ECM Mechano-Sensing Regulates Cytoskeleton Assembly and Receptor-Mediated Endocytosis of Nanoparticles (original) (raw)
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Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion
Cell Motility and the Cytoskeleton, 2004
The morphology and cytoskeletal structure of fibroblasts, endothelial cells, and neutrophils are documented for cells cultured on surfaces with stiffness ranging from 2 to 55,000 Pa that have been laminated with fibronectin or collagen as adhesive ligand. When grown in sparse culture with no cell-cell contacts, fibroblasts and endothelial cells show an abrupt change in spread area that occurs at a stiffness range around 3,000 Pa. No actin stress fibers are seen in fibroblasts on soft surfaces, and the appearance of stress fibers is abrupt and complete at a stiffness range coincident with that at which they spread. Upregulation of ␣5 integrin also occurs in the same stiffness range, but exogenous expression of ␣5 integrin is not sufficient to cause cell spreading on soft surfaces. Neutrophils, in contrast, show no dependence of either resting shape or ability to spread after activation when cultured on surfaces as soft as 2 Pa compared to glass. The shape and cytoskeletal differences evident in single cells on soft compared to hard substrates are eliminated when fibroblasts or endothelial cells make cell-cell contact. These results support the hypothesis that mechanical factors impact different cell types in fundamentally different ways, and can trigger specific changes similar to those stimulated by soluble ligands. Cell Motil. Cytoskeleton 60: 24 -34, 2005.
Acta Biomaterialia, 2015
Mechanical properties of materials strongly influence cell fate and functions. Focal adhesions are involved in the extremely important processes of mechanosensing and mechanotransduction. To address the relationship between the mechanical properties of cell substrates, focal adhesion/cytoskeleton assembly and cell functions, we investigated the behavior of NIH/3T3 cells over a wide range of stiffness (3-1000 kPa) using two of the most common synthetic polymers for cell cultures: polyacrylamide and polydimethylsiloxane. An overlapping stiffness region was created between them to compare focal adhesion characteristics and cell functions, taking into account their different time-dependent behavior. Indeed, from a rheological point of view, polyacrylamide behaves like a strong gel (elastically), whereas polydimethylsiloxane like a viscoelastic solid. First, focal adhesion characteristics and dynamics were addressed in terms of material stiffness, then cell spreading area, migration rate and cell mechanical properties were correlated with focal adhesion size and assembly. Focal adhesion size was found to increase in the whole range of stiffness and to be in agreement in the overlapping rigidity region for the investigated materials. Cell mechanics directly correlated with focal adhesion lengths, whereas migration rate followed an inverse correlation. Cell spreading correlated with the substrate stiffness on polyacrylamide hydrogel, while no specific trend was found on polydimethylsiloxane. Substrate mechanics can be considered as a key physical cue that regulates focal adhesion assembly, which in turn governs important cellular properties and functions.
Cell Movement Is Guided by the Rigidity of the Substrate
Biophysical Journal, 2000
Directional cell locomotion is critical in many physiological processes, including morphogenesis, the immune response, and wound healing. It is well known that in these processes cell movements can be guided by gradients of various chemical signals. In this study, we demonstrate that cell movement can also be guided by purely physical interactions at the cell-substrate interface. We cultured National Institutes of Health 3T3 fibroblasts on flexible polyacrylamide sheets coated with type I collagen. A transition in rigidity was introduced in the central region of the sheet by a discontinuity in the concentration of the bis-acrylamide cross-linker. Cells approaching the transition region from the soft side could easily migrate across the boundary, with a concurrent increase in spreading area and traction forces. In contrast, cells migrating from the stiff side turned around or retracted as they reached the boundary. We call this apparent preference for a stiff substrate "durotaxis." In addition to substrate rigidity, we discovered that cell movement could also be guided by manipulating the flexible substrate to produce mechanical strains in the front or rear of a polarized cell. We conclude that changes in tissue rigidity and strain could play an important controlling role in a number of normal and pathological processes involving cell locomotion.
