Vinculin is essential for sustaining normal levels of endogenous forces at cell-cell contacts.

Transmission of cell-generated (i.e., endogenous) tension at cell-cell contacts is crucial for tissue shape changes during morphogenesis and adult tissue repair in tissues such as epithelia. E-cadherin-based adhesions at cell-cell contacts are the primary means by which endogenous tension is transmitted between cells. The E-cadherin-ß-catenin-α-catenin complex mechanically couples to the actin cytoskeleton (and thereby the cell's contractile machinery) both directly and indirectly. However, the key adhesion constituents required for substantial endogenous force transmission at these adhesions in cell-cell contacts are unclear. Due to the role of α-catenin as a mechanotransducer that recruits vinculin at cell-cell contacts, we expected α-catenin to be essential for sustaining normal levels of force transmission. Instead, using the traction force imbalance method to determine the inter-cellular force at a single cell-cell contact between cell pairs, we found that it is vinculin that is essential for sustaining normal levels of endogenous force transmission, with absence of vinculin decreasing the inter-cellular tension by over 50%. Our results constrain the potential mechanical pathways of force transmission at cell-cell contacts and suggest that vinculin can transmit forces at E-cadherin adhesions independent of α-catenin, possibly through ß-catenin. Furthermore, we tested the ability of lateral cell-cell contacts to withstand external stretch and found that both vinculin and α-catenin are essential to maintain cell-cell contact stability under external forces.

E-cadherin adhesion dynamics as revealed by an accelerated force ramp are dependent upon the presence of α-catenin.

Tissue remodeling and shape changes often rely on force-induced cell rearrangements occurring via cell-cell contact dynamics. Epithelial cell-cell contact shape changes are particularly dependent upon E-cadherin adhesion dynamics which are directly influenced by cell-generated and external forces. While both the mobility of E-cadherin adhesions and their adhesion strength have been reported before, it is not clear how these two aspects of E-cadherin adhesion dynamics are related. Here, using magnetic pulling cytometry, we applied an accelerated force ramp on the E-cadherin adhesion between an E-cadherin-coated magnetic microbead and an epithelial cell to ascertain this relationship. Our approach enables the determination of the adhesion strength and force-dependent mobility of individual adhesions, which revealed a direct correlation between these key characteristics. Since α-catenin has previously been reported to play a role in both E-cadherin mobility and adhesion strength when studied independently, we also probed epithelial cells in which α-catenin has been knocked out. We found that, in the absence of α-catenin, E-cadherin adhesions not only had lower adhesion strength, as expected, but were also more mobile. We observed that α-catenin was required for the recovery of strained cell-cell contacts and propose that the adhesion strength and force-dependent mobility of E-cadherin adhesions act in tandem to regulate cell-cell contact homeostasis. Our approach introduces a method which relates the force-dependent adhesion mobility to adhesion strength and highlights the morphological role played by α-catenin in E-cadherin adhesion dynamics.

Fibrillar force generation by fibroblasts depends on formin.

Fibroblasts in the extra-cellular matrix (ECM) often adopt a predominantly one-dimensional fibrillar geometry by virtue of their adhesion to the fibrils in the ECM. How much forces such fibrillar fibroblasts exert and how they respond to the extended stiffness of their micro-environment comprising of other ECM components and cells are not clear. We use fibroblasts adherent on fibronectin lines micropatterned onto soft polyacrylamide gels as an in vitro experimental model that maintains fibrillar cell morphology while still letting the cell mechanically interact with a continuous micro-environment of specified stiffness. We find that the exerted traction, quantified as the strain energy or the maximum exerted traction stress, is not a function of cell length. Both the strain energy and the maximum traction stress exerted by fibrillar cells are similar for low (13 kPa) or high (45 kPa) micro-environmental stiffness. Furthermore, we find that fibrillar fibroblasts exhibit prominent linear actin structures. Accordingly, inhibition of the formin family of nucleators strongly decreases the exerted traction forces. Interestingly, fibrillar cell migration is, however, not affected under formin inhibition. Our results suggest that fibrillar cell migration in such soft microenvironments is not dependent on high cellular force exertion in the absence of other topological constraints.

Spatial Proliferation of Epithelial Cells Is Regulated by E-Cadherin Force.

