Elastomeric Pillar Cages Modulate Actomyosin Contractility of Epithelial Microtissues by Substrate Stiffness and Topography.

Cell contractility regulates epithelial tissue geometry development and homeostasis. The underlying mechanobiological regulation circuits are poorly understood and experimentally challenging. We developed an elastomeric pillar cage (EPC) array to quantify cell contractility as a mechanoresponse of epithelial microtissues to substrate stiffness and topography. The spatially confined EPC geometry consisted of 24 circularly arranged slender pillars (1.2 MPa, height: 50 µm; diameter: 10 µm, distance: 5 µm). These high-aspect-ratio pillars were confined at both ends by planar substrates with different stiffness (0.15-1.2 MPa). Analytical modeling and finite elements simulation retrieved cell forces from pillar displacements. For evaluation, highly contractile myofibroblasts and cardiomyocytes were assessed to demonstrate that the EPC device can resolve static and dynamic cellular force modes. Human breast (MCF10A) and skin (HaCaT) cells grew as adherence junction-stabilized 3D microtissues within the EPC geometry. Planar substrate areas triggered the spread of monolayered clusters with substrate stiffness-dependent actin stress fiber (SF)-formation and substantial single-cell actomyosin contractility (150-200 nN). Within the same continuous microtissues, the pillar-ring topography induced the growth of bilayered cell tubes. The low effective pillar stiffness overwrote cellular sensing of the high substrate stiffness and induced SF-lacking roundish cell shapes with extremely low cortical actin tension (11-15 nN). This work introduced a versatile biophysical tool to explore mechanobiological regulation circuits driving low- and high-tensional states during microtissue development and homeostasis. EPC arrays facilitate simultaneously analyzing the impact of planar substrate stiffness and topography on microtissue contractility, hence microtissue geometry and function.

A Combined AFM and Lateral Stretch Device Enables Microindentation Analyses of Living Cells at High Strains.

Mechanical characterization of living cells undergoing substantial external strain promises insights into material properties and functional principles of mechanically active tissues. However, due to the high strains that occur in physiological situations (up to 50%) and the complex structure of living cells, suitable experimental techniques are rare. In this study, we introduce a new system composed of an atomic force microscope (AFM), a cell stretching system based on elastomeric substrates, and light microscopy. With this system, we investigated the influence of mechanical stretch on monolayers of keratinocytes. In repeated indentations at the same location on one particular cell, we found significant stiffening at 25% and 50% strain amplitude. To study the contribution of intermediate filaments, we used a mutant keratinocyte cell line devoid of all keratins. For those cells, we found a softening in comparison to the wild type, which was even more pronounced at higher strain amplitudes.

Nanoscale Topography and Poroelastic Properties of Model Tissue Breast Gland Basement Membranes.

Basement membranes (BMs) are thin layers of condensed extracellular matrix proteins serving as permeability filters, cellular anchoring sites, and barriers against cancer cell invasion. It is believed that their biomechanical properties play a crucial role in determining cellular behavior and response, especially in mechanically active tissues like breast glands. Despite this, so far, relatively little attention has been dedicated to their analysis because of the difficulty of isolating and handling such thin layers of material. Here, we isolated BMs derived from MCF10A spheroids-three-dimensional breast gland model systems mimicking in vitro the most relevant phenotypic characteristics of human breast lobules-and characterized them by atomic force microscopy, enhanced resolution confocal microscopy, and scanning electron microscopy. By performing atomic force microscopy height-clamp experiments, we obtained force-relaxation curves that offered the first biomechanical data on isolated breast gland BMs to our knowledge. Based on enhanced resolution confocal microscopy and scanning electron microscopy imaging data, we modeled the system as a polymer network immersed in liquid and described it as a poroelastic material. Finite-element simulations matching the experimental force-relaxation curves allowed for the first quantification, to our knowledge, of the bulk and shear moduli of the membrane as well as its water permeability. These results represent a first step toward a deeper understanding of the mechanism of tensional homeostasis regulating mammary gland activity as well as its disruption during processes of membrane breaching and metastatic invasion.

Nanometer-Resolved Mapping of Cell-Substrate Distances of Contracting Cardiomyocytes Using Surface Plasmon Resonance Microscopy.

