Dynamic Fibroblast Cultures: Response to Mechanical Stretching (original) (raw)
Related papers
Experimental Cell Research, 2001
Cell lines derived from rat aorta and frog kidney were cultured on elastic membrane, and mechanical stress was given to the cells by stretching the membrane periodically. Cell reorientation oblique to the direction of stretching occurred as a result of the rapid withdrawal of cell periphery located along the direction of stretching and gradual extension of the cell membrane toward the direction oblique to the direction of stretching. Dynamic reorganization of stress fibers in living cells was visualized by labeling stress fibers with TRITC 3-actin or EGFP-tagged moesin fragments with actin-binding ability. Stress fibers aligned in the direction of stretching disappeared soon after the start of stretching and then obliquely reoriented stress fibers appeared. The stretchinduced reorientation of cultured cells was suppressed by an inhibitor of stretch-activated (SA) cation channels and by a Ca 2؉ chelator. However, the rearrangement of stress fibers was not affected by these agents. From these results, we suggest that Ca 2؉ influx via SA channels is involved in stretch-induced cell reorientation but stress fiber rearrangement is independent of SA channels. Therefore, cell reorientation does not simply depend on the arrangement of stress fibers but may be controlled by some additional mechanism(s) which is regulated by calcium signaling.
The Direction of Stretch-Induced Cell and Stress Fiber Orientation Depends on Collagen Matrix Stress
PLoS ONE, 2014
Cell structure depends on both matrix strain and stiffness, but their interactive effects are poorly understood. We investigated the interactive roles of matrix properties and stretching patterns on cell structure by uniaxially stretching U2OS cells expressing GFP-actin on silicone rubber sheets supporting either a surface-adsorbed coating or thick hydrogel of type-I collagen. Cells and their actin stress fibers oriented perpendicular to the direction of cyclic stretch on collagen-coated sheets, but oriented parallel to the stretch direction on collagen gels. There was significant alignment parallel to the direction of a steady increase in stretch for cells on collagen gels, while cells on collagen-coated sheets did not align in any direction. The extent of alignment was dependent on both strain rate and duration. Stretch-induced alignment on collagen gels was blocked by the myosin light-chain kinase inhibitor ML7, but not by the Rho-kinase inhibitor Y27632. We propose that active orientation of the actin cytoskeleton perpendicular and parallel to direction of stretch on stiff and soft substrates, respectively, are responses that tend to maintain intracellular tension at an optimal level. Further, our results indicate that cells can align along directions of matrix stress without collagen fibril alignment, indicating that matrix stress can directly regulate cell morphology. Citation: Tondon A, Kaunas R (2014) The Direction of Stretch-Induced Cell and Stress Fiber Orientation Depends on Collagen Matrix Stress. PLoS ONE 9(2): e89592.
Cellular and Matrix Mechanics of Bioartificial Tissues During Continuous Cyclic Stretch
Annals of Biomedical Engineering, 2006
Bioartificial tissues are useful model systems for studying cell and extra-cellular matrix mechanics. These tissues provide a 3D environment for cells and allow tissue components to be easily modified and quantified. In this study, we fabricated bioartificial tissue rings from a 1 ml solution containing one million cardiac fibroblasts and 1 mg collagen. After 8 days, rings compacted to <1% of original volume and cell number increased 2.4 fold. We initiated continuous cyclic stretching of the rings after 2, 4, or 8 days of incubation, while monitoring the tissue forces. Peak tissue force during each cycle decreased rapidly after initiating stretch, followed by further slow decline. We added 2 µM Cytochalasin-D to some rings prior to initiation of stretch to determine the force contributed by the matrix. Cell force was estimated by subtracting matrix force from tissue force. After 12 h, matrix force-strain curves were highly nonlinear. Cell force-strain curves were linear during loading and showed hysteresis indicating viscoelastic behavior. Cell stiffness increased with stretching frequency from 0.001-0.25 Hz. Cell stiffness decreased with stretch amplitude (5-25%) at 0.1 Hz. The trends in cell stiffness do not fit simple viscoelastic models previously proposed, and suggest possible strain-amplitude related changes during cyclic stretch.
