Probing compression versus stretch activated recruitment of cortical actin and apical junction proteins using mechanical stimulations of suspended doublets (original) (raw)
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Cell Stiffening in Response to External Stress is Correlated to Actin Recruitment
Biophysical Journal, 2008
We designed a micromanipulation device that allows the local application of a constant force on living cells, and the measurement of their stiffness. The force is applied through an Arg-Gly-Asp-coated bead adhering on the cell and trapped in optical tweezers controlled by a feedback loop. Epifluorescence observations of green fluorescent protein-actin in the cells are made during force application. We observe a stiffening of cells submitted to a constant force within a few minutes, coupled to actin recruitment both at the bead-cell contact and up to several micrometers from the stress application zone. Moreover, kinetics of stiffening and actin recruitment exhibit a strong correlation. This work presents the first quantification of the dynamics of cell mechanical reinforcement under stress, which is a novel insight into the elucidation of the more general phenomenon of cell adaptation to stress.
Biophysical Journal, 2009
We investigate the dynamic response of single cells to weak and local rigidities, applied at controlled adhesion sites. Using multiple latex beads functionalized with fibronectin, and each trapped in its own optical trap, we study the reaction in real time of single 3T3 fibroblast cells to asymmetrical tensions in the tens of pN $ mm À1 range. We show that the cell feels a rigidity gradient even at this low range of tension, and over time develops an adapted change in the force exerted on each adhesion site. The rate at which force increases is proportional to trap stiffness. Actomyosin recruitment is regulated in space and time along the rigidity gradient, resulting in a linear relationship between the amount of recruited actin and the force developed independently in trap stiffness. This time-regulated actomyosin behavior sustains a constant and rigidity-independent velocity of beads inside the traps. Our results show that the strengthening of extracellular matrix-cytoskeleton linkages along a rigidity gradient is regulated by controlling adhesion area and actomyosin recruitment, to maintain a constant deformation of the extracellular matrix.
Probing cell structure by controlling the mechanical environment with cell–substrate interactions
Journal of Biomechanics, 2009
Recent results demonstrate the exquisite sensitivity of cell morphology and structure to mechanical stimulation. Mechanical stimulation is often coupled with cell-substrate interactions that can, in turn, influence molecular response and determine cellular fates including apoptosis, proliferation, and differentiation. To understand these effects as they specifically relate to compressive mechanical stimulation and topographic control, we developed a microfabricated system to grow cells on polydimethylsiloxane (PDMS) microchannel surfaces where we maintained compression stimulation. We also probed cellular response following compressive mechanical stimulation to PDMS substrates of varying stiffness. In these instances, we examined cytoskeletal and morphologic changes in living cells attached to our substrate following the application of localized compressive stimulation. We found that the overall morphology and cell structure, including the actin cytoskeleton, oriented in the direction of the compressive strain applied and along the topographic microchannels. Furthermore by comparing topographic response to material stiffness, we found a 40% increase in cell area for cells cultured on the microchannels versus softer PDMS as well as a decreased cell area of 30% when using softer PDMS over unmodified PDMS. These findings have implications for research in a diversity of fields including cell-material interactions, mechanotransduction, and tissue engineering.
Actomyosin Cortical Mechanical Properties in Nonadherent Cells Determined by Atomic Force Microscopy
Biophysical Journal, 2016
The organization of filamentous actin and myosin II molecular motor contractility is known to modify the mechanical properties of the cell cortical actomyosin cytoskeleton. Here we describe a novel method, to our knowledge, for using force spectroscopy approach curves with tipless cantilevers to determine the actomyosin cortical tension, elastic modulus, and intracellular pressure of nonadherent cells. We validated the method by measuring the surface tension of water in oil microdrops deposited on a glass surface. We extracted an average tension of T~20.25 nN/mm, which agrees with macroscopic experimental methods. We then measured cortical mechanical properties in nonadherent human foreskin fibroblasts and THP-1 human monocytes before and after pharmacological perturbations of actomyosin activity. Our results show that myosin II activity and actin polymerization increase cortex tension and intracellular pressure, whereas branched actin networks decreased them. Interestingly, myosin II activity stiffens the cortex and branched actin networks soften it, but actin polymerization has no effect on cortex stiffness. Our method is capable of detecting changes in cell mechanical properties in response to perturbations of the cytoskeleton, allowing characterization with physically relevant parameters. Altogether, this simple method should be of broad application for deciphering the molecular regulation of cell cortical mechanical properties.
