in vitro manipulation with cantilevers. SFs were isolated from cultured vascular smooth muscle cells with a combination of low ionic-strength extraction and detergent extraction and were stretched until breaking. The breaking force and the Young's modulus (assuming that SFs were isotropic) were, on average, 377 nN and 1.45 MPa, which were approximately 600-fold greater and three orders of magnitude lower, respectively, than those of actin filaments reported previously. Strain-induced stiffening was observed in the force-strain curve. We also found that the extracted SFs shortened to approximately 80% of the original length in an ATP-independent manner after they were dislodged from the substrate, suggesting that SFs had preexisting strain in the cytoplasm. The force required for stretching the single SFs from the zero-stress length back to the original length was approximately 10 nN, which was comparable with the traction force level applied by adherent cells at single adhesion sites to maintain cell integrity. These results suggest that SFs can bear intracellular stresses that may affect overall cell mechanical properties and will impact interpretation of intracellular stress distribution and force-transmission mechanism in adherent cells.     

Strain waveform dependence of stress fiber reorientation in cyclically stretched osteoblastic cells: effects of viscoelastic compression of stress fibers

Actin stress fibers (SFs) of cells cultured on cyclically stretched substrate tend to reorient in the direction in which a normal strain of substrate becomes zero. However, little is known about the mechanism of this reorientation. Here we investigated the effects of cyclic stretch waveform on SF reorientation in osteoblastic cells. Cells adhering to silicone membranes were subjected to cyclic uniaxial stretch, having one of the following waveforms with an amplitude of 8% for 24 h: triangular, trapezoid, bottom hold, or peak hold. SF reorientation of these cells was then analyzed. No preferential orientation was observed for the triangular and the peak-hold waveforms, whereas SFs aligned mostly in the direction with zero normal strain (~55°) with other waveforms, especially the trapezoid waveform, which had a hold time both at loaded and unloaded states. Viscoelastic properties of SFs were estimated in a quasi-in situ stress relaxation test using intact and SF-disrupted cells that maintained their shape on the substrate. The dynamics of tension F(SFs) acting on SFs during cyclic stretching were simulated using these properties. The simulation demonstrated that F(SFs) decreased gradually during cyclic stretching and exhibited a compressive value (F(SFs) < 0). The magnitude and duration time of the compressive forces were relatively larger in the group with a trapezoid waveform. The frequency of SF orientation had a significant negative correlation with the applied compressive forces integrated with time in a strain cycle, and the integrated value was largest with the trapezoid waveform. These results may indicate that the applied compressive forces on SFs have a significant effect on the stretch-induced reorientation of SFs, and that SFs realigned to avoid their compression. Stress relaxation of SFs might be facilitated during the holding period in the trapezoid waveform, and depolymerization and reorientation of SFs were significantly accelerated by their viscoelastic compression.     

Model of coupled transient changes of Rac, Rho, adhesions and stress fibers alignment in endothelial cells responding to shear stress

Interactions of cell adhesions, Rho GTPases and actin in the endothelial cells' response to external forces are complex and not fully understood, but a qualitative understanding of the mechanosensory response begins to emerge. Here, we formulate a mathematical model of the coupled dynamics of cell adhesions, small GTPases Rac and Rho and actin stress fibers guiding a directional reorganization of the actin cytoskeleton. The model is based on the assumptions that the interconnected cytoskeleton transfers the shear force to the adhesion sites, which in turn transduce the force into a chemical signal that activates integrins at the basal surface of the cell. Subsequently, activated and ligated integrins signal and transiently de-activate Rho, causing the disassembly of actin stress fibers and inhibiting the maturation of focal complexes into focal contacts. Focal complexes and ligated integrins activate Rac, which in turn enhances focal complex assembly. When Rho activity recovers, stress fibers re-assemble and promote the maturation of focal complexes into focal contacts. Merging stress fibers self-align, while the elevated level of Rac activity at the downstream edge of the cell is translated into an alignment of the cells and the newly forming stress fibers in the flow direction. Numerical solutions of the model equations predict transient changes in Rac and Rho that compare well with published experimental results. We report quantitative data on early alignment of the stress fibers and its dependence on cell shape that agrees with the model.     

A novel method for measuring tension generated in stress fibers by applying external forces

The distribution of contractile forces generated in cytoskeletal stress fibers (SFs) contributes to cellular dynamic functions such as migration and mechanotransduction. Here we describe a novel (to our knowledge) method for measuring local tensions in SFs based on the following procedure: 1), known forces of different magnitudes are applied to an SF in the direction perpendicular to its longitudinal axis; 2), force balance equations are used to calculate the resulting tensions in the SF from changes in the SF angle; and 3), the relationship between tension and applied force thus established is extrapolated to an applied force of zero to determine the preexisting tension in the SF. In this study, we measured tensions in SFs by attaching magnetic particles to them and applying known forces with an electromagnetic needle. Fluorescence microscopy was used to capture images of SFs fluorescently labeled with myosin II antibodies, and analysis of these images allowed the tension in the SFs to be measured. The average tension measured in this study was comparable to previous reports, which indicates that this method may become a powerful tool for elucidating the mechanisms by which cytoskeletal tensions affect cellular functions.     