Cell proliferation and contact inhibition play a major role in maintaining epithelial cell homeostasis. Prior experiments have shown that externally applied forces, such as stretch, result in increased proliferation in an E-cadherin force-dependent manner. In this study, the spatial regulation of cell proliferation in large epithelial colonies was examined. Surprisingly, cells at the center of the colony still had increased proliferation as compared to cells in confluent monolayers. E-cadherin forces were found to be elevated for both cells at the edge and center of these larger colonies when compared to confluent monolayers. To determine if high levels of E-cadherin force were necessary to induce proliferation at the center of the colony, a lower-force mutant of E-cadherin was developed. Cells with lower E-cadherin force had significantly reduced proliferation for cells at the center of the colony but minimal differences for cells at the edges of the colony. Similarly, increasing substrate stiffness was found to increase E-cadherin force and increase the proliferation rate across the colony. Taken together, these results show that forces through cell-cell junctions regulate proliferation across large groups of epithelial cells. In addition, an important finding of this study is that junction forces are dynamic and modulate cellular function even in the absence of externally applied loads.

Effect of pharmacological modulation of actin and myosin on collective cell electrotaxis.

Electrotaxis-the directional migration of cells in response to an electric field-is most evident in multicellular collectives and plays an important role in physiological contexts. While most cell types respond to applied electric fields of the order of a Volt per centimeter, our knowledge of the factors influencing this response is limited. This is especially true for collective cell electrotaxis, in which the subcellular migration response within a cell has to be coordinated with coupled neighboring cells. Here, we investigated the effect of the level of actin cytoskeleton polymerization and myosin activity on collective cell electrotaxis of Madin-Darby Canine Kidney (MDCK) cells in response to a weak electric field of physiologically relevant magnitude. We modulated the polymerization state of the actin cytoskeleton using the depolymerizing agent cytochalasin D or the polymerizing agent jasplakinolide. We also modulated the contractility of the cell using the myosin motor inhibitor blebbistatin or the phosphatase inhibitor calyculin A. While all the above pharmacological treatments altered cell speed to various extents, we found that only increasing the contractility and a high level of increase/stabilization of polymerized actin had a strong inhibitory effect specifically on the directedness of collective cell electrotaxis. On the other hand, even as the effect of the actin modulators on collective cell migration was varied, most conditions of actin and myosin pharmacological modulation-except for high level of actin polymerization/stabilization-resulted in cell speeds that were similar in the absence or presence of the electric field. Our results led us to speculate that the applied electric field may largely impact the cellular apparatus specifying the polarity of collective cell migration, rather than the functioning of the migratory apparatus. Bioelectromagnetics. 39:289-298, 2018. © 2018 Wiley Periodicals, Inc.

Endogenous Sheet-Averaged Tension Within a Large Epithelial Cell Colony.

Epithelial cells form quasi-two-dimensional sheets that function as contractile media to effect tissue shape changes during development and homeostasis. Endogenously generated intrasheet tension is a driver of such changes, but has predominantly been measured in the presence of directional migration. The nature of epithelial cell-generated forces transmitted over supracellular distances, in the absence of directional migration, is thus largely unclear. In this report, we consider large epithelial cell colonies which are archetypical multicell collectives with extensive cell-cell contacts but with a symmetric (circular) boundary. Using the traction force imbalance method (TFIM) (traction force microscopy combined with physical force balance), we first show that one can determine the colony-level endogenous sheet forces exerted at the midline by one half of the colony on the other half with no prior assumptions on the uniformity of the mechanical properties of the cell sheet. Importantly, we find that this colony-level sheet force exhibits large variations with orientation-the difference between the maximum and minimum sheet force is comparable to the average sheet force itself. Furthermore, the sheet force at the colony midline is largely tensile but the shear component exhibits significantly more variation with orientation. We thus show that even an unperturbed epithelial colony with a symmetric boundary shows significant directional variation in the endogenous sheet tension and shear forces that subsist at the colony level.

Protrusive activity guides changes in cell-cell tension during epithelial cell scattering.

Knowing how epithelial cells regulate cell-matrix and cell-cell adhesions is essential to understand key events in morphogenesis as well as pathological events such as metastasis. During epithelial cell scattering, epithelial cell islands rupture their cell-cell contacts and migrate away as single cells on the extracellular matrix (ECM) within hours of growth factor stimulation, even as adhesion molecules such as E-cadherin are present at the cell-cell contact. How the stability of cell-cell contacts is modulated to effect such morphological transitions is still unclear. Here, we report that in the absence of ECM, E-cadherin adhesions continue to sustain substantial cell-generated forces upon hepatocyte growth factor (HGF) stimulation, consistent with undiminished adhesion strength. In the presence of focal adhesions, constraints that preclude the spreading and movement of cells at free island edges also prevent HGF-mediated contact rupture. To explore the role of cell motion and cell-cell contact rupture, we examine the biophysical changes that occur during the scattering of cell pairs. We show that the direction of cell movement with respect to the cell-cell contact is correlated with changes in the average intercellular force as well as the initial direction of cell-cell contact rupture. Our results suggest an important role for protrusive activity resulting in cell displacement and force redistribution in guiding cell-cell contact rupture during scattering.