Cells are known to respond to physical cues from their microenvironment such as matrix rigidity. Discrete adhesive ligands within flexible strands of fibronectin connect cell surface integrins to the broader extracellular matrix and are thought to mediate mechanosensing through the cytoskeleton-integrin-ECM linkage. We set out to determine if adhesive ligand tether length is another physical cue that cells can sense. Substrates were covalently modified with adhesive arginylglycylaspartic acid (RGD) ligands coupled with short (9.5 nm), medium (38.2 nm) and long (318 nm) length inert polyethylene glycol tethers. The size and length of focal adhesions of human foreskin fibroblasts gradually decreased from short to long tethers. Furthermore, we found cell adhesion varies in a linker length dependent manner with a remarkable 75% reduction in the density of cells on the surface and a 50% reduction in cell area between the shortest and longest linkers. We also report the interplay between RGD ligand concentration and tether length in determining cellular spread area. Our findings show that without varying substrate rigidity or ligand density, tether length alone can modulate cellular behaviour. It is known that cells are able to sense the rigidity of their microenvironment as illustrated by their differential behaviour when cultured on soft versus stiff substrates. Cell differentiation 1–3 , rate of DNA synthesis 4 , apoptosis 4 , traction forces 4 , motility 5–8 and spread area 2,4,5,7–11 have all been shown to be modulated by changes in substrate rigidity. It has also been demonstrated that transformed cancer cells respond differently to substrate stiffness compared to normal cells 4,12–14. Furthermore, the density of extracellular matrix (ECM) proteins such as collagen and fibronectin play a major role in determining cell behaviour 9,15–17. In order for cells to sense the rigidity of the ECM, they must first form linkages with ECM proteins via trans-membrane integrin receptors 18. Fibronectin is an ECM protein that connects to cell surface integrins via a discrete section along its length containing the adhesive RGD ligand 19,20. With the rest of the fibronectin protein playing a passive role in adhesion, a picture emerges of cells tethered to the matrix via thin and flexible strands of varying length. Within this scenario, we considered the possibility that cells are receptive not only to ECM rigidity and adhesive ligand density but also to the length of the local tether to which the ligand attaches to the broader ECM microenvironment. It was previously argued that, on poly(acrylamide) and poly(dimethylsiloxane) substrates functionalized with ECM proteins, cellular responses due to modulation of substrate stiffness were due to concomitantly modifying the fibronectin or collagen tether density, which resulted in substantial changes in nanoscale mechanical properties 11. Subsequently, it was found that varying the apparent porosity of poly(acrylamide) gels in order to control such tethering density did not result in changes in cell behaviour 17. This indicates that apparent porosity alone is not sufficient to account for the observations made; however, the approach developed did not permit the direct measurement of the density of tethers between the matrix and deposited ECM proteins. Notably, it was not possible to vary the apparent porosity without altering the density of polymer chains in the sample, which is expected
Cytoplasmic Force Gradient in Migrating Adhesive Cells
Biophysical Journal, 2008
Amoeboid movement is believed to involve a pressure gradient along the cell length, with contractions in the posterior region driving cytoplasmic streaming forward. However, a parallel mechanism has yet to be demonstrated in migrating adhesive cells. To probe the distribution of intracellular forces, we microinjected high molecular weight linear polyacrylamide (PAA) as a passive force sensor into migrating NIH3T3 fibroblasts. Injected PAA appeared as amorphous aggregates that underwent shape change and directional movement in response to differential forces exerted by the surrounding environment. PAA injected into the posterior region moved toward the front, whereas PAA in the anterior region never moved to the posterior region. This preferential forward movement was observed only in migrating cells with a defined polarity. Disruption of myosin II activity by blebbistatin inhibited the forward translocation of PAA while cell migration persisted in a disorganized fashion. These results suggest a myosin II-dependent force gradient in migrating cells, possibly as a result of differential cortical contractions between the anterior and posterior regions. This gradient may be responsible for the forward transport of cellular components and for maintaining the directionality during cell migration.
