Stress Fiber Contraction Research Articles

Simultaneous Stretching and Contraction of Stress Fibers In Vivo

Simultaneous Stretching and Contraction of Stress Fibers In Vivo

Mechanisms of force generation and transmission by myofibroblasts.

The myofibroblast has been shown to have a key role in tissue reconstruction after injury and pathological changes characterized by fibrosis. Force generation by the myofibroblast depends on the isometric contraction of stress fibers containing alpha-smooth muscle actin, and is mediated through Rho/Rho-kinase. The force is transmitted by vinculin and tensin-containing "supermature" focal adhesions, which connect stress fibers with the extracellular matrix. Force production and transmission by the myofibroblast are modulated by the coordinated action of cytokines, extracellular matrix components and mechanical tension. Regulation of these phenomena will be important for therapeutic strategies aimed at influencing fibrocontractive diseases.

Change of the Topography of Ventral Cell Surface during Spreading of Fibroblasts as Revealed by Evanescent Wave-Excited Fluorescence Microscopy: Effect of Contractility and Microtubule Integrity

Evanescent wave-excited fluorescence microscopy, which selectively probes the ventral membranes of cells adhered to glass substrate, was utilized to observe the change in the topography of the ventral plasma membranes of Swiss 3T3 fibroblasts during spreading. In the initial stage of the spreading (up to 2 hours after seeding), the ventral membrane was close (<100nm) to the substrate in the peripheral and the central regions. About 4 hrs after seeding, the ventral surface assumed a flat topography for a short period and then gradually became uneven, displaying streak pattern of cell-to-substrate contact (6-8 hours after seeding). By 24 hours after seeding, cells gained polygonal shape and most regions except for the focal adhesions were separated from the substrate. Within these well-spread cells actin stress fibers were found to emanate obliquely from the focal adhesions, as previously reported. When cells were grown in the presence of 2, 3-butanedione monoxime (BDM), an inhibitor of actomyosin-based contraction of stress fibers and the cell, the ventral membranes in majority of the cells displayed flat topography, and the tilt of the stress fibers decreased. Cells grown in the presence of colchicine, a microtubule-depolymerizing agent also possessed flat ventral membrane and less tilted stress fibers. These results suggest that the contraction of stress fibers and integrity of microtubules are important in the formation of the uneven topography of ventral membrane and the tilt of stress fibers.

Stress fiber organization regulated by MLCK and Rho-kinase in cultured human fibroblasts.

To understand the roles of Rho-kinase and myosin light chain kinase (MLCK) for the contraction and organization of stress fibers, we treated cultured human foreskin fibroblasts with several MLCK, Rho-kinase, or calmodulin inhibitors and analyzed F-actin organization in the cells. Some cells were transfected with green fluorescent protein (GFP)-labeled actin, and the effects of inhibitors were also studied in these living cells. The Rho-kinase inhibitors Y-27632 and HA1077 caused disassembly of stress fibers and focal adhesions in the central portion of the cell within 1 h. However, stress fibers located in the periphery of the cell were not severely affected by the Rho-kinase inhibitors. When these cells were washed with fresh medium, the central stress fibers and focal adhesions gradually reformed, and within 3 h the cells were completely recovered. ML-7 and KT5926 are specific MLCK inhibitors and caused disruption and/or shortening of peripheral stress fibers, leaving the central fibers relatively intact even though their number was reduced. The calmodulin inhibitors W-5 and W-7 gave essentially the same results as the MLCK inhibitors. The MLCK and calmodulin inhibitors, but not the Rho-kinase inhibitors, caused cells to lose the spread morphology, indicating that the peripheral fibers play a major role in keeping the flattened state of the cell. When stress fiber models were reactivated, the peripheral fibers contracted before the central fibers. Thus our study shows that there are at least two different stress fiber systems in the cell. The central stress fiber system is dependent more on the activity of Rho-kinase than on that of MLCK, while the peripheral stress fiber system depends on MLCK.

Rho-kinase--mediated contraction of isolated stress fibers.

