Stress Fiber Contraction Research Articles

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.

Contraction of Stress Fibers Extracted from Smooth Muscle Cells: Effects of Varying Ionic Strength

Contraction of Stress Fibers Extracted from Smooth Muscle Cells: Effects of Varying Ionic Strength

A constrained mixture approach to mechano-sensing and force generation in contractile cells

Biological tissues are very particular types of materials that have the ability to change their structure, properties and chemistry in response to external cues. Contractile cells, i.e. fibroblasts, are key players of tissue adaptivity as they are capable of reorganizing their surrounding extra-cellular matrix (ECM) by contracting and generating mechanical forces. This contractile behavior is attributed to the development of a stress-fiber (SF) network within the cell’s cytoskeleton, a process that is known to be highly dependent of the nature of the mechanical environment (such as ECM stiffness or the presence of stress and strain). To describe these processes in a consistent manner, the present paper introduces a mutiphasic formulation (fluid/solid/solute mixture) that accounts for four major elements of cell contraction: cytoskeleton, cytosol, SF and actin monomers, as well as their interactions. The model represents the cross-talks between mechanics and chemistry through various means: (a) a mechano-sensitive formation and dissociation of an anisotropic SF network described by mass exchange between actin monomer and polymers, (b) a bio-mechanical model for SF contraction that captures the well-known length–tension and velocity–tension relation for muscles cells and (c) a convection/diffusion description for the transport of fluid and monomers within the cell. Numerical investigations show that the multiphasic model is able to capture the dependency of cell contraction on the stiffness of the mechanical environment and accurately describes the development of an oriented SF network observed in contracting fibroblasts.

Viscoelastic response of contractile filament bundles

The actin cytoskeleton of adherent tissue cells often condenses into filament bundles contracted by myosin motors, so-called stress fibers, which play a crucial role in the mechanical interaction of cells with their environment. Stress fibers are usually attached to their environment at the endpoints, but possibly also along their whole length. We introduce a theoretical model for such contractile filament bundles which combines passive viscoelasticity with active contractility. The model equations are solved analytically for two different types of boundary conditions. A free boundary corresponds to stress fiber contraction dynamics after laser surgery and results in good agreement with experimental data. Imposing cyclic varying boundary forces allows us to calculate the complex modulus of a single stress fiber.

Abstract 1436: The Cdk5 activator p39 specifically links muskelin to myosin and regulates stress fiber organization and contraction

Abstract Cyclin-dependent kinase 5 (Cdk5), a proline-directed serine/threonine protein kinase, regulates signaling pathways implicated in the adhesion and migration of various cell types. Activation of Cdk5 requires its association with its activators p35 or p39. We have recently shown, in lens epithelial cells, that Cdk5 kinase activity regulates rho-dependent myosin phosphorylation, cytoskeletal contraction, and cell migration by controlling Src-p190RhoGAP signaling. In a separate line of investigation, we demonstrated that the N-terminus of p39 binds myosin essential light chain (MLC), while the C-terminus of p39 binds muskelin, a multi-domain scaffolding protein. As both muskelin and MLC have been associated with cytoskeletal organization, the present study seeks to determine whether p39 might serve as an adaptor that links muskelin to myosin and to investigate the possible biological relevance of such an interaction. Immunoprecipitation studies demonstrated that muskelin forms a protein complex with p39, MLC, myosin regulatory light chain (MRLC), and myosin heavy chain II (MHC II). Suppressing expression of p39 abrogated the interaction between muskelin and the myosin subunits, demonstrating that p39 is required to link muskelin to myosin. Immunofluorescence staining showed that muskelin colocalized with MRLC and actin-containing stress fibers. Suppressing muskelin expression with either of two different siRNAs significantly reduced MRLC phosphorylation and disrupted stress fiber organization. Similar effects were observed when Cdk5 activity was inhibited or suppressed. Moreover, suppressing muskelin expression prevented any further reduction of MRLC phosphorylation when Cdk5 activity was blocked, indicating that muskelin plays an important role in Cdk5-dependent regulation of MRLC phosphorylation. The Cdk5 activating protein p39 was necessary and sufficient for Cdk5-dependent regulation of myosin phosphorylation, as suppression of p39, but not p35, significantly reduced MRLC phosphorylation. Together, these results demonstrate that the Cdk5 activating protein p39 specifically links muskelin to myosin and thus, to stress fibers. Therefore, p39 has a dual role in cytoskeletal regulation: on one hand, it is an integral subunit of Cdk5 kinase; on the other, it acts as an adaptor protein to recruit muskelin to stress fibers. Each of these p39 functions is required for myosin phosphorylation, cytoskeletal contraction, and efficient cell migration. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1436. doi:10.1158/1538-7445.AM2011-1436

