Physically-Induced Cytoskeleton Remodeling of Cells in Three-Dimensional Culture (original) (raw)
Related papers
Dynamic Fibroblast Cultures: Response to Mechanical Stretching
Cell Adhesion & Migration, 2007
Mechanical forces play an important role in the organization, growth and function of tissues. Dynamic extracellular environment affects cellular behavior modifying their orientation and their cytoskeleton. In this work, human fibroblasts have been subjected for three hours to increasing substrate deformations (1-25%) applied as cyclic uniaxial stretching at different frequencies (from 0.25 Hz to 3 Hz). Our objective was to identify whether and in which ranges the different deformations magnitude and rate were the factors responsible of the cell alignment and if actin cytoskeleton modification was involved in these responses. After three hours of cyclically stretched substrate, results evidenced that fibroblasts aligned perpendicularly to the stretch direction at 1% substrate deformation and reached statistically higher orientation at 2% substrate deformation with unmodified values at 5-20%, while 25% substrate deformation induced cellular death. It was also shown that a percentage of cells oriented perpendicularly to the deformation were not influenced by increased frequency of cyclical three hours deformations (0.25-3 Hz). Cyclic substrate deformation was shown also to involve actin fibers which orient perpendicularly to the stress direction as well. Thus, we argue that a substrate deformation induces a dynamic change in cytoskeleton able to modify the entire morphology of the cells.
Journal of Biomechanics, 2008
The stress fiber network within contractile fibroblasts structurally reinforces and provides tension, or "tone", to tissues such as those found in healing wounds. Stress fibers have previously been observed to polymerize in response to mechanical forces. We observed that, when stretched sufficiently, contractile fibroblasts diminished the mechanical tractions they exert on their environment through depolymerization of actin filaments then restored tissue tension and rebuilt actin stress fibers through staged Ca ++ -dependent processes. These staged Ca ++ -modulated contractions consisted of a rapid phase that ended less than a minute after stretching, a plateau of inactivity, and a final gradual phase that required several minutes to complete. Active contractile forces during recovery scaled with the degree of rebuilding of the actin cytoskeleton. This complementary action demonstrates a programmed regulatory mechanism that protects cells from excessive stretch through choreographed active mechanical and biochemical healing responses.
Mapping Cell-Matrix Stresses during Stretch Reveals Inelastic Reorganization of the Cytoskeleton
Biophysical Journal, 2008
The mechanical properties of the living cell are intimately related to cell signaling biology through cytoskeletal tension. The tension borne by the cytoskeleton (CSK) is in part generated internally by the actomyosin machinery and externally by stretch. Here we studied how cytoskeletal tension is modified during stretch and the tensional changes undergone by the sites of cell-matrix interaction. To this end we developed a novel technique to map cell-matrix stresses during application of stretch. We found that cell-matrix stresses increased with imposition of stretch but dropped below baseline levels on stretch release. Inhibition of the actomyosin machinery resulted in a larger relative increase in CSK tension with stretch and in a smaller drop in tension after stretch release. Cell-matrix stress maps showed that the loci of cell adhesion initially bearing greater stress also exhibited larger drops in traction forces after stretch removal. Our results suggest that stretch partially disrupts the actinmyosin apparatus and the cytoskeletal structures that support the largest CSK tension. These findings indicate that cells use the mechanical energy injected by stretch to rapidly reorganize their structure and redistribute tension.
Biophysical Journal, 2006
Cells change their form and function by assembling actin stress fibers at their base and exerting traction forces on their extracellular matrix (ECM) adhesions. Individual stress fibers are thought to be actively tensed by the action of actomyosin motors and to function as elastic cables that structurally reinforce the basal portion of the cytoskeleton; however, these principles have not been directly tested in living cells, and their significance for overall cell shape control is poorly understood. Here we combine a laser nanoscissor, traction force microscopy, and fluorescence photobleaching methods to confirm that stress fibers in living cells behave as viscoelastic cables that are tensed through the action of actomyosin motors, to quantify their retraction kinetics in situ, and to explore their contribution to overall mechanical stability of the cell and interconnected ECM. These studies reveal that viscoelastic recoil of individual stress fibers after laser severing is partially slowed by inhibition of Rho-associated kinase and virtually abolished by direct inhibition of myosin light chain kinase. Importantly, cells cultured on stiff ECM substrates can tolerate disruption of multiple stress fibers with negligible overall change in cell shape, whereas disruption of a single stress fiber in cells anchored to compliant ECM substrates compromises the entire cellular force balance, induces cytoskeletal rearrangements, and produces ECM retraction many microns away from the site of incision; this results in large-scale changes of cell shape (. 5% elongation). In addition to revealing fundamental insight into the mechanical properties and cell shape contributions of individual stress fibers and confirming that the ECM is effectively a physical extension of the cell and cytoskeleton, the technologies described here offer a novel approach to spatially map the cytoskeletal mechanics of living cells on the nanoscale.
