Nano-mechanical single-cell sensing of cell-matrix contacts (original) (raw)
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Measurement of cellular forces at focal adhesions using elastic micro-patterned substrates
Materials Science and Engineering: C, 2003
Mechanical force is known to play an important role in the regulation of cellular behaviour, including adhesion, motility, differentiation and proliferation. For stationary, mechanically active cells like fibroblasts, adhesion to flat substrates occurs mainly at sites of focal adhesions, which are micron-sized protein aggregates at the plasma membrane, which on the cytoplasmic side connect to the actin cytoskeleton. In recent years, evidence has been growing that focal adhesions act as mechanosensors which convert mechanical force into biochemical signalling. We have investigated the relationship between force and aggregation at focal adhesions by a new method which combines elastic micro-patterned substrates (to record substrate deformation), fluorescence labelling of focal adhesion proteins (to monitor aggregation) and numerical solution of the inverse problem of linear elasticity theory (to calculate forces at focal adhesions). We have found that force correlates linearly with lateral size of aggregation, with a stress constant of 5.5 nN/Am 2. This finding indicates that mechanosensing involves regulation of aggregation.
Cells lying on a bed of microneedles: An approach to isolate mechanical force
Proceedings of The National Academy of Sciences, 2003
We describe an approach to manipulate and measure mechanical interactions between cells and their underlying substrates by using microfabricated arrays of elastomeric, microneedle-like posts. By controlling the geometry of the posts, we varied the compliance of the substrate while holding other surface properties constant. Cells attached to, spread across, and deflected multiple posts. The deflections of the posts occurred independently of neighboring posts and, therefore, directly reported the subcellular distribution of traction forces. We report two classes of force-supporting adhesions that exhibit distinct force-size relationships. Force increased with size of adhesions for adhesions larger than 1 m 2 , whereas no such correlation existed for smaller adhesions. By controlling cell adhesion on these micromechanical sensors, we showed that cell morphology regulates the magnitude of traction force generated by cells. Cells that were prevented from spreading and flattening against the substrate did not contract in response to stimulation by serum or lysophosphatidic acid, whereas spread cells did. Contractility in the unspread cells was rescued by expression of constitutively active RhoA. Together, these findings demonstrate a coordination of biochemical and mechanical signals to regulate cell adhesion and mechanics, and they introduce the use of arrays of mechanically isolated sensors to manipulate and measure the mechanical interactions of cells.
Review of Scientific Instruments
The adhesion of cells to the extracellular matrix is a hierarchical, force-dependent, multistage process that evolves at several temporal scales. An understanding of this complex process requires a precise measurement of forces and its correlation with protein responses in living cells. We present a method to quantitatively assess live cell responses to a local and specific mechanical stimulus. Our approach combines atomic force microscopy with fluorescence imaging. Using this approach, we evaluated the recruitment of adhesion proteins such as vinculin, focal adhesion kinase, paxillin, and zyxin triggered by applying forces in the nN regime to live cells. We observed in real time the development of nascent adhesion sites, evident from the accumulation of early adhesion proteins at the position where the force was applied. We show that the method can be used to quantify the recruitment characteristic times for adhesion proteins in the formation of focal complexes. We also found a spatial remodeling of the mature focal adhesion protein zyxin as a function of the applied force. Our approach allows the study of a variety of complex biological processes involved in cellular mechanotransduction.
Micropatterning tractional forces in living cells
Cell Motility and the Cytoskeleton, 2002
Here we describe a method for quantifying traction in cells that are physically constrained within micron-sized adhesive islands of defined shape and size on the surface of flexible polyacrylamide gels that contain fluorescent microbeads (0.2-m diameter). Smooth muscle cells were plated onto square (50 ϫ 50 m) or circular (25or 50-m diameter) adhesive islands that were created on the surface of the gels by applying a collagen coating through microengineered holes in an elastomeric membrane that was later removed. Adherent cells spread to take on the size and shape of the islands and cell tractions were quantitated by mapping displacement fields of the fluorescent microbeads within the gel. Cells on round islands did not exhibit any preferential direction of force application, but they exerted their strongest traction at sites where they formed protrusions. When cells were confined to squares, traction was highest in the corners both in the absence and presence of the contractile agonist, histamine, and cell protrusions were also observed in these regions. Quantitation of the mean traction exerted by cells cultured on the different islands revealed that cell tension increased as cell spreading was promoted. These results provide a mechanical basis for past studies that demonstrated a similar correlation between spreading and growth within various anchorage-dependent cells. This new approach for analyzing the spatial distribution of mechanical forces beneath individual cells that are experimentally constrained to defined sizes and shapes may provide additional insight into the biophysical basis of cell regulation. Cell Motil. Cytoskeleton 52: 97-106, 2002.