Mechanical properties of actin stress fibers in living cells

Actin stress fibers (SFs) play an important role in many cellular functions, including morphological stability, adhesion, and motility. Because of their central role in force transmission, it is important to characterize the mechanical properties of SFs. However, most of the existing studies focus on properties of whole cells or of actin filaments isolated outside cells. In this study, we explored the mechanical properties of individual SFs in living endothelial cells by nanoindentation using an atomic force microscope. Our results demonstrate the pivotal role of SF actomyosin contractile level on mechanical properties. In the same SF, decreasing contractile level with 10 μM blebbistatin decreased stiffness, whereas increasing contractile level with 2 nM calyculin A increased stiffness. Incrementally stretching and indenting SFs made it possible to determine stiffness as a function of strain level and demonstrated that SFs have nearly linear stress-stain properties in the baseline state but nonlinear properties at a lower contractile level. The stiffnesses of peripheral and central portions of the same SF, which were nearly the same in the baseline state, became markedly different after contractile level was increased with calyculin A. Because these results pertain to effects of interventions in the same SF in a living cell, they provide important new understanding about cell mechanics.     

The assembly and function of perinuclear actin cap in migrating cells

Stress fibers are actin bundles encompassing actin filaments, actin-crosslinking, and actin-associated proteins that represent the major contractile system in the cell. Different types of stress fibers assemble in adherent cells, and they are central to diverse cellular processes including establishment of the cell shape, morphogenesis, cell polarization, and migration. Stress fibers display specific cellular organization and localization, with ventral fibers present at the basal side, and dorsal fibers and transverse actin arcs rising at the cell front from the ventral to the dorsal side and toward the nucleus. Perinuclear actin cap fibers are a specific subtype of stress fibers that rise from the leading edge above the nucleus and terminate at the cell rear forming a dome-like structure. Perinuclear actin cap fibers are fixed at three points: both ends are anchored in focal adhesions, while the central part is physically attached to the nucleus and nuclear lamina through the linker of nucleoskeleton and cytoskeleton (LINC) complex. Here, we discuss recent work that provides new insights into the mechanism of assembly and the function of these actin stress fibers that directly link extracellular matrix and focal adhesions with the nuclear envelope.     

Actin stress fibre subtypes in mesenchymal-migrating cells

Mesenchymal cell migration is important for embryogenesis and tissue regeneration. In addition, it has been implicated in pathological conditions such as the dissemination of cancer cells. A characteristic of mesenchymal-migrating cells is the presence of actin stress fibres, which are thought to mediate myosin II-based contractility in close cooperation with associated focal adhesions. Myosin II-based contractility regulates various cellular activities, which occur in a spatial and temporal manner to achieve directional cell migration. These myosin II-based activities involve the maturation of integrin-based adhesions, generation of traction forces, establishment of the front-to-back polarity axis, retraction of the trailing edge, extracellular matrix remodelling and mechanotransduction. Growing evidence suggests that actin stress fibre subtypes, namely dorsal stress fibres, transverse arcs and ventral stress fibres, could provide this spatial and temporal myosin II-based activity. Consistent with their functional differences, recent studies have demonstrated that the molecular composition of actin stress fibre subtypes differ significantly. This present review focuses on the current view of the molecular composition of actin stress fibre subtypes and how these fibre subtypes regulate mesenchymal cell migration.     

Tension is required but not sufficient for focal adhesion maturation without a stress fiber template

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.     

A kinematic model coupling stress fiber dynamics with JNK activation in response to matrix stretching

The role of the actin cytoskeleton in regulating mechanotransduction in response to external forces is complex and incompletely understood. Here, we develop a mathematical model coupling the dynamic disassembly and reassembly of actin stress fibers and associated focal adhesions to the activation of c-jun N-terminal kinase (JNK) in cells attached to deformable matrices. The model is based on the assumptions that stress fibers are pre-extended to a preferred level under static conditions and that perturbations from this preferred level destabilize the stress fibers. The subsequent reassembly of fibers upregulates the rate of JNK activation as a result of the formation of new integrin bonds within the associated focal adhesions. Numerical solutions of the model equations predict that different patterns of matrix stretch result in distinct temporal patterns in JNK activation that compare well with published experimental results. In the case of cyclic uniaxial stretching, stretch-induced JNK activation slowly subsides as stress fibers gradually reorient perpendicular to the stretch direction. In contrast, JNK activation is chronically elevated in response to cyclic equibiaxial stretch. A step change in either uniaxial or equibiaxial stretch results in a short, transient upregulation in JNK that quickly returns to the basal level as overly stretched stress fibers disassemble and are replaced by fibers assembled at the preferred level of stretch. In summary, the model describes a mechanism by which the dynamic properties of the actin cytoskeleton allow cells to adapt to applied forces through turnover and reorganization to modulate intracellular signaling.     