Cell-ECM traction force modulates endogenous tension at cell-cell contacts.

Cells in tissues are mechanically coupled both to the ECM and neighboring cells, but the coordination and interdependency of forces sustained at cell-ECM and cell-cell adhesions are unknown. In this paper, we demonstrate that the endogenous force sustained at the cell-cell contact between a pair of epithelial cells is approximately 100 nN, directed perpendicular to the cell-cell interface and concentrated at the contact edges. This force is stably maintained over time despite significant fluctuations in cell-cell contact length and cell morphology. A direct relationship between the total cellular traction force on the ECM and the endogenous cell-cell force exists, indicating that the cell-cell tension is a constant fraction of the cell-ECM traction. Thus, modulation of ECM properties that impact cell-ECM traction alters cell-cell tension. Finally, we show in a minimal model of a tissue that all cells experience similar forces from the surrounding microenvironment, despite differences in the extent of cell-ECM and cell-cell adhesion. This interdependence of cell-cell and cell-ECM forces has significant implications for the maintenance of the mechanical integrity of tissues, mechanotransduction, and tumor mechanobiology.

Regulation of cell motile behavior by crosstalk between cadherin- and integrin-mediated adhesions.

During normal development and in disease, cohesive tissues undergo rearrangements that require integration of signals from cell adhesions to neighboring cells and to the extracellular matrix (ECM). How a range of cell behaviors is coordinated by these different adhesion complexes is unknown. To analyze epithelial cell motile behavior in response to combinations of cell-ECM and cell-cell adhesion cues, we took a reductionist approach at the single-cell scale by using unique, functionalized micropatterned surfaces comprising alternating stripes of ECM (collagenIV) and adjustable amounts of E-cadherin-Fc (EcadFc). On these surfaces, individual cells spatially segregated integrin- and cadherin-based complexes between collagenIV and EcadFc surfaces, respectively. Cell migration required collagenIV and did not occur on surfaces functionalized with only EcadFc. However, E-cadherin adhesion dampened lamellipodia activity on both collagenIV and EcadFc surfaces and biased the direction of cell migration without affecting the migration rate, all in an EcadFc concentration-dependent manner. Traction force microscopy showed that spatial confinement of integrin-based adhesions to collagenIV stripes induced anisotropic cell traction on collagenIV and migration directional bias. Selective depletion of different pools of alphaE-catenin, an E-cadherin and actin binding protein, identified a membrane-associated pool required for E-cadherin-mediated adhesion and down-regulation of lamellipodia activity and a cytosolic pool that down-regulated the migration rate in an E-cadherin adhesion-independent manner. These results demonstrate that there is crosstalk between E-cadherin- and integrin-based adhesion complexes and that E-cadherin regulates lamellipodia activity and cell migration directionality, but not cell migration rate.

α-Catenin Dependent E-cadherin Adhesion Dynamics as Revealed by an Accelerated Force Ramp.

Tissue remodeling and shape changes often rely on force-induced cell rearrangements occurring via cell-cell contact dynamics. Epithelial cell-cell contact shape changes are particularly dependent upon E-cadherin adhesion dynamics which are directly influenced by cell-generated and external forces. While both the mobility of E-cadherin adhesions and their adhesion strength have been reported before, it is not clear how these two aspects of E-cadherin adhesion dynamics are related. Here, using magnetic pulling cytometry, we applied an accelerated force ramp on the E-cadherin adhesion between an E-cadherin-coated magnetic microbead and an epithelial cell to ascertain this relationship. Our approach enables the determination of the adhesion strength and force-dependent mobility of individual adhesions, which revealed a direct correlation between these key characteristics. Since α-catenin has previously been reported to play a role in both E-cadherin mobility and adhesion strength when studied independently, we also probed epithelial cells in which α-catenin has been knocked out. We found that, in the absence of α-catenin, E-cadherin adhesions not only had lower adhesion strength, as expected, but were also more mobile. We observed that α-catenin was required for the recovery of strained cell-cell contacts and propose that the adhesion strength and force-dependent mobility of E-cadherin adhesions act in tandem to regulate cell-cell contact homeostasis. Our approach introduces a method which relates the force-dependent adhesion mobility to adhesion strength and highlights the morphological role played by α-catenin in E-cadherin adhesion dynamics.