It has been shown that quantitative measurements of the cell-substrate distance of steady cells are possible with scanning surface plasmon resonance microscopy setups in combination with an angle resolved analysis. However, the accuracy of the determined cell-substrate distances as well as the capabilities for the investigation of cell dynamics remained limited due to the assumption of a homogeneous refractive index of the cytosol. Strong spatial or temporal deviations between the local refractive index and the average value can result in errors in the calculated cell-substrate distance of around 100 nm, while the average accuracy was determined to 37 nm. Here, we present a combination of acquisition and analysis techniques that enables the measurement of the cell-substrate distance of contractile cells as well as the study of intracellular processes through changes in the refractive index at the diffraction limit. By decoupling the measurement of the cell-substrate distance and the refractive index of the cytoplasm, we could increase the accuracy of the distance measurement on average by a factor of 25 reaching 1.5 nm under ideal conditions. We show a temporal and spatial mapping of changes in the refractive index and the cell-substrate distance which strongly correlate with the action potentials and reconstruct the three-dimensional profile of the basal cell membrane and its dynamics, while we reached an actual measurement accuracy of 2.3 nm.

An excitable Rho GTPase signaling network generates dynamic subcellular contraction patterns.

Rho GTPase-based signaling networks control cellular dynamics by coordinating protrusions and retractions in space and time. Here, we reveal a signaling network that generates pulses and propagating waves of cell contractions. These dynamic patterns emerge via self-organization from an activator-inhibitor network, in which the small GTPase Rho amplifies its activity by recruiting its activator, the guanine nucleotide exchange factor GEF-H1. Rho also inhibits itself by local recruitment of actomyosin and the associated RhoGAP Myo9b. This network structure enables spontaneous, self-limiting patterns of subcellular contractility that can explore mechanical cues in the extracellular environment. Indeed, actomyosin pulse frequency in cells is altered by matrix elasticity, showing that coupling of contractility pulses to environmental deformations modulates network dynamics. Thus, our study reveals a mechanism that integrates intracellular biochemical and extracellular mechanical signals into subcellular activity patterns to control cellular contractility dynamics.

Measuring cellular traction forces on non-planar substrates.

Animal cells use traction forces to sense the mechanics and geometry of their environment. Measuring these traction forces requires a workflow combining cell experiments, image processing and force reconstruction based on elasticity theory. Such procedures have already been established mainly for planar substrates, in which case one can use the Green's function formalism. Here we introduce a workflow to measure traction forces of cardiac myofibroblasts on non-planar elastic substrates. Soft elastic substrates with a wave-like topology were micromoulded from polydimethylsiloxane and fluorescent marker beads were distributed homogeneously in the substrate. Using feature vector-based tracking of these marker beads, we first constructed a hexahedral mesh for the substrate. We then solved the direct elastic boundary volume problem on this mesh using the finite-element method. Using data simulations, we show that the traction forces can be reconstructed from the substrate deformations by solving the corresponding inverse problem with an L1-norm for the residue and an L2-norm for a zeroth-order Tikhonov regularization. Applying this procedure to the experimental data, we find that cardiac myofibroblast cells tend to align both their shapes and their forces with the long axis of the deformable wavy substrate.

Matrix elasticity, replicative senescence and DNA methylation patterns of mesenchymal stem cells.

Matrix elasticity guides differentiation of mesenchymal stem cells (MSCs) but it is unclear if these effects are only transient - while the cells reside on the substrate - or if they reflect persistent lineage commitment. In this study, MSCs were continuously culture-expanded in parallel either on tissue culture plastic (TCP) or on polydimethylsiloxane (PDMS) gels of different elasticity to compare impact on replicative senescence, in vitro differentiation, gene expression, and DNA methylation (DNAm) profiles. The maximal number of cumulative population doublings was not affected by matrix elasticity. Differentiation towards adipogenic and osteogenic lineage was increased on soft and rigid biomaterials, respectively - but this propensity was no more evident if cells were transferred to TCP. Global gene expression profiles and DNAm profiles revealed relatively few differences in MSCs cultured on soft or rigid matrices. Furthermore, only moderate DNAm changes were observed upon culture on very soft hydrogels of human platelet lysate. Our results support the notion that matrix elasticity influences cellular behavior while the cells reside on the substrate, but it does not have major impact on cell-intrinsic lineage determination, replicative senescence or DNAm patterns.

Defined 2-D microtissues on soft elastomeric silicone rubber using lift-off epoxy-membranes for biomechanical analyses.

Surface patterning with complex molecules has become a valuable tool in cell biology and biotechnology, as it enables one to control cell shape and function in culture. However, this technique for micro-contact printing is normally performed on rigid substrates, e.g. Petri dishes or glass. Despite the fact that these substrates can easily be patterned they are artificially stiff environments for cells affecting their morphology and function. Those artifacts can be avoided on tissue elasticity resembling substrates, leading to a nature like cell morphology and behavior. However, reproducible patterning of very soft elastomeric substrates is challenging. Here, we describe a simple and highly accurate method through cavities of lift-off membranes for protein patterning of silicone rubber substrates in an elasticity range down to 1.5 kPa without altering their mechanical properties. Membranes are made of epoxy resin with feature sizes that can be chosen almost arbitrarily including widths down to 5 µm and aspect ratios of 100 and more. Different feature shapes were used to actively manipulate cell adhesion, cell morphology and the actin cytoskeleton on soft substrates. Manipulation of cytoskeletal organization furthermore allowed the comparison of myofibril alignment and cellular forces of cardiac myocytes. These data could show that cell forces are largely unaffected upon active disordering of overall myofibril alignment on a single cell level while aligned multicellular systems generate cell forces in an additive manner.