It is widely accepted that actin filaments and the conventional double-headed myosin interact to generate force for many types of nonmuscle cell motility, and that this interaction occurs when the myosin regulatory light chain (MLC) is phosphorylated by MLC kinase (MLCK) together with calmodulin and Ca(2+). However, recent studies indicate that Rho-kinase is also involved in regulating the smooth muscle and nonmuscle cell contractility. We have recently isolated reactivatable stress fibers from cultured cells and established them as a model system for actomyosin-based contraction in nonmuscle cells. Here, using isolated stress fibers, we show that Rho-kinase mediates MLC phosphorylation and their contraction in the absence of Ca(2+). More rapid and extensive stress fiber contraction was induced by MLCK than was by Rho-kinase. When the activity of Rho-kinase but not MLCK was inhibited, cells not only lost their stress fibers and focal adhesions but also appeared to lose cytoplasmic tension. Our study suggests that actomyosin-based nonmuscle contractility is regulated by two kinase systems: the Ca(2+)-dependent MLCK and the Rho-kinase systems. We propose that Ca(2+) is used to generate rapid contraction, whereas Rho-kinase plays a major role in maintaining sustained contraction in cells.

Isolation and in vitro contraction of stress fibers

Isolated stress fibers are useful for studying their properties and functions, but they are also useful as a nonmuscle contraction model system for investigating the regulatory mechanism for the actomyosin-based contractility in nonmuscle cells. This chapter discusses the isolation of stress fibers en masse from cultured cells. It also describes the way to observe contraction of stress fibers. The isolation procedure is simple and provides stress fibers that are pure enough for biochemical as well as structural and other studies. The contraction of stress fibers is best observed by using those still attached to the substrate surface. The contraction is an actomyosin-based, ATP-driven contraction. Rho kinase is involved in stress fiber contraction. Both RhoA and Rho kinase are present in isolated stress fibers. The chapter explains that the stress fiber model can contract in the absence of Ca 2+ and that this contraction is inhibited by a Rho kinase inhibitor. In this case, the myosin regulatory light chain is phosphorylated, not by myosin light chain kinase, but by Rho kinase. The studies presented in the chapter indicate that stress fiber contraction is regulated by two independent systems: one by the Ca 2+ - dependent myosin light chain kinase system and the other by the Ca 2+ - independent Rho kinase system.

Properties of Filament-bound Myosin Light Chain Kinase

Myosin light chain kinase binds to actin-containing filaments from cells with a greater affinity than to F-actin. However, it is not known if this binding in cells is regulated by Ca2+/calmodulin as it is with F-actin. Therefore, the binding properties of the kinase to stress fibers were examined in smooth muscle-derived A7r5 cells. Full-length myosin light chain kinase or a truncation mutant lacking residues 2-142 was expressed as chimeras containing green fluorescent protein at the C terminus. In intact cells, the full-length kinase bound to stress fibers, whereas the truncated kinase showed diffuse fluorescence in the cytoplasm. After permeabilization with saponin, the fluorescence from the truncated kinase disappeared, whereas the fluorescence of the full-length kinase was retained on stress fibers. Measurements of fluorescence intensities and fluorescence recovery after photobleaching of the full-length myosin light chain kinase in saponin-permeable cells showed that Ca2+/calmodulin did not dissociate the kinase from these filaments. However, the filament-bound kinase was sufficient for Ca2+-dependent phosphorylation of myosin regulatory light chain and contraction of stress fibers. Thus, dissociation of myosin light chain kinase from actin-containing thin filaments is not necessary for phosphorylation of myosin light chain in thick filaments. We note that the distance between the N terminus and the catalytic core of the kinase is sufficient to span the distance between thin and thick filaments.

Isolation and contraction of the stress fiber.

Stress fibers were isolated from cultured human foreskin fibroblasts and bovine endothelial cells, and their contraction was demonstrated in vitro. Cells in culture dishes were first treated with a low-ionic-strength extraction solution and then further extracted using detergents. With gentle washes by pipetting, the nucleus and the apical part of cells were removed. The material on the culture dish was scraped, and the freed material was forced through a hypodermic needle and fractionated by sucrose gradient centrifugation. Isolated, free-floating stress fibers stained brightly with fluorescently labeled phalloidin. When stained with anti-alpha-actinin or anti-myosin, isolated stress fibers showed banded staining patterns. By electron microscopy, they consisted of bundles of microfilaments, and electron-dense areas were associated with them in a semiperiodic manner. By negative staining, isolated stress fibers often exhibited gentle twisting of microfilament bundles. Focal adhesion-associated proteins were also detected in the isolated stress fiber by both immunocytochemical and biochemical means. In the presence of Mg-ATP, isolated stress fibers shortened, on the average, to 23% of the initial length. The maximum velocity of shortening was several micrometers per second. Polystyrene beads on shortening isolated stress fibers rotated, indicating spiral contraction of stress fibers. Myosin regulatory light chain phosphorylation was detected in contracting stress fibers, and a myosin light chain kinase inhibitor, KT5926, inhibited isolated stress fiber contraction. Our study demonstrates that stress fibers can be isolated with no apparent loss of morphological features and that they are truly contractile organelle.