Integrating Shear Flow and Trypsin Treatment to Assess Cell Adhesion Strength.

Cell adhesion is of fundamental importance in cell and tissue organization, and for designing cell-laden constructs for tissue engineering. Prior methods to assess cell adhesion strength for strongly adherent cells using hydrodynamic shear flow either involved the use of specialized flow devices to generate high shear stress or used simpler implementations like larger height parallel plate chambers that enable multi-hour cell culture but generate low shear stress and are hence more applicable for weakly adherent cells. Here, we propose a shear flow assay for adhesion strength assessment of strongly adherent cells that employs off-the-shelf parallel plate chambers for shear flow as well as simultaneous trypsin treatment to tune down the adhesion strength of cells. We implement the assay with a strongly adherent cell type and show that shear stress in the 0.07 to 7 Pa range is sufficient to dislodge the cells with simultaneous trypsin treatment. Imaging of cells over a square centimeter area allows cell morphological analysis of hundreds of cells. We show that the cell area of cells that are dislodged, on average, does not monotonically increase with shear stress at the higher end of shear stresses used and suggest that this can be explained by the likely higher resistance of high circularity cells to trypsin digestion. The adhesion strength assay proposed can be easily adapted by labs to assess the adhesion strength of both weakly and strongly adherent cell types and has the potential to be adapted for substrate stiffness-dependent adhesion strength assessment in mechanobiology studies.

Epithelial Cell-Like Elasticity Modulates Actin-Dependent E-Cadherin Adhesion Organization.

E-cadherin adhesions are essential for cell-to-cell cohesion and mechanical coupling between epithelial cells and reside in a microenvironment that comprises the adjoining epithelial cells. While E-cadherin has been shown to be a mechanosensor, it is unknown if E-cadherin adhesions can differentially sense stiffness within the range of that of epithelial cells. A survey of literature shows that epithelial cells' Young's moduli of elasticity lie predominantly in the sub-kPa to few-kPa range, with cancer cells often being softer than noncancerous ones. Here, we devised oriented E-cadherin-coated soft silicone substrates with sub-kPa or few-kPa elasticity but with similar viscous moduli and found that E-cadherin adhesions differentially organize depending on the magnitude of epithelial cell-like elasticity. Our results show that the actin cytoskeleton organizes E-cadherin adhesions in two ways─by supporting irregularly shaped adhesions at localized regions of high actin density and linear shaped adhesions at the end of linear actin bundles. Linearly shaped E-cadherin adhesions associated with radially oriented actin─but not irregularly shaped E-cadherin adhesions associated with circumferential actin foci─were much more numerous on 2.4 kPa E-cadherin substrates compared to 0.3 kPa E-cadherin substrates. However, the total amount of E-cadherin in both types of adhesions taken together was similar on the 0.3 and 2.4 kPa E-cadherin substrates across many cells. Our results show how the distribution of E-cadherin adhesions, supported by actin density and architecture, is modulated by epithelial cell-like elasticity and have significant implications for disease states like carcinomas characterized by altered epithelial cell elasticity.

Integrating shear flow and trypsin treatment to assess cell adhesion strength.

Cell adhesion is of fundamental importance in cell and tissue organization and for designing cell-laden constructs for tissue engineering. Prior methods to assess cell adhesion strength for strongly adherent cells using hydrodynamic shear flow either involved the use of specialized flow devices to generate high shear stress or used simpler implementations like larger height parallel plate chambers that enable multihour cell culture but generate low wall shear stress and are, hence, more applicable for weakly adherent cells. Here, we propose a shear flow assay for adhesion strength assessment of strongly adherent cells that employs off-the-shelf parallel plate chambers for shear flow as well as simultaneous trypsin treatment to tune down the adhesion strength of cells. We implement the assay with a strongly adherent cell type and show that wall shear stress in the 0.07-7 Pa range is sufficient to dislodge the cells with simultaneous trypsin treatment. Imaging of cells over a square centimeter area allows cell morphological analysis of hundreds of cells. We show that the cell area of cells that are dislodged, on average, does not monotonically increase with wall shear stress at the higher end of wall shear stresses used and suggest that this can be explained by the likely higher resistance of high circularity cells to trypsin digestion. The adhesion strength assay proposed can be used to assess the adhesion strength of both weakly and strongly adherent cell types and has the potential to be adapted for substrate stiffness-dependent adhesion strength assessment in mechanobiology studies.