Active force generation in cross-linked filament bundles without motor proteins

Cytoskeletal filaments often interact laterally through cross-linking proteins, contributing to passive cellular viscoelasticity and, perhaps surprisingly, active force generation. We present a theory, based on the formation and rupture of cross-linker bonds, that relates molecular properties of those interactions to the macroscale mechanics of filament bundles. Computing the force-velocity relation for such a bundle, we demonstrate significant contractile forces in the absence of molecular motors. This theory provides insight into cytokinesis, cytoskeletal mechanics, and stress-fiber contraction.

T0201-3-4 アクチンストレスファイバのねじり収縮([T0201-3]細胞の構造と流れにおけるマイクロ・ナノスケール解析(3))

Nonmuscle actomyosin or actin stress fibers play an essential role in cellular adaptation to physical environment, yet the relation between their function and structure remains poorly understood. Herein, we provide the first evidence that stress fibers twist during contraction or stretching. We directly pulled or induced MgATP-based contraction of stress fibers isolated from the substrate. Twisting motion was observed in both cases, suggesting that stress fibers have a helical structure. Given that stress fibers are dynamic organelles that bear tensile stress in appropriate places inside cells, the helical structure seems to be advantageous as it is easy to disintegrate when compressive force is applied, serving as a ready source of actomyosin to reassemble new stress fibers.

Cdk5-Dependent Regulation of Rho Activity, Cytoskeletal Contraction, and Epithelial Cell Migration via Suppression of Src and p190RhoGAP

Cdk5 regulates adhesion and migration in a variety of cell types. We previously showed that Cdk5 is strongly activated during stress fiber formation and contraction in spreading cells. Here we determine the mechanism linking Cdk5 to stress fiber contractility and its relevance to cell migration. Immunofluorescence showed that Cdk5 colocalized with phosphorylated myosin regulatory light chain (pMRLC) on contracting stress fibers. Inhibiting Cdk5 activity by various means significantly reduced pMRLC level and cytoskeletal contraction, with loss of central stress fibers. Blocking Cdk5 activity also reduced Rho-Rho kinase (ROCK) signaling, which is the principal pathway of myosin phosphorylation under these conditions. Next, we examined the effect of Cdk5 activity on Src, a known regulator of Rho. Inhibiting Cdk5 activity increased Src activation and phosphorylation of its substrate, p190RhoGAP, an upstream inhibitor of Rho. Inhibiting both Cdk5 and Src activity completely reversed the effect of Cdk5 inhibition on Rho and prevented the loss of central stress fibers, demonstrating that Cdk5 exerts its effects on Rho-ROCK signaling by suppressing Src activity. Moreover, inhibiting either Cdk5 or ROCK activity increased cell migration to an equal extent, while inhibiting both kinases produced no additional effect, demonstrating that Cdk5-dependent regulation of ROCK activity is a physiological determinant of migration rate.

Advanced glycation end products increase endothelial permeability through the RAGE/Rho signaling pathway

Although increased vascular permeability is known to be a major characteristic of diabetic vasculopathy, the precise mechanisms and relevance of advanced glycation end products (AGE) to hyperpermeability of vessels remains unclear. Here, we studied changes in cytoskeletal configuration and the signaling mechanism induced by AGE in human endothelial cells. AGE-BSA stimulation induced Rho activation, intercellular gap formation, prominent actin stress fiber and cell contraction without changing VE-cadherin, and subsequently transendothelial diffusion of FITC-labeled dextran. These processes induced by AGE-BSA were inhibited by either Rho-kinase inhibitor Y27632 or anti-RAGE antibody. We also showed that RhoA and RAGE spontaneously formed a complex. These findings suggest that activation of RAGE/Rho is involved in AGE-BSA-induced hyperpermeability through gap formation and actin reorganization in diabetes. Structured summary MINT- 7301170: Rhotekin (uniprotkb: Q9BST9) physically interacts (MI: 0915) with RhoA (uniprotkb: P61586) by pull down (MI: 0096) MINT- 7301204, MINT- 7301186: RhoA (uniprotkb: P61586) physically interacts (MI: 0915) with RAGE (uniprotkb: Q15109) by anti bait coimmunoprecipitation (MI: 0006)