Fluorescence anisotropy imaging microscopy maps calmodulin binding during cellular contraction and locomotion.

Calmodulin is a calcium transducer that activates key regulatory and structural proteins through calcium-induced binding to the target proteins. A fluorescent analog of calmodulin in conjunction with ratio imaging, relative to a volume indicator, has demonstrated that calmodulin is uniformly distributed in serum-deprived fibroblasts and there is no immediate change in the distribution upon stimulation with complete serum. The same fluorescent analog of calmodulin together with steady state fluorescence anisotropy imaging microscopy has been used to define the temporal and spatial changes in calmodulin binding to cellular targets during stimulation of serum-deprived fibroblasts and in polarized fibroblasts during wound healing. In serum-deprived fibroblasts, which exhibit a low free calcium ion concentration, a majority of the fluorescent analog of calmodulin remained unbound (fraction bound, fB < 10%). However, upon stimulation of the serum-deprived cells with complete serum, calmodulin binding (maximum fB approximately 95%) was directly correlated with the time course of the elevation and decline of the free calcium ion concentration, while the contraction of stress fibers continued for an hour or more. Calmodulin binding was also elevated in the leading lamellae of fibroblasts (maximum FB approximately 50%) during the lamellar contraction phase of wound healing and was spatially correlated with the contraction of transverse fibers containing myosin II. Highly polarized and motile fibroblasts exhibited the highest anisotropy (calmodulin binding) in the retracting tails and in association with contracting transverse fibers in the cortex of the cell. These results suggest that local activation of myosin II-based contractions involves the local binding of calmodulin to target proteins. The results also demonstrate a powerful yet simple mode of light microscopy that will be valuable for mapping molecular binding of suitably labeled macromolecules in living cells.

Relative distribution of actin, myosin I, and myosin II during the wound healing response of fibroblasts.

Myosin I is present in Swiss 3T3 fibroblasts and its localization reflects a possible involvement in the extension and/or retraction of protrusions at the leading edge of locomoting cells and the transport of vesicles, but not in the contraction of stress fibers or transverse fibers. An affinity-purified polyclonal antibody to brush border myosin I colocalizes with a polypeptide of 120 kD in fibroblast extracts. Within initial protrusions of polarized, migrating fibroblasts, myosin I exhibits a punctate distribution, whereas actin is diffuse and myosin II is absent. Myosin I also exists in linear arrays parallel to the direction of migration in filopodia and microspikes, established protrusions, and within the leading lamellae of migrating cells. Myosin II and actin colocalize along transverse fibers in the lamellae of migrating cells, while myosin I displays no definitive organization along these fibers. During contractions of actin-based fibers, myosin II is concentrated in the center of the cell, while the distribution of myosin I does not change. Thus, myosin I is found at the correct location and time to be involved in the extension and/or retraction of protrusions and the transport of vesicles. Myosin II-based contractions in more posterior cellular regions could generate forces to separate cells, maintain a polarized cell shape, maintain the direction of locomotion, maximize the rate of locomotion, and/or aid in the delivery of cytoskeletal/contractile subunits to the leading edge.

Temporal and Spatial Dynamics of the Actin-Based Cytoskeleton

There has been a renaissance and revolution in the use of light microscopy in the biomedical sciences. The renaissance has been due to the importance of studying the temporal and spatial dynamics of ions, metabolites and macromolecules in living cells and tissues. The revolution has been due to the integration of developments in molecular biology, fluorescent probe chemistry, machine vision, and imaging technology. It is now possible to use the living cell as a microcuvette and to explore the chemical and molecular dynamics responsible for cellular functions.We have been investigating the formation, transport and contraction of stress fibers in Swiss 3T3 cells. Fluorescent analogs of actin, myosin, vinculin and profilin have been investigated in serum deprived cells before, during and after stimulation with thrombin. The activities of these components of the actin-based cytoskeleton have been quantified using time-lapse imaging, fluorescence redistribution after photobleaching, video-enhanced contrast and reflection interference contrast microscopy.