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.
Scientific Reports, 2022
wound contraction, and finally tissue remodeling. During the last phase of tissue remodeling, a systematic degradation of granulation tissue occurs and is replaced with a more organized and elastic ECM. This process may be induced by the secretion of matrix metalloproteinases (MMPs) during mechanical stimulation 10,11. Furthermore, mechanical stimulus directly applied to the site of injury has been shown to facilitate scar healing by decreasing blood flow and edema and increasing collagen turnover and ECM remodeling 12. The reductionist two-dimensional (2D) membrane models, in which mechanical strain is applied to a fibroblast monolayer have been instrumental in understanding the mechano-responsiveness of FMT 13-17. Additionally, 2D in vitro models studying the role of mechanical strain and stiffness on cell behavior provides the ability to quantify traction forces using time-lapse microscopy. However, these models fail to accurately mimic the interstitial feedback conditions from the surrounding ECM and adhesion fields experienced by the cells in vivo 18. Moreover, fibroblast culture in 2D models tends to resemble a pancake shape, spreading to cellular extensions and constraining its migration as compared to the spindle or stellate shape as observed in 3D or in vivo models. Thus, failing to accurately replicate cell migration, adhesion, proliferation, and further differentiation response to mechanical forces compared to models grown in 3D tissue analogues 19. Currently, 3D floated cell-encapsulated collagen lattices are the most utilized in vitro model to understand the interaction between fibroblast cells and the ECM and whether this interaction promotes myofibroblast differentiation 20-22. In the collagen lattice model, cells within the collagen create small tractional forces during the attachment and migration within the matrix which leads to a reduction in floated collagen lattice diameter 20-22. Besides collagen lattice models, other prominent studies created a 3D matrix model to investigate the role of changing ECM mechanical stiffness on fibrotic tissue formation and myofibroblast differentiation 23-26. Although these models enable fibroblast cells to interact with 3D ECM to study FMT, they ignore the extracellular mechanical loading exerted on the ECM and the residing cells. Yet, in clinical settings, the importance of extracellular mechanical loading on fibrotic tissue formation has been recognized for fibrosis-associated disorders. Mechanical loading-induced reorientation of the wound and pressure therapy are commonly used in the treatment of fibrotic tissue formation 10,27,28. Thus, understanding the pathophysiology of mechanical loading mediated fibroblast-tomyoblast transition is crucial for creating efficient therapies for musculoskeletal tissues operating under constant mechanical loading conditions. There remains an unanswered question regarding how the degree of mechanical loading applied to a fibroblast-laden 3D matrix and associated changes in structural and mechanical properties of the matrix affect the shift from normal tissue repair to fibroblast-to-myofibroblast transition. To this end, in this study, our objective was to investigate the dose-response relationship between various mechanical strain amplitudes applied to a 3D tissue analogue, subsequent structural changes within the tissue, and the molecular changes in residing fibroblasts that regulate the fibroblast transition from the homeostatic state to differentiated myofibroblasts. Methods and materials Synthesizing of fibroblast-laden three-dimensional tissue analogue and mechanical loading. The three-dimensional (3D) tissue analogue material was prepared using the most important structural
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.
Buckling of actin stress fibers: A new wrinkle in the cytoskeletal tapestry
Cell Motility and The Cytoskeleton, 2002
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. Cell Motil. Cytoskeleton 52:266–274, 2002. © 2002 Wiley-Liss, Inc.
Direct Observations of the Mechanical Behaviors of the Cytoskeleton in Living Fibroblasts
The Journal of Cell Biology, 1999
Cytoskeletal proteins tagged with green fluorescent protein were used to directly visualize the mechanical role of the cytoskeleton in determining cell shape. Rat embryo (REF 52) fibroblasts were deformed using glass needles either uncoated for purely physical manipulations, or coated with laminin to induce attachment to the cell surface. Cells responded to uncoated probes in accordance with a three-layer model in which a highly elastic nucleus is surrounded by cytoplasmic microtubules that behave as a jelly-like viscoelastic fluid. The third, outermost cortical layer is an elastic shell under sustained tension. Adhesive, laminin-coated needles caused focal recruitment of actin filaments to the contacted surface region and increased the cortical layer stiffness. This direct visualization of actin recruit-ment confirms a widely postulated model for mechanical connections between extracellular matrix proteins and the actin cytoskeleton. Cells tethered to laminintreated needles strongly resisted elongation by actively contracting. Whether using uncoated probes to apply simple deformations or laminin-coated probes to induce surface-to-cytoskeleton interaction we observed that experimentally applied forces produced exclusively local responses by both the actin and microtubule cytoskeleton. This local accomodation and dissipation of force is inconsistent with the proposal that cellular tensegrity determines cell shape.