113 Effects of Cyclic Strain at Focal Adhesions on a Cell
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Strain (injury)
Focal point
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Mechanotransduction
mechanobiology
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Cells sense the mechanical properties of their surrounding environment and activate intracellular signaling pathways that play important roles in cell survival, proliferation, differentiation, and migration. Migration of cells into an injury site is crucial for repair after injury and requires cytoskeletal reorganization and remodeling of focal adhesions that connect the cytoskeleton to the extracellular matrix. Thus, it is possible that a directional cyclic stretch stimulation of cells may facilitate the wound healing process and establish ordered tissue formation. Here, we investigated the effects of directional cyclic uniaxial stretch on wound repair processes of monolayer epithelial-like cells that was scratch wounded. We controlled the direction of scratched wound in cell tissue to be i) perpendicular to the stretch direction (perpendicular stretch), ii) parallel to the direction of the zero normal strain in the substrate θ0 (～60º) (oblique stretch), and iii) parallel to stretch direction (parallel stretch). We found that cyclic stretching perpendicular to the scratched wound direction did not improve cell migration, whereas oblique stretching, by which cells were induced to align in the zero normal strain direction θ0, significantly facilitated cell migration for wound closure even though the migration direction was varied. We further found that cell migration for wound closure was improved most efficiently by cyclic stretching parallel to the wound direction, which facilitated polymerization of actin cytoskeleton aligning in the migration direction and vinculin–actin interactions. These results indicate that cell migration for wound healing is significantly influenced not only by the normal strain applied to cells but also by shear strain under cyclic strain fields, and cells for wound healing preferentially migrate to the direction in which both the normal and shear strains applied to them become smaller.
Vinculin
Mechanotransduction
Stress fiber
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This paper describes a micro device which applies cyclic strain to focal adhesions of a cell. In recent years, evidence has been growing that focal adhesions act as mechanosensors of cells which convert mechanical force into biomechanical signaling. However, there are no effective micro devices which can directly apply mechanical stimulation to each focal adhesion. Here we develop a micropillar substrate embedding micron-sized magnetic particles and enabling the micropillars to be deflected by external magnetic field. The top of the micropillars were coated with a fibronectin, a kind of protein which promotes the adhesion of cells. Moreover, we stained this protein to check the condition of the coating and to detect the position of pillars. Using the magnetic micropillar substrate, we observed the deformation of an osteoblast cell at its focal adhesions. The findings indicate that the present micro device can be used to investigate mechanosensing systems of a cell.
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Hemodynamic shear stress regulates endothelial cell biochemical processes that govern cytoskeletal contractility, focal adhesion dynamics, and extracellular matrix (ECM) assembly. Since shear stress causes rapid strain focusing at discrete locations in the cytoskeleton, we hypothesized that shear stress coordinately alters structural dynamics in the cytoskeleton, focal adhesion sites, and ECM on a time scale of minutes. Using multiwavelength four-dimensional fluorescence microscopy, we measured the displacement of rhodamine-fibronectin and green fluorescent protein-labeled actin, vimentin, paxillin, and/or vinculin in aortic endothelial cells before and after onset of steady unidirectional shear stress. In the cytoskeleton, the onset of shear stress increased actin polymerization into lamellipodia, altered the angle of lateral displacement of actin stress fibers and vimentin filaments, and decreased centripetal remodeling of actin stress fibers in subconfluent and confluent cell layers. Shear stress induced the formation of new focal complexes and reduced the centripetal remodeling of focal adhesions in regions of new actin polymerization. The structural dynamics of focal adhesions and the fibronectin matrix varied with cell density. In subconfluent cell layers, shear stress onset decreased the displacement of focal adhesions and fibronectin fibrils. In confluent monolayers, the direction of fibronectin and focal adhesion displacement shifted significantly toward the downstream direction within 1 min after onset of shear stress. These spatially coordinated rapid changes in the structural dynamics of cytoskeleton, focal adhesions, and ECM are consistent with focusing of mechanical stress and/or strain near major sites of shear stress-mediated mechanotransduction.