Formation, transport, contraction, and disassembly of stress fibers in fibroblasts

Swiss mouse 3T3 fibroblasts grown on a solid substrate in the presence of 10% serum exhibit cell movement, organelle transport, and cytokinesis. When the serum concentration in the culture medium is decreased to 0.2% for 48 h the serum-deprived cells virtually stop locomoting, spread, decreased organelle transport, and exhibit extensive arrays of stress fibers that are visible with video-enhanced differential interference contrast microscopy and that also incorporate fluorescent analogs of actin and conventional myosin (myosin II). The stress fibers form in a constitutive manner at the cytoplasm-membrane interface, transport toward the nucleus, and then disappear. The rate of transport of these fibers is quite heterogeneous with average rates in the range of 10-20 microns/h. When serum-deprived cells are stimulated with mitogens such as 10% serum or 10 nM thrombin, many of the stress fibers immediately begin to shorten, suggesting a contraction. The rate of shortening is approximately two orders of magnitude slower than that of unloaded smooth muscle cells. The fiber shortening is often accompanied by retraction of the edges of the cell and continues for at least the 1st hour post-stimulation.

Reorganization of α-actinin and vinculin in living cells following ATP depletion and replenishment

Although it is known that the depletion of cellular ATP induces a dramatic, reversible disruption of microfilament structures, the morphological pathway remains obscure. I have studied this process by following directly the dynamic redistribution of fluorescently labeled α-actinin and vinculin which had been microinjected into living mouse 3T3 fibroblasts. Before treatment, microinjected α-actinin displayed characteristic distribution along stress fibers, whereas vinculin was localized predominantly at adhesion plaques. The first response after adding NaN 3 and 2-deoxyglucose was the retraction of lamellipodia, followed, over a period of 2 h, by a dramatic contraction of stress fibers and loosening of focal contacts. Vinculin plaques shrank from an elongated shape to small aggregates. During recovery, which was initiated by removing NaN 3 and 2-deoxyglucose from the medium, lamellipodia appeared rapidly and α-actinin dispersed from contracted aggregates. Some partially dispersed aggregates later served as initiation sites for the formation of stress fibers. The recovery of vinculin plaques occurred predominantly through direct elongation, and focal contacts developed concomitantly. A small fraction of vinculin aggregates, however, moved into the perinuclear region without developing into adhesion plaques, and some new vinculin plaques formed de novo. Possible mechanisms involved and relationships to disruptions induced by other agents are discussed.

Stress fibers in the splenic sinus endothelium in situ: molecular structure, relationship to the extracellular matrix, and contractility.

In the present study, we investigated structural and functional aspects of stress fibers in a cell type in situ, i.e., the sinus endothelium of the human spleen. In this cell type, stress fibers extend underneath the basal plasma membrane and are arranged parallel to the cellular long axis. Ultrastructurally, the stress fibers were found to be composed of thin actin-like filaments (5-8 nm) and thick myosin-like filaments (10-15 nm X 300 nm). Actin filaments displayed changes in polarity (determined by S-1-myosin subfragment decoration), which may allow a sliding filament mechanism. At their plasmalemmal attachment sites, actin filaments exhibited uniform polarity with the S-1-arrowhead complexes pointing away from the plasma membrane. Fluorescence microscopy showed that the stress fibers have a high affinity for phalloidin and antibodies to actin, myosin, tropomyosin, and alpha-actinin. Vinculin was confined to the cytoplasmic aspect of the plasmalemmal termination sites of stress fibers, while laminin, fibronectin, and collagens were located at the extracellular aspect of these stress fiber-membrane associations. Western blot analysis revealed polypeptide bands that contained actin, myosin, and alpha-actinin to be major components of isolated cells. Exposure of permeabilized cells to MgATP results in prominent changes in cellular shape caused by stress fiber contraction. It is concluded that the stress fibers in situ anchored to cell-to-extracellular matrix contacts can create tension that might allow the endothelium to resist the fluid shear forces of blood flow.