Recoil after Severing Reveals Stress Fiber Contraction Mechanisms

Stress fibers are cellular contractile actomyosin machines central to wound healing, shear stress response, and other processes. Contraction mechanisms have been difficult to establish because stress fibers in cultured cells typically exert isometric tension and present little kinetic activity. In a recent study, living cell stress fibers were severed with laser nanoscissors and recoiled several μm over ∼5 s. We developed a quantitative model of stress fibers based on known components and available structural information suggesting periodic sarcomeric organization similar to striated muscle. The model was applied to the severing assay and compared to the observed recoil. We conclude that the sarcomere force-length relation is similar to that of muscle with two distinct regions on the ascending limb and that substantial external drag forces act on the recoiling fiber corresponding to effective cytosolic viscosity ∼104 times that of water. This may originate from both nonspecific and specific interactions. The model predicts highly nonuniform contraction with caps of collapsed sarcomeres growing at the severed ends. A directly measurable signature of external drag is that cap length and recoil distance increase at intermediate times as t1/2. The severing data is consistent with this prediction.

Intracellular Particles Involved in Stress Fiber Formation through Remodeling of Actin Filament Networks

Formation of stress fibers, the actin filament (F-actin) bundles that align in a highly-ordered manner, is crucial for cell migration. The migrating cells retract their tails by contraction of stress fibers. The alignment angle of stress fibers controls the direction of cell migration. In the process of cell migration, stress fibers formed in lamellae, compensating for the contracted stress fibers. Although the alignment angle of the newly forming stress fibers is important for the regulation of cell migration, the mechanism that determines the direction of stress fiber formation is poorly understood.To elucidate the mechanism of direction determination of stress fiber formation, we observed dynamics of actin cytoskeleton in lamellae of living fibroblasts. By using the scanning probe microscopy (SPM), we established the method to visualize actin cytoskeleton in living cells with the nanometer-scale spatial resolution and the second-scale temporal resolution. As a result of the time-lapse SPM observation, we found the submicron-size particles included in the mesh-like F-actin networks were replaced by the newly-forming stress fibers. The particles moved in the opposite direction of stress fiber formation. Further observation revealed that the new stress fibers formed in the region between the pre-existing stress fibers and the particles. We also revealed that the stress fiber formation resulted from the remodeling of the pre-existing F-actin networks. Inhibitory studies showed that actomyosin and Rho-kinase, both essential for stress fiber formation, regulated the movement of the particles. Immunofluorescent studies showed that vinculin, a focal adhesion protein, and F-actin were localized at some particles. From these results, we propose a model for the direction determination of stress fiber formation induced by the particles.

Biomechanical properties of actin stress fibers of non-motile cells

The structure of stress fibers, contractile actin bundles, differs between motile and non-motile cells, though the same term "stress fiber" is used to refer to the structurally as well as functionally different actin bundles. Stress fibers in non-motile cells run between separate focal adhesions, producing isometric tension due to actomyosin contraction. The stress fiber contraction is maintained through dynamic molecular exchanges between preformed and cytoplasmic components. This isometric contraction has been recognized as being essential for extracellular matrix assembly and resultant wound healing. In addition, there have recently been increasing data suggesting that stress fibers under contraction by themselves work as a mechanosensitive element. In this review we discuss, from molecular and physical viewpoints, biomechanical properties of the stress fiber of non-motile cells such as contraction force, resistance to stretching, and their roles in keeping a mechanical homeostasis, which play vital roles in the mechanosensing process.

Role for stress fiber contraction in surface tension development and stretch-activated channel regulation in C2C12 myoblasts

Membrane-cytoskeleton interaction regulates transmembrane currents through stretch-activated channels (SACs); however, the mechanisms involved have not been tested in living cells. We combined atomic force microscopy, confocal immunofluorescence, and patch-clamp analysis to show that stress fibers (SFs) in C2C12 myoblasts behave as cables that, tensed by myosin II motor, activate SACs by modifying the topography and the viscoelastic (Young's modulus and hysteresis) and electrical passive (membrane capacitance, C(m)) properties of the cell surface. Stimulation with sphingosine 1-phosphate to elicit SF formation, the inhibition of Rho-dependent SF formation by Y-27632 and of myosin II-driven SF contraction by blebbistatin, showed that not SF polymerization alone but the generation of tensional forces by SF contraction were involved in the stiffness response of the cell surface. Notably, this event was associated with a significant reduction in the amplitude of the cytoskeleton-mediated corrugations in the cell surface topography, suggesting a contribution of SF contraction to plasma membrane stretching. Moreover, C(m), used as an index of cell surface area, showed a linear inverse relationship with cell stiffness, indicating participation of the actin cytoskeleton in plasma membrane remodeling and the ability of SF formation to cause internalization of plasma membrane patches to reduce C(m) and increase membrane tension. SF contraction also increased hysteresis. Together, these data provide the first experimental evidence for a crucial role of SF contraction in SAC activation. The related changes in cell viscosity may prevent SAC from abnormal activation.