Nature Cell Biology, 2015
During cell migration, the forces generated in the actin cytoskeleton are transmitted across transmembrane receptors to the extracellular matrix or other cells through a series of mechanosensitive, regulable protein-protein interactions termed the molecular clutch. In integrinbased focal adhesions, the proteins forming this linkage are organized into a conserved threedimensional nano-architecture. Here we discuss how the physical interactions between the actin cytoskeleton and focal-adhesion-associated molecules mediate force transmission from the molecular clutch to the extracellular matrix. Cell migration is important during embryonic development, immune responses and wound healing, and can lead to inflammation and cancer metastasis when misregulated 1. Migration can occur through different mechanisms, including lamellipodia or pressure-driven bleb formation 2 , water permeation 3 and other processes 4,5 , depending on the cell type and tissue environment-a plasticity that facilitates robust migration in many contexts 1. However, the common feature of all these scenarios is that cells must be able to apply forces to generate traction against, and move themselves relative to, their immediate surroundings. The actin cytoskeleton is the major source of internally generated force that regulates cell shape and drives migration 6. Actin-based cellular forces must somehow be transmitted through the cell membrane to generate friction that induces traction against the extracellular environment. Friction between the cell and its environment can either be non-specific or mediated by specific surface receptors that bind to the extracellular matrix (ECM) or other cells. Nonspecific friction can be generated when cells are held under confinement, and is thought to drive non-haptotactic, bleb-based amoeboid motility during immune responses and cancer metastasis 7,8. Specific interactions between cells and their surroundings, such as integrin-ECM and cadherin-cadherin receptor-ligand interactions, drive haptotactic 'mesenchymal' motility during wound healing and development. This Review will focus on the physical mechanisms of cell-ECM traction generation during lamellipodia-and integrin-dependent mesenchymal cell migration.
Nature Cell Biology, 2001
Mechanical forces play a major role in the regulation of cell adhesion and cytoskeletal organization. In order to explore the molecular mechanism underlying this regulation, we have investigated the relationship between local force applied by the cell to the substrate and the assembly of focal adhesions. A novel approach was developed for real-time, high-resolution measurements of forces applied by cells at single adhesion sites. This method combines micropatterning of elastomer substrates and fluorescence imaging of focal adhesions in live cells expressing GFPtagged vinculin. Local forces are correlated with the orientation, total fluorescence intensity and area of the focal adhesions, indicating a constant stress of 5.5 ± 2 nNµm-2. The dynamics of the force-dependent modulation of focal adhesions were characterized by blocking actomyosin contractility and were found to be on a time scale of seconds. The results put clear constraints on the possible molecular mechanisms for the mechanosensory response of focal adhesions to applied force. C ell adhesion plays a critical role in many fundamental processes such as embryonic morphogenesis, angiogenesis, inflammation and wound healing 1-3 . One of the unique features of adhesion to a rigid surface, unlike binding to soluble ligands, is the ability of forces to develop at the contact site. Such forces can be external, such as shear flow in blood vessels, or can be generated by the cell's own contractile apparatus. There is a close relationship between the abilities of cells such as fibroblasts to generate force and to assemble matrix adhesions 4-7 .
Nanomechanics of a fibroblast suspended using point-like anchors reveal cytoskeleton formation
In an attempt to better elucidate the material-cytoskeleton crosstalk during the initial stage of cell adhesion, here we report how suspended cells anchored to point-like bonds are able to assemble their cytoskeleton when subjected to mechanical stress. The combination of holographic optical tweezers and digital holography gives the cell footholds for adhesion and mechanical stimulation, and at the same time, acts as a label-free, force-revealing system over time, detecting the cell nanomechanical response in the pN range. To confirm the formation of the cytoskeleton structures after the stimulation, a fluorescence imaging system was added as a control. The strategy here proposed portends broad applicability to investigate the correlation between the forces applied to cells and their cytoskeleton assembly process in this or other complex configurations with multiple anchor points.
Intracellular forces during guided cell growth on micropatterns using FRET measurement
Journal of Biomechanics, 2015
Interaction of cells with extracellular matrix (ECM) regulates cell shape, differentiation and polarity. This effect has been widely observed in cells grown on substrates with various patterned features, stiffness and surface chemistry. It has been postulated that mechanical confinement of cells by the substrate causes a redistribution of tension in cytoskeletal proteins resulting in cytoskeletal reorganization through force sensitive pathways. However, the mechanisms for force transduction during reorganization remain unclear. In this study, using FRET based force sensors we have measured tension in an actin cross-linking protein, α-actinin, and followed reorganization of actin cytoskeleton in real time in HEK cells grown on patterned substrates. We show that the patterned substrates cause a redistribution of tension in α-actinin that coincides with cytoskeleton reorganization. Higher tension was observed in portions of cells where they form bridges across inhibited regions of the patterned substrates; the attachment to the substrate is found to release tension. Real time measurements of α-actinin tension and F-actin arrangement show that an increase in tension coincides with formation of F-actin bundles at the cell periphery during cellspreading across inhibited regions, suggesting that mechanical forces stimulate cytoskeleton enhancement. Rho-ROCK inhibitor (Y27632) causes reduction in actinin tension followed by retraction of bridged regions. Our results demonstrate that changes in cell shape and expansion over patterned surfaces is a force sensitive process that requires actomyosin contractile force involving Rho-ROCK pathway.
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.