Cellular mechanotransduction relies on tension-induced and chaperone-assisted autophagy.

Mechanical tension is an ever-present physiological stimulus essential for the development and homeostasis of locomotory, cardiovascular, respiratory, and urogenital systems. Tension sensing contributes to stem cell differentiation, immune cell recruitment, and tumorigenesis. Yet, how mechanical signals are transduced inside cells remains poorly understood. Here, we identify chaperone-assisted selective autophagy (CASA) as a tension-induced autophagy pathway essential for mechanotransduction in muscle and immune cells. The CASA complex, comprised of the molecular chaperones Hsc70 and HspB8 and the cochaperone BAG3, senses the mechanical unfolding of the actin-crosslinking protein filamin. Together with the chaperone-associated ubiquitin ligase CHIP, the complex initiates the ubiquitin-dependent autophagic sorting of damaged filamin to lysosomes for degradation. Autophagosome formation during CASA depends on an interaction of BAG3 with synaptopodin-2 (SYNPO2). This interaction is mediated by the BAG3 WW domain and facilitates cooperation with an autophagosome membrane fusion complex. BAG3 also utilizes its WW domain to engage in YAP/TAZ signaling. Via this pathway, BAG3 stimulates filamin transcription to maintain actin anchoring and crosslinking under mechanical tension. By integrating tension sensing, autophagosome formation, and transcription regulation during mechanotransduction, the CASA machinery ensures tissue homeostasis and regulates fundamental cellular processes such as adhesion, migration, and proliferation.

Cyclic stretch induces reorientation of cells in a Src family kinase- and p130Cas-dependent manner.

Recognition of external mechanical signals by cells is an essential process for life. One important mechanical signal experienced by various cell types, e.g. around blood vessels, within the lung epithelia or around the intestine, is cyclic stretch. As a response, many cell types reorient their actin cytoskeleton and main cell axis almost perpendicular to the direction of stretch. Despite the vital necessity of cellular adaptation to cyclic stretch, the underlying mechanosensory signal cascades are far from being understood. Here we show an important function of Src-family kinase activity in cellular reorientation upon cyclic stretch. Deletion of all three family members, namely c-Src, Yes and Fyn (SYF), results in a strongly impaired cell reorientation of mouse embryonic fibroblasts with an only incomplete reorientation upon expression of c-Src. We further demonstrate that this reorientation phenotype of SYF-depleted cells is not caused by affected protein exchange dynamics within focal adhesions or altered cell force generation. Instead, Src-family kinases regulate the reorientation in a mechanotransduction-dependent manner, since knock-down and knock-out of p130Cas, a putative stretch sensor known to be phosphorylated by Src-family kinases, also reduce cellular reorientation upon cyclic stretch. This impaired reorientation is identical in intensity upon mutating stretch-sensitive tyrosines of p130Cas only. These statistically highly significant data pinpoint early events in a Src family kinase- and p130Cas-dependent mechanosensory/mechanotransduction pathway.

Cyclic stress at mHz frequencies aligns fibroblasts in direction of zero strain.

Recognition of external mechanical signals is vital for mammalian cells. Cyclic stretch, e.g. around blood vessels, is one such signal that induces cell reorientation from parallel to almost perpendicular to the direction of stretch. Here, we present quantitative analyses of both, cell and cytoskeletal reorientation of umbilical cord fibroblasts. Cyclic strain of preset amplitudes was applied at mHz frequencies. Elastomeric chambers were specifically designed and characterized to distinguish between zero strain and minimal stress directions and to allow accurate theoretical modeling. Reorientation was only induced when the applied stretch exceeded a specific amplitude, suggesting a non-linear response. However, on very soft substrates no mechanoresponse occurs even for high strain. For all stretch amplitudes, the angular distributions of reoriented cells are in very good agreement with a theory modeling stretched cells as active force dipoles. Cyclic stretch increases the number of stress fibers and the coupling to adhesions. We show that changes in cell shape follow cytoskeletal reorientation with a significant temporal delay. Our data identify the importance of environmental stiffness for cell reorientation, here in direction of zero strain. These in vitro experiments on cultured cells argue for the necessity of rather stiff environmental conditions to induce cellular reorientation in mammalian tissues.