A Model for Stress Fiber Realignment Caused by Cytoskeletal Fluidization During Cyclic Stretching
Cellular and Molecular Bioengineering, 2011
Uniaxial cyclic substrate stretching results in a concerted change of cytoskeletal organization such that stress fibers (SFs) realign away from the direction of stretching. Recent experiments revealed that brief transient stretch promptly ablates cellular contractile stress by means of cytoskeletal fluidization, followed by a slow stress recovery by means of resolidification. This, in turn, suggests that fluidization, resolidification and SF realignment may be linked together during stretching. We propose a mathematical model that simulates the effects of fluidization and resolidification on cytoskeletal contractile stress in order to investigate how these phenomena affect cytoskeletal realignment in response to pure uniaxial stretching of the substrate. The model comprises of individual elastic SFs anchored at the endpoints to an elastic substrate. Employing the global stability convention, the model predicts that in response to repeated stretch–unstretch cycles, SFs tend to realign in the direction perpendicular to stretching, consistent with data from the literature. The model is used to develop a computational scheme for predicting changes in cell orientation and polarity during stretching and how they relate to the underlying alterations in the cytoskeletal organization. We conclude that depletion of cytoskeletal contractile stress by means of fluidization and subsequent stress recovery by means of resolidification may play a key role in reorganization of cytoskeletal SFs in response to uniaxial stretching of the substrate.
Cell shape-dependent early responses of fibroblasts to cyclic strain
Randomly spread fibroblasts on fibronectin-coated elastomeric membranes respond to cyclic strain by a varying degree of focal adhesion assembly and actin reorganization. We speculated that the individual shape of the cells, which is linked to cytoskeletal structure and pre-stress, might tune these integrin-dependent mechanotransduction events. To this aim, fibronectin circles, squares and rectangles of identical surface area (2000 μm 2 ) were microcontact printed onto elastomeric substrates. Fibroblasts plated on these patterns occupied the corresponding shapes. Cyclic 10% equibiaxial strain was applied to patterned cells for 30 min, and changes in cytoskeleton and cell-matrix adhesions were quantified after fluorescence staining. After strain, megakaryocytic leukemia-1 protein translocated to the nucleus in most cells, indicating efficient RhoA activation independently of cell shape. However, circular and square cells (with radial symmetry) showed a significantly greater increase in the number of actin stress fibers and vinculin-positive focal adhesions after cyclic strain than rectangular (bipolar) cells of identical size. Conversely, cyclic strain induced larger changes in pY397-FAK positive focal complexes and zyxin relocation from focal adhesions to stress fibers in bipolar compared to symmetric cells. Thus, radially symmetric cells responded to cyclic strain with a larger increase in assembly, whereas bipolar cells reacted with more pronounced reorganization of actin stress fibers and matrix contacts. We conclude that integrin-mediated responses to external mechanical strain are differentially modulated in cells that have the same spreading area but different geometries, and do not only depend on mere cell size.
PLoS ONE, 2013
The actin cytoskeleton plays a crucial role for the spreading of cells, but is also a key element for the structural integrity and internal tension in cells. In fact, adhesive cells and their actin stress fiber-adhesion system show a remarkable reorganization and adaptation when subjected to external mechanical forces. Less is known about how mechanical forces alter the spreading of cells and the development of the actin-cell-matrix adhesion apparatus. We investigated these processes in fibroblasts, exposed to uniaxial cyclic tensile strain (CTS) and demonstrate that initial cell spreading is stretch-independent while it is directed by the mechanical signals in a later phase. The total temporal spreading characteristic was not changed and cell protrusions are initially formed uniformly around the cells. Analyzing the actin network, we observed that during the first phase the cells developed a circumferential arc-like actin network, not affected by the CTS. In the following orientation phase the cells elongated perpendicular to the stretch direction. This occurred simultaneously with the de novo formation of perpendicular mainly ventral actin stress fibers and concurrent realignment of cell-matrix adhesions during their maturation. The stretch-induced perpendicular cell elongation is microtubule-independent but myosin II-dependent. In summary, a CTS-induced cell orientation of spreading cells correlates temporary with the development of the actomyosin system as well as contact to the underlying substrate by cell-matrix adhesions.