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Cells respond to fluid shear stress through dynamic processes involving changes in actomyosin and other cytoskeletal stresses, remodeling of cell adhesions, and cytoskeleton reorganization. In this study we simultaneously measured focal adhesion dynamics and cytoskeletal stress and reorganization in MDCK cells under fluid shear stress. The measurements used co-expression of fluorescently labeled paxillin and force sensitive FRET probes of α-actinin. A shear stress of 0.74 dyn/cm(2) for 3 hours caused redistribution of cytoskeletal tension and significant focal adhesion remodeling. The fate of focal adhesions is determined by the stress state and stability of the linked actin stress fibers. In the interior of the cell, the mature focal adhesions disassembled within 35-40 min under flow and stress fibers disintegrated. Near the cell periphery, the focal adhesions anchoring the stress fibers perpendicular to the cell periphery disassembled, while focal adhesions associated with peripheral fibers sustained. The diminishing focal adhesions are coupled with local cytoskeletal stress release and actin stress fiber disassembly whereas sustaining peripheral focal adhesions are coupled with an increase in stress and enhancement of actin bundles. The results show that flow induced formation of peripheral actin bundles provides a favorable environment for focal adhesion remodeling along the cell periphery. Under such condition, new FAs were observed along the cell edge under flow. Our results suggest that the remodeling of FAs in epithelial cells under flow is orchestrated by actin cytoskeletal stress redistribution and structural reorganization.
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Vinculin
mechanobiology
Paxillin
Mechanotransduction
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Endothelial cell spreading is mediated by the extracellular matrix (ECM), and it is the thought to be regulated by the binding of integrin receptors and the associated focal adhesions (FA). In the present study, we examined the relationship between FA formation and endothelial cell shape changes using microfabricated substrates patterned with islands of immobilized fibronectin (FN) surrounded by nonadhesive borders to control the degree of cell spreading. The amount of FA formation, as detected by the localized presence of vinculin and phosphotyrosine, increased in direct proportion to the degree of cell spreading. To determine whether the observed increase in FA formation was a direct result of more fibronectin in contact with spread cells, single cells were spread across multiple subcellular sized islands such that the size and spacing of the islands could vary cell spreading independently from the amount of cell-ECM contact. We found that total FA increased with cell spreading, regardless of the amount of ECM in contact with cells. The FAs formed on these substrates developed a structural directionality and quantity which correlated to the direction and magnitude of tensional stress in the actin cytoskeleton. Thus, global changes in cell shape regulate the local cytoskeletal tensions that drives FA formation.
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Although the roles of focal adhesion that adhere to the surface and sense cellular environment in order to regulate cellular behavior have been reported, the topographical effects of cell substrates on focal adhesion are not entirely understood. Here, we investigated the spatial property of focal adhesion when culturing on the substrates with microtopography. Polydimethylsiloxane (PDMS) substrates with grid micropatterns fabricated by soft lithography were used as substrates for culturing cells. We found that on the grid micropattern 1μm high, focal adhesion complex were aggregated on the top surface of the grid, whereas they formed on the bottom areas of the substrate over the height of 2μm. On the other hand, focal adhesion sites were not changed by myosin II and the rigidity of substrates and actin filament formation. These results show that the location of focal adhesion formation on the grid micropatterned substrates is critically regulated by the height of topography.
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Endothelial cells (ECs) adapt to mechanical environments such as cyclic stretching by altering their morphology and cytoskeletal structure. The detailed mechanism underlying EC remodeling in response to cyclic stretching, however, remains unclear. To understand the contribution of strain in contact area between focal adhesions (FAs) and the substrate to morphological and cytoskeletal changes in cells, we applied cyclic stretching to ECs using a microsubstrate with arrays of micropillars on which cells were selectively stretched between FAs but FA-substrate contact area were hardly stretched. Bovine aortic ECs were seeded on a silicone elastomer micropillar substrate in a silicone chamber. ECs were then subjected to 20% stretching at 0.5 Hz for up to 6 h using a stretching apparatus. Cells stretched on a flat substrate were also observed. Under static conditions, no significant difference was seen in EC morphology between flat and micropillar substrates. After exposure to cyclic stretching for 3 h, ECs on both flat and micropillar substrates were aligned perpendicular to the direction of stretching. Stress fibers were oriented about 60° to the direction of stretching on the flat substrate, while stress fibers were not aligned in any direction for the micropillar substrate. After 6 h of stretching, stress fibers on the micropillar substrate were oriented approximately 90° to the direction of stretching. These results suggest that strain in contact area between FAs and the substrate may have an impact on reorientation rates of stress fibers in ECs in response to cyclic stretching.
Strain (injury)
Stress fiber
Morphology
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