Plexin-B1 Utilizes RhoA and Rho Kinase to Promote the Integrin-dependent Activation of Akt and ERK and Endothelial Cell Motility

The semaphorins are a family of proteins originally identified as axon-guiding molecules in the developing nervous system that have been recently shown to regulate many cellular functions, including motility, in a variety of cell types. We have previously shown that in endothelial cells Semaphorin 4D acts through its receptor, Plexin-B1, to elicit a pro-angiogenic phenotype that involves the activation of the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway. Here we show through the use of a receptor chimeric approach, Plexin-B1 mutants, and dominant negative and pharmacological inhibitors that this response is dependent upon the activation of RhoA and its downstream target, Rho kinase (ROK). Indeed, we demonstrate that in endothelial cells, Semaphorin 4D promotes the formation of focal adhesion complexes, stress fibers, and the phosphorylation of myosin light chain, a response that was abolished by the use of ROK inhibitors and absent from cells expressing Plexin-B1 mutant constructs incapable of signaling to RhoA. Stress fiber polymerization and contraction are in turn necessary for RhoA-dependent pro-angiogenic signaling through Plexin-B1. Furthermore, we observed that in endothelial cells Plexin-B1 promotes the integrin-mediated activation of Pyk2, resulting in the stimulation of PI3K, Akt, and ERK. These findings provide evidence that Plexin-B1 promotes endothelial cell motility through RhoA and ROK by regulating the integrin-dependent signaling networks that result in the activation of PI3K and Akt.

Coupling biochemistry and mechanics in cell adhesion: a model for inhomogeneous stress fiber contraction

Biochemistry and mechanics are closely coupled in cell adhesion. At sites of cell-matrix adhesion, mechanical force triggers signaling through the Rho-pathway, which leads to structural reinforcement and increased contractility in the actin cytoskeleton. The resulting force acts back to the sites of adhesion, resulting in a positive feedback loop for mature adhesion. Here, we model this biochemical–mechanical feedback loop for the special case when the actin cytoskeleton is organized in stress fibers, which are contractile bundles of actin filaments. Activation of myosin II molecular motors through the Rho-pathway is described by a system of reaction–diffusion equations, which are coupled into a viscoelastic model for a contractile actin bundle. We find strong spatial gradients in the activation of contractility and in the corresponding deformation pattern of the stress fiber, in good agreement with experimental findings.

Colocalization of Glial Fibrillary Acidic Protein, Metallothionein, and MHC II in Human, Rat, NOD/SCID, and Nude Mouse Skin Keratinocytes and Fibroblasts

The expression of glial fibrillary acidic protein (GFAP) by perivascular cells of many mammalian organs suggests an as yet unknown function of this intermediate filament protein in the maintenance of homeostasis and vascular permeability at the blood-tissue interface. Although a similar situation may exist at the air-tissue interface, the cellular distribution of GFAP in skin tissue has never been demonstrated. To approach this issue, we have employed immunofluorescence and Western blotting techniques to detect GFAP in skin sections of young and adult humans, normal rodents, and two types of mutant mice, as well as in rat lung sections, and in cultured human keratinocytes and fibroblasts. Colocalization with antigens known to be associated with GFAP in other tissues was also tested. Epidermal and hair follicle keratinocytes and dermal fibroblasts showed distinct staining for GFAP as well as colocalization with alpha-actin, metallothionein, and antigens of the class-II major histocompatibility complex (MHC II). GFAP was also identified in rat alveolar fibroblasts which, in common with keratinocytes, form part of the air-tissue interface. GFAP was upregulated together with MHC II in nude mice but was barely detectable in the skin of non-obese diabetic severe combined immunodeficiency mice, suggesting a possible involvement in antigen-presenting functions. The intriguing distribution of a common set of antigens both in certain cells of the integumentary system and at the blood-tissue interfaces of internal organs suggests the involvement of these proteins in universal mechanisms controlling tissue homeostasis and protection.