Study of Cell Orientation Alignment in Response to Cyclic Mechanical Stresses
2013
A number of studies have been performed on the subject of ‘Cell Orientation Alignment’ caused by mechanical stresses applied to sample substrates. Cells in most solid tissues (muscles, tendons, skin) have a characteristic alignment. How does this develop? Current research indicates that when cells on a two-dimensional substrate are subject to stress, either by stretching the substrate, by subjecting them to a well-aligned flow, or by other means, they reorient and align themselves in preferred directions. Figure 1: Cells before stretching Figure 2: Cells after stretching (Images 1-3 courtesy of Simon Jungbauer and Ralf Kemkemer, Max Planck Institut fuer Metallforschung, Stuttgart) An understanding of these responses of cells to mechanical stimuli will have implications for mechanical inhibition of cancer cells and promotion of stem cell activity. If the behavior of cells can be formulated mathematically it would make it would aid in detecting cancerous cells, making it easier for me...
Mechanical strain increases cell stiffness through cytoskeletal filament reorganization
American journal of physiology. Lung cellular and molecular physiology, 2003
We tested the hypothesis that cytoskeletal reorganization induced by cyclic strain increases cytoskeletal stiffness (G'). G' was measured by optical magnetic twisting cytometry in control cells and cells that had received mechanical strain for 10-12 days. G' was measured before and after both contractile and relaxant agonists, and in the strained cells both parallel (Para) and perpendicular (Perp) to the aligned cytoskeleton. Before activation, G' Para was 24 +/- 5% (+/- SE) greater compared with Perp (P < 0.05), and 35% +/- 6 greater compared with control (Cont, P…
Scientific Reports, 2011
It was our objective to study the role of mechanical stimulation on fibronectin (FN) reorganization and recruitment by exposing fibroblasts to shear fluid flow and equibiaxial stretch. Mechanical stimulation was also combined with a Rho inhibitor to probe their coupled effects on FN. Mechanically stimulated cells revealed a localization of FN around the cell periphery as well as an increase in FN fibril formation. Mechanical stimulation coupled with chemical stimulation also revealed an increase in FN fibrils around the cell periphery. Complimentary to this, fibroblasts exposed to fluid shear stress structurally rearranged pre-coated surface FN, but unstimulated and stretched cells did not. These results show that mechanical stimulation directly affected FN reorganization and recruitment, despite perturbation by chemical stimulation. Our findings will help elucidate the mechanisms of FN biosynthesis and organization by furthering the link of the role of mechanics with FN. F ibronectin (FN) is an abundant ECM protein secreted by cells as a soluble dimer and assembled into an insoluble fibrillar network at the cell surface, forming a FN matrix 1,2 . The complex, dynamic process of FN matrix assembly is a cell-mediated process that involves interactions between receptors on the cell surface and FN 2,3 . FN initially exists as a compact, folded structure that contains sets of binding domains for a variety of extracellular and cell surface molecules such as collagen, glycosaminoglycans, fibrin, integrins, and FN itself 2,4,5 . Although FN contains binding domains for many proteins, FN matrix assembly is believed to be induced by a conformational change through binding of FN to a5b1integrins on the cell surface 2,4,6,7 . This conformational change induces focal adhesion-integrin co-localization and subsequent integrin clustering and syndecan colocalization, which recruits signaling and cytoskeletal proteins . Integrin -cytoskeleton interactions have proved to be requisite for matrix assembly and mediated by the Rho family of GTPases, specifically Rho, Rac, and Cdc42 10-12 . For example lysophosphatidic acid (LPA)-induced Rho activation has been shown to induce actin stress fiber formation and enhance FN matrix assembly 13,14 while Rho inhibition has been shown to inhibit LPAinduced stress fiber formation and matrix assembly 10,13 . Therefore FN matrix assembly depends on the precise coordination and synergy between extracellular events and intracellular pathways.