α-Catenin-dependent vinculin recruitment to adherens junctions is antagonistic to focal adhesions.

Vinculin is a protein found in both focal adhesions (FAs) and adherens junctions (AJs) which regulates actin connectivity to these structures. Many studies have demonstrated that mechanical perturbations of cells result in enhanced recruitment of vinculin to FAs and/or AJs. Likewise, many other studies have shown "cross-talk" between FAs and AJs. Vinculin itself has been suggested to be a probable regulator of this adhesion cross-talk. In this study we used MDCK as a model system of epithelia, developing cell lines in which vinculin recruitment was reduced or enhanced at AJs. Careful analysis of these cells revealed that perturbing vinculin recruitment to AJs resulted in a reduction of detectable FAs. Interestingly the cross-talk between these two structures was not due to a limited pool of vinculin, as increasing expression of vinculin did not rescue FA formation. Instead, we demonstrate that vinculin translocation between AJs and FAs is necessary for actin cytoskeleton rearrangements that occur during cell migration, which is necessary for large, well-formed FAs. Last, we show using a wound assay that collective cell migration is similarly hindered when vinculin recruitment is reduced or enhanced at AJs, highlighting that vinculin translocation between each compartment is necessary for efficient collective migration.

Allosteric cross talk between cadherin extracellular domains.

Atomic force microscopy and surface force apparatus measurements determined the functional impact of the cadherin point mutation W2A and domain deletion mutations on C-cadherin binding signatures. Direct comparison of results obtained using both experimental approaches demonstrates that C-cadherin ectodomains form multiple independent bonds that require different structural regions. The results presented reveal significant interdomain cross talk. They further demonstrate that the mutation W2A not only abolishes adhesion between N-terminal domains, but allosterically modulates other binding states that require functional domains distal to the N-terminal binding site. Such allosteric effects may play a prominent role in modulating adhesion by Type I classic cadherins, cadherin oligomerization at junctional contacts, and propagation of binding information to the cytoplasmic region.

Essential role of the Crk family-dosage in DiGeorge-like anomaly and metabolic homeostasis.

CRK and CRKL (CRK-like) encode adapter proteins with similar biochemical properties. Here, we show that a 50% reduction of the family-combined dosage generates developmental defects, including aspects of DiGeorge/del22q11 syndrome in mice. Like the mouse homologs of two 22q11.21 genes CRKL and TBX1, Crk and Tbx1 also genetically interact, thus suggesting that pathways shared by the three genes participate in organogenesis affected in the syndrome. We also show that Crk and Crkl are required during mesoderm development, and Crk/Crkl deficiency results in small cell size and abnormal mesenchyme behavior in primary embryonic fibroblasts. Our systems-wide analyses reveal impaired glycolysis, associated with low Hif1a protein levels as well as reduced histone H3K27 acetylation in several key glycolysis genes. Furthermore, Crk/Crkl deficiency sensitizes MEFs to 2-deoxy-D-glucose, a competitive inhibitor of glycolysis, to induce cell blebbing. Activated Rapgef1, a Crk/Crkl-downstream effector, rescues several aspects of the cell phenotype, including proliferation, cell size, focal adhesions, and phosphorylation of p70 S6k1 and ribosomal protein S6. Our investigations demonstrate that Crk/Crkl-shared pathways orchestrate metabolic homeostasis and cell behavior through widespread epigenetic controls.

Elasticity and rupture of a multi-domain neural cell adhesion molecule complex.

The neural cell adhesion molecule (NCAM) plays an important role in nervous system development. NCAM forms a complex between its terminal domains Ig1 and Ig2. When NCAM of cell A and of cell B connect to each other through complexes Ig12(A)/Ig12(B), the relative mobility of cells A and B and membrane tension exerts a force on the Ig12(A)/Ig12(B) complex. In this study, we investigated the response of the complex to force, using steered molecular dynamics. Starting from the structure of the complex from the Ig1-Ig2-Ig3 fragment, we first demonstrated that the complex, which differs in dimensions from a previous structure from the Ig1-Ig2 fragment in the crystal environment, assumes the same extension when equilibrated in solvent. We then showed that, when the Ig12(A)/Ig12(B) complex is pulled apart with forces 30-70 pN, it exhibits elastic behavior (with a spring constant of approximately 0.03 N/m) because of the relative reorientation of domains Ig1 and Ig2. At higher forces, the complex ruptures; i.e., Ig12(A) and Ig12(B) separate. The interfacial interactions between Ig12(A) and Ig12(B), monitored throughout elastic extension and rupture, identify E16, F19, K98, and L175 as key side chains stabilizing the complex.