1∕fruffle oscillations in plasma membranes of amphibian epithelial cells under normal and inverted gravitational orientations

Membrane ruffle fluctuations of amphibian epithelial cells A6 (CCL102) cultured in normal and upside down oriented plates have been analyzed through video microscopy. Our results reveal that their edge ruffle fluctuations exhibit a stochastic dynamics with 1/f(alpha) power spectrum over at least two decades at low frequencies and long range correlated, self-affine lateral border profiles. In a few and small areas of the membrane, probably nearby focal contacts, we found periodic oscillations which could be induced by myosin driven contraction of stress fibers. Furthermore, whereas the different gravitational orientations had none or little effect on the structure (power spectra and surface roughness) of these membrane ruffle fluctuations, their dynamic parameters were differentially affected. Indeed, the decay time of ruffles remained unchanged, but the period of lamellipodia oscillations near the focal adhesion points was significantly altered in A6 cells cultured upside down.

LIM Kinase 1 Coordinates Microtubule Stability and Actin Polymerization in Human Endothelial Cells

Microtubule (MT) destabilization promotes the formation of actin stress fibers and enhances the contractility of cells; however, the mechanism involved in the coordinated regulation of MTs and the actin cytoskeleton is poorly understood. LIM kinase 1 (LIMK1) regulates actin polymerization by phosphorylating the actin depolymerization factor, cofilin. Here we report that LIMK1 is also involved in the MT destabilization. In endothelial cells endogenous LIMK1 co-localizes with MTs and forms a complex with tubulin via the PDZ domain. MT destabilization induced by thrombin or nocodazole resulted in a decrease of LIMK1 colocalization with MTs. Overexpression of wild type LIMK1 resulted in MT destabilization, whereas the kinase-dead mutant of LIMK1 (KD) did not affect MT stability. Importantly, down-regulation of endogenous LIMK1 by small interference RNA resulted in abrogation of the thrombin-induced MTs destabilization and the inhibition of thrombin-induced actin polymerization. Expression of Rho kinase 2, which phosphorylates and activates LIMK1, dramatically decreases the interaction of LIMK1 with tubulin but increases its interaction with actin. Interestingly, expression of KD-LIMK1 or small interference RNA-LIMK1 prevents thrombin-induced microtubule destabilization and F-actin formation, suggesting that LIMK1 activity is required for thrombin-induced modulation of microtubule destabilization and actin polymerization. Our findings indicate that LIMK1 may coordinate microtubules and actin cytoskeleton.

Presynaptic and Postsynaptic Roles of NO, cGK, and RhoA in Long-Lasting Potentiation and Aggregation of Synaptic Proteins

Recent results suggest that long-lasting potentiation at hippocampal synapses involves the rapid formation of clusters or puncta of presynaptic as well as postsynaptic proteins, both of which are blocked by antagonists of NMDA receptors and an inhibitor of actin polymerization. We have investigated whether the increase in puncta involves retrograde signaling through the NO-cGMP-cGK pathway and also examined the possible roles of two classes of molecules that regulate the actin cytoskeleton: Ena/VASP proteins and Rho GTPases. Our results suggest that NO, cGMP, cGK, actin, and Rho GTPases including RhoA play important roles in the potentiation and act directly in both the presynaptic and postsynaptic neurons, where they contribute to the increase in puncta of synaptic proteins. cGK phosphorylates synaptic VASP during the potentiation, whereas Rho GTPases act both in parallel and upstream of cGMP, in part by maintaining the synaptic localization of soluble guanylyl cyclase.

Simultaneous stretching and contraction of stress fibers in vivo.

To study the dynamics of stress fiber components in cultured fibroblasts, we expressed alpha-actinin and the myosin II regulatory myosin light chain (MLC) as fusion proteins with green fluorescent protein. Myosin activation was stimulated by treatment with calyculin A, a serine/threonine phosphatase inhibitor that elevates MLC phosphorylation, or with LPA, another agent that ultimately stimulates phosphorylation of MLC via a RhoA-mediated pathway. The resulting contraction caused stress fiber shortening and allowed observation of changes in the spacing of stress fiber components. We have observed that stress fibers, unlike muscle myofibrils, do not contract uniformly along their lengths. Although peripheral regions shortened, more central regions stretched. We detected higher levels of MLC and phosphorylated MLC in the peripheral region of stress fibers. Fluorescence recovery after photobleaching revealed more rapid exchange of myosin and alpha-actinin in the middle of stress fibers, compared with the periphery. Surprisingly, the widths of the myosin and alpha-actinin bands in stress fibers also varied in different regions. In the periphery, the banding patterns for both proteins were shorter, whereas in central regions, where stretching occurred, the bands were wider.