Journal of Biomechanics, 2012
A membrane potential change in cells is accompanied with mechanical deformation. This electromechanical response can play a significant role in regulating action potential in neurons and in controlling voltage-gated ion channels. However, measuring this subtle deformation in mammalian cells has been a difficult task. We show a plasmonic imaging method to image mechanical deformation in single cells upon a change in the membrane potential. Using this method, we have studied the electromechanical response in mammalian cells and have observed the local deformation within the cells that are associated with cell-substrate interactions. By analyzing frequency dependence of the response, we have further examined the electromechanical deformation in terms of mechanical properties of cytoplasm and cytoskeleton. We demonstrate a plasmonic imaging approach to quantify the electromechanical responses of single mammalian cells and determine local variability related to cell-substrate interactions.
Mechanotransduction at Cell-Matrix and Cell-Cell Contacts
Annual Review of Biomedical Engineering, 2004
▪ Mechanical forces play an important role in the organization, growth, maturation, and function of living tissues. At the cellular level, many of the biological responses to external forces originate at two types of specialized microscale structures: focal adhesions that link cells to their surrounding extracellular matrix and adherens junctions that link adjacent cells. Transmission of forces from outside the cell through cell-matrix and cell-cell contacts appears to control the maturation or disassembly of these adhesions and initiates intracellular signaling cascades that ultimately alter many cellular behaviors. In response to externally applied forces, cells actively rearrange the organization and contractile activity of the cytoskeleton and redistribute their intracellular forces. Recent studies suggest that the localized concentration of these cytoskeletal tensions at adhesions is also a major mediator of mechanical signaling. This review summarizes the role of mechanical ...
Combining Dynamic Stretch and Tunable Stiffness to Probe Cell Mechanobiology In Vitro
PLoS ONE, 2011
Cells have the ability to actively sense their mechanical environment and respond to both substrate stiffness and stretch by altering their adhesion, proliferation, locomotion, morphology, and synthetic profile. In order to elucidate the interrelated effects of different mechanical stimuli on cell phenotype in vitro, we have developed a method for culturing mammalian cells in a two-dimensional environment at a wide range of combined levels of substrate stiffness and dynamic stretch. Polyacrylamide gels were covalently bonded to flexible silicone culture plates and coated with monomeric collagen for cell adhesion. Substrate stiffness was adjusted from relatively soft (G9 = 0.3 kPa) to stiff (G9 = 50 kPa) by altering the ratio of acrylamide to bis-acrylamide, and the silicone membranes were stretched over circular loading posts by applying vacuum pressure to impart near-uniform stretch, as confirmed by strain field analysis. As a demonstration of the system, porcine aortic valve interstitial cells (VIC) and human mesenchymal stem cells (hMSC) were plated on soft and stiff substrates either statically cultured or exposed to 10% equibiaxial or pure uniaxial stretch at 1Hz for 6 hours. In all cases, cell attachment and cell viability were high. On soft substrates, VICs cultured statically exhibit a small rounded morphology, significantly smaller than on stiff substrates (p,0.05). Following equibiaxial cyclic stretch, VICs spread to the extent of cells cultured on stiff substrates, but did not reorient in response to uniaxial stretch to the extent of cells stretched on stiff substrates. hMSCs exhibited a less pronounced response than VICs, likely due to a lower stiffness threshold for spreading on static gels. These preliminary data demonstrate that inhibition of spreading due to a lack of matrix stiffness surrounding a cell may be overcome by externally applied stretch suggesting similar mechanotransduction mechanisms for sensing stiffness and stretch.