In situ determination of exerted forces in magnetic pulling cytometry.

Localized application of exogenous forces on soft biomaterials and cells is often essential for the study of their response to external mechanical stimuli. Magnetic means of applying forces, particularly those based on permanent magnets and magnetic beads coupled to substrates or cells provide an accessible means of exerting forces of appropriate magnitude. The amount of force exerted, however, is often inferred from calibration performed ex situ, with typically similar but different magnetic beads. Here, we construct a simple magnetic tweezer by coupling a pencil-shaped stainless-steel probe to permanent neodymium magnets using a 3D printed adapter. We then demonstrate the in situ determination of magnetic bead pulling forces on a super-paramagnetic micro-bead coupled to a soft substrate using traction force microscopy. We determine the force exerted on the magnetic bead by the magnet probe - and thus exerted by the magnetic bead on the soft polyacrylamide substrate - as a function of the distance between the probe tip and the magnetic bead. We also show that we can determine the force exerted on a magnetic bead coupled to a cell by the changes in the traction force exerted by the cell on the soft substrate beneath. We thus demonstrate that forces of nanonewton magnitude can be locally exerted on soft substrates or cells and simultaneously determined using traction force microscopy. Application of this method for the in situ measurement of localized exogenous forces exerted on cells can also enable dissection of cellular force transmission pathways.

Mechanical response of an epithelial island subject to uniaxial stretch on a hybrid silicone substrate.

INTRODUCTION: The mechanical response of large multi-cellular collectives to external stretch has remained largely unexplored, despite its relevance to normal function and to external challenges faced by some tissues. Here, we introduced a simple hybrid silicone substrate to enable external stretch while providing a physiologically relevant physical micro-environment for cells. METHODS: We micropatterned epithelial islands on the substrate using a stencil to allow for a circular island shape without restraining island edges. We then used traction force microscopy to determine the strain energy and the inter-cellular sheet tension within the island as a function of time after stretch. RESULTS: While the strain energy stored in the substrate for unstretched cell islands stayed constant over time, a uniaxial 10% stretch resulted in an abrupt increase, followed by sustained increase in the strain energy of the islands over tens of minutes, indicating slower dynamics than for single cells reported previously. The sheet tension at the island mid-line perpendicular to the stretch direction also more than doubled compared to unstretched islands. Interestingly, the sheet tension at the island mid-line parallel to the stretch direction also reached similar levels over tens of minutes indicating the tendency of the island to homogenize its internal stress. CONCLUSIONS: We found that the sheet tension within large epithelial islands depends on its direction relative to that of the stretch initially, but not at longer times. We suggest that the hybrid silicone substrate provides for an accessible substrate for studying the mechanobiology of large epithelial cell islands.

Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope.

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been proven to be useful substrates for culturing cells in a physical microenvironment that partially mimics in vivo conditions. Here, we present a simple protocol for characterizing the Young's moduli of isotropic linear elastic substrates typically used for mechanobiology studies. The protocol consists of preparing a soft silicone substrate on a Petri dish or stiff silicone, coating the top surface of the silicone substrate with fluorescent beads, using a millimeter-scale sphere to indent the top surface (by gravity), imaging the fluorescent beads on the indented silicone surface using a fluorescence microscope, and analyzing the resultant images to calculate the Young's modulus of the silicone substrate. Coupling the substrate's top surface with a moduli extracellular matrix protein (in addition to the fluorescent beads) allows the silicone substrate to be readily used for cell plating and subsequent studies using traction force microscopy experiments. The use of stiff silicone, instead of a Petri dish, as the base of the soft silicone, enables the use of mechanobiology studies involving external stretch. A specific advantage of this protocol is that a widefield fluorescence microscope, which is commonly available in many labs, is the major equipment necessary for this procedure. We demonstrate this protocol by measuring the Young's modulus of soft silicone substrates of different elastic moduli.