Buckling of actin stress fibers: a new wrinkle in the cytoskeletal tapestry

Intracellular tension is considered an important determinant of cytoskeletal architecture and cell function. However, many details about cytoskeletal tension remain poorly understood because these forces cannot be directly measured in living cells. Therefore, we have developed a method to characterize the magnitude and distribution of pre-extension of actin stress fibers (SFs) due to resting tension in the cytoskeleton. Using a custom apparatus, human aortic endothelial cells (HAECs) were cultured on a pre-stretched silicone substrate coated with a fibronectin-like polymer. Release of the substrate caused SFs aligned in the shortening direction in adhered cells to buckle when compressed rapidly (5% shortening per second or greater) beyond their unloaded slack length. Subsequently, the actin cytoskeleton completely disassembled in 5 sec and reassembled within 60 sec. Quantification of buckling in digital fluorescent micrographs of cells fixed and stained with rhodamine phalloidin indicated a nonuniform distribution of 0-26% pre-extension of SFs in non-locomoting HAECs. Local variability suggests heterogeneity of cytoskeletal tension and/or stiffness within individual cells. These findings provide new information about the magnitude and distribution of cytoskeletal tension and the dynamics of actin stress fibers, and the approach offers a novel method to elucidate the role of specific cytoskeletal elements and crosslinking proteins in the force generating apparatus of non-muscle cells.     

Orientation of apical and basal actin stress fibers in isolated and subconfluent endothelial cells as an early response to cyclic stretching

We investigated the response of apical and basal actin stress fibers (SFs) and its dependency on cell confluency for endothelial cells subjected to cyclic stretching. Porcine aortic endothelial cells from the 2nd and 5th passages were transferred to a fibronectin-coated silicone chamber with 5000-8000 cells/cm2 (isolated condition), positioning the cells apart, or with 25,000-27,000 cells/cm2 (subconfluent condition), allowing cell-to-cell contact. The substrate was stretched cyclically by 0.5 Hz for 2 h with a peak strain on the substrate that was 15% in the stretch direction and -4% in the transverse direction. The actin filaments (AFs) were stained with rhodamine phalloidin and their orientations were examined under a confocal laser scanning microscope. In the basal region, SFs formed in all of the cells under both the isolated and subconfluent conditions. We observed an average of 5 and 9 SFs per cell under the isolated and subconfluent conditions, respectively, in the fluorescent images of the apical region. We also observed cells that were bush-like without apical AFs or apical SFs. On average, the SFs in the subconfluent cells oriented in the direction of minimal strain, while the SFs in the isolated cells oriented in the direction of a 2% compressive strain. These results suggest that such differential response may be due to differences in the transmission of mechanical stretching to the central and apical regions of the cell through the SFs. We also speculate that cell-to-cell contact might change the strength, orientation, and anchorage of apical AFs and play a critical role in mechanical signal transduction.     

Simultaneous contraction and buckling of stress fibers in individual cells

It has been proposed that buckling of actin stress fibers (SFs) may be associated with their disassembly. However, much of the detail remains unknown partly because the use of an elastic membrane sheet, conventionally necessary for inducing SF buckling with a mechanical compression to adherent cells, may limit high quality and quick imaging of the dynamic cellular events. Here, we present an alternate approach to induce buckling behavior of SFs on a readily observable glass plate. Actin SFs were extracted from cells, and constituent myosin II (MII) molecules were partially photo-inactivated in contractility. An addition of Mg-ATP allowed actin-myosin cross-bridge cycling and resultant contraction of only thick SFs that still contained active MII in the large volume. Meanwhile, thin SFs with virtually no active motor protein in the small volume had no choice but to buckle with the shortening movement of nearby thick SFs functioning as a compression-inducing element. This novel technique, thus allowing for selective inductions of contraction and buckling of SFs and measurements of the cellular prestress, may be applicable to not only investigations on their disassembly mechanisms but also to measurements of the relative thickness of individual SFs in each cell.     

Nonmuscle myosin II is responsible for maintaining endothelial cell basal tone and stress fiber integrity

Cultured confluent endothelial cells exhibit stable basal isometric tone associated with constitutive myosin II regulatory light chain (RLC) phosphorylation. Thrombin treatment causes a rapid increase in isometric tension concomitant with myosin II RLC phosphorylation, actin polymerization, and stress fiber reorganization while inhibitors of myosin light chain kinase (MLCK) and Rho-kinase prevent these responses. These findings suggest a central role for myosin II in the regulation of endothelial cell tension. The present studies examine the effects of blebbistatin, a specific inhibitor of myosin II activity, on basal tone and thrombin-induced tension development. Although blebbistatin treatment abolished basal tension, this was accompanied by an increase in myosin II RLC phosphorylation. The increase in RLC phosphorylation was Ca²⁺ dependent and mediated by MLCK. Similarly, blebbistatin inhibited thrombin-induced tension without interfering with the increase in RLC phosphorylation or in F-actin polymerization. Blebbistatin did prevent myosin II filament incorporation and association with polymerizing or reorganized actin filaments leading to the disappearance of stress fibers. Thus the inhibitory effects of blebbistatin on basal tone and induced tension are consistent with a requirement for myosin II activity to maintain stress fiber integrity. actin; blebbistatin; isometric tension; myosin light chain kinase; regulatory light chain phosphorylation; focal adhesions