The regulation of traction force in relation to cell shape and focal adhesions
Biomaterials, 2011
Mechanical forces provide critical inputs for proper cellular functions. The interplay between the generation of, and response to, mechanical forces regulate such cellular processes as differentiation, proliferation, and migration. We postulate that adherent cells respond to a number of physical and topographical factors, including cell size and shape, by detecting the magnitude and/or distribution of traction forces under different conditions. To address this possibility we introduce a new simple method for precise micropatterning of hydrogels, and then apply the technique to systematically investigate the relationship between cell geometry, focal adhesions, and traction forces in cells with a series of spread areas and aspect ratios. Contrary to previous findings, we find that traction force is not determined primarily by the cell spreading area but by the distance from cell center to the perimeter. This distance in turn controls traction forces by regulating the size of focal adhesions, such that constraining the size of focal adhesions by micropatterning can override the effect of geometry. We propose that the responses of traction forces to center-periphery distance, possibly through a positive feedback mechanism that regulates focal adhesions, provide the cell with the information on its own shape and size. A similar positive feedback control may allow cells to respond to a variety of physical or topographical signals via a unified mechanism.
Cell morphology and migration linked to substrate rigidity
Soft Matter, 2007
A mathematical model, based on thermodynamics, was developed to demonstrate how substrate rigidity influences cell morphology and migration. The mechanisms by which substrate rigidity are translated into cell-morphological changes and cell movement are described. The model takes into account the competition between the elastic energies in the cell-substrate system and work of adhesion at the cell periphery. The cell morphology and migration are dictated by the minimum of the total free energy of the cell-substrate system. By using this model, reported experimental observations on cell morphological changes and migration can be better understood with a theoretical basis. In addition, these observations can be more accurately correlated with the variation of substrate rigidity. This study indicates that the activity of the adherent cell is dependent not only on the substrate rigidity but also is correlated with the relative rigidity between the cell and substrate. Moreover, the study suggests that the cell stiffness can be estimated based on the substrate stiffness corresponding to the change of trend in morphological stability.
Biophysical Journal, 2015
In cancer metastasis and other physiological processes, cells migrate through the three-dimensional (3D) extracellular matrix of connective tissue and must overcome the steric hindrance posed by pores that are smaller than the cells. It is currently assumed that low cell stiffness promotes cell migration through confined spaces, but other factors such as adhesion and traction forces may be equally important. To study 3D migration under confinement in a stiff (1.77 MPa) environment, we use soft lithography to fabricate polydimethylsiloxane (PDMS) devices consisting of linear channel segments with 20 mm length, 3.7 mm height, and a decreasing width from 11.2 to 1.7 mm. To study 3D migration in a soft (550 Pa) environment, we use self-assembled collagen networks with an average pore size of 3 mm. We then measure the ability of four different cancer cell lines to migrate through these 3D matrices, and correlate the results with cell physical properties including contractility, adhesiveness, cell stiffness, and nuclear volume. Furthermore, we alter cell adhesion by coating the channel walls with different amounts of adhesion proteins, and we increase cell stiffness by overexpression of the nuclear envelope protein lamin A. Although all cell lines are able to migrate through the smallest 1.7 mm channels, we find significant differences in the migration velocity. Cell migration is impeded in cell lines with larger nuclei, lower adhesiveness, and to a lesser degree also in cells with lower contractility and higher stiffness. Our data show that the ability to overcome the steric hindrance of the matrix cannot be attributed to a single cell property but instead arises from a combination of adhesiveness, nuclear volume, contractility, and cell stiffness.
2000
Many research and commercial applications use a synthetic substrate which is seeded with cells in a serum-containing medium. The surface properties of the material influence the composition of the adsorbed protein layer, which subsequently regulates a variety of cell behaviors such as attachment, spreading, proliferation, migration, and differentiation. In this study, we examined the relationships among cell attachment, spreading, cytoskeletal organization, and migration rate for MC3T3-E1 osteoblasts on glass surfaces modified with-SO x ,-NH 2 ,-N + (CH 3) 3 ,-SH, and-CH 3 terminal silanes. We also studied the relationship between cell spread area and migration rate for a variety of anchorage-dependent cell types on a model polymeric biomaterial, poly(acrylonitrilevinylchloride). Our results indicated that MC3T3-E1 osteoblast behavior was surface chemistry dependent, and varied with individual functional groups rather than general surface properties such as wettability. In addition, cell migration rate was inversely related to cell spread area for MC3T3-E1 osteoblasts on a variety of silane-modified surfaces as well as for different anchoragedependent cell types on a model polymeric biomaterial. Furthermore, the data revealed significant differences in migration rate among different cell types on a common polymeric substrate, suggesting that cell type-specific differences must be considered when using, selecting, or designing a substrate for research and therapeutic applications.