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Microarchitectural cues drive aligned fibrillar collagen deposition in vivo and in biomaterial scaffolds, but the cell-signaling events that underlie this process are not well understood. Utilizing a multicellular patterning model system that allows for observation of intracellular signaling events during collagen matrix assembly, we investigated the role of calcium (Ca2+) signaling in human mesenchymal stem cells (MSCs) during this process. We observed spontaneous Ca2+ oscillations in MSCs during fibrillar collagen assembly, and hypothesized that the transient receptor potential vanilloid 4 (TRPV4) ion channel, a mechanosensitive Ca2+-permeable channel, may regulate this signaling. Inhibition of TRPV4 nearly abolished Ca2+ signaling at initial stages of collagen matrix assembly, while at later times had reduced but significant effects. Importantly, blocking TRPV4 activity dramatically reduced aligned collagen fibril assembly; conversely, activating TRPV4 accelerated aligned collagen formation. TRPV4-dependent Ca2+ oscillations were found to be independent of pattern shape or subpattern cell location, suggesting this signaling mechanism is necessary for aligned collagen formation but not sufficient in the absence of physical (microarchitectural) cues that force multicellular alignment. As cell-generated mechanical forces are known to be critical to the matrix assembly process, we examined the role of TRPV4-mediated Ca2+ signaling in force generated across the load-bearing focal adhesion protein vinculin within MSCs using an FRET-based tension sensor. Inhibiting TRPV4 decreased tensile force across vinculin, whereas TRPV4 activation caused a dynamic unloading and reloading of vinculin. Together, these findings suggest TRPV4 activity regulates forces at cell-matrix adhesions and is critical to aligned collagen matrix assembly by MSCs.

Intracellular forces defined The ability of cells to respond to physical forces is fundamental to development and physiology, including regulation of blood pressure, cell adhesion and migration. A major limitation to the study of these phenomena has been the difficulty of measuring molecular forces in cells in vivo . Grashoff et al . now report the development of a genetically encoded, fluorescent tension-sensing module capable of measuring mechanical forces across specific proteins in vivo . The sensor was tested on vinculin, a membrane-cytoskeletal protein that is recruited to focal adhesions and connects cell-adhesion molecules (integrins) to actin filaments. The data reveal a regulatory mechanism in which the ability of vinculin to bear force determines whether focal adhesions assemble or disassemble under force. This new biosensor should be applicable to other proteins involved in mechanotransduction. The ability of cells to respond to physical forces is central to development and physiology, but until now it has been difficult to directly measure forces across proteins in vivo . Here, however, a calibrated biosensor is described that can measure forces with high sensitivity across specific proteins in cells. This is applied to the vinculin protein, and a regulatory mechanism is revealed in which the force applied to vinculin determines whether focal adhesions assemble or disassemble. Mechanical forces are central to developmental, physiological and pathological processes 1 . However, limited understanding of force transmission within sub-cellular structures is a major obstacle to unravelling molecular mechanisms. Here we describe the development of a calibrated biosensor that measures forces across specific proteins in cells with piconewton (pN) sensitivity, as demonstrated by single molecule fluorescence force spectroscopy 2 . The method is applied to vinculin, a protein that connects integrins to actin filaments and whose recruitment to focal adhesions (FAs) is force-dependent 3 . We show that tension across vinculin in stable FAs is ∼2.5 pN and that vinculin recruitment to FAs and force transmission across vinculin are regulated separately. Highest tension across vinculin is associated with adhesion assembly and enlargement. Conversely, vinculin is under low force in disassembling or sliding FAs at the trailing edge of migrating cells. Furthermore, vinculin is required for stabilizing adhesions under force. Together, these data reveal that FA stabilization under force requires both vinculin recruitment and force transmission, and that, surprisingly, these processes can be controlled independently.

Molecular tension sensors have contributed to a growing understanding of mechanobiology. However, the limited dynamic range and inability to specify the mechanical sensitivity of these sensors has hindered their widespread use in diverse contexts. Here, we systematically examine the components of tension sensors that can be altered to improve their functionality. Guided by the development of a first principles model describing the mechanical behavior of these sensors, we create a collection of sensors that exhibit predictable sensitivities and significantly improved performance in cellulo. Utilized in the context of vinculin mechanobiology, a trio of these new biosensors with distinct force- and extension-sensitivities reveal that an extension-based control paradigm regulates vinculin loading in a variety of mechanical contexts. To enable the rational design of molecular tension sensors appropriate for diverse applications, we predict the mechanical behavior, in terms of force and extension, of additional 1020 distinct designs. Cells must sense signals from their surroundings to play their roles within the body. These signals can be biochemical, such as growth-promoting substances, or mechanical, for example the stiffness or softness of the environment. Mechanical signals can be detected by load-bearing proteins, which stretch like tiny springs in response to forces. In animals, these proteins span the membrane separating the interior of the cell from the exterior. Externally, the proteins attach to structures around the cell; internally, they connect to the machinery that both generates forces and allows cells to respond to signals from outside. As such, load-bearing proteins form a direct mechanical link between cell and environment. Scientists use tools called molecular tension sensors to measure how much a load-bearing protein stretches in response to changes, and the force that is being applied to it. However, just like any other type of scale, these sensors only work over a certain range, which happens to be limited. This means that, for example, they cannot measure forces in tissues that are too soft (like the brain), or too stiff (such as bones). New sensors that can assess forces in these contexts are therefore needed, but so far research in this area has been slow due to a reliance on ‘trial-and-error’ approaches. Here, LaCroix et al. developed a new method to predict the sensitivity of molecular tension sensors inside cells. This was accomplished by examining several existing sensors, and identifying which components could be altered to change the properties of the sensors. Then, this information was used to create a computer model that could predict how new sensors would behave, and which range of forces they could measure. Finally, the sensors designed following this method were tested in mouse cells grown in the laboratory, and they worked better than their predecessors. The next step was for LaCroix et al. to use a trio of new sensors with different sensitivities to study the load-bearing protein vinculin in mouse cells. The goal was to figure out exactly how cells manage their load-bearing proteins. Indeed, it was widely assumed that a cell acts on a load-bearing protein by applying a force on it. In response, the protein would stretch by a certain amount, which can change depending on its properties – a ‘stiffer’ protein would stretch less. Unexpectedly, the new sensors showed that cells instead manipulate how much vinculin stretches, applying varying forces to achieve the same length of the protein in different environments. Improved molecular tension sensors will give scientists a better insight into how cells respond to their mechanical environment, which could help to direct cell behavior in tissues engineered in the laboratory. This knowledge is also directly relevant to human health, as the mechanical properties of many tissues change during disease, such as tumors stiffening during cancer.

The ability of cells and tissues to respond differentially to mechanical forces applied in distinct directions is mediated by the ability of load-bearing proteins to preferentially maintain physical linkages in certain directions. However, the molecular basis and biological consequences of directional force-sensitive binding remain unclear. Vinculin (Vcn) is a load-bearing linker protein that exhibits directional catch bonding due to interactions between the Vcn tail domain (Vt) and filamentous (F)-actin. We developed a computational approach to predict Vcn residues involved in directional catch bonding and produced a set of associated Vcn variants with unaltered Vt structure, actin binding, or phospholipid interactions. Incorporation of the variants did not affect Vcn activation but reduced Vcn loading and altered exchange dynamics, consistent with the loss of directional catch bonding. Expression of Vcn variants perturbed the coordination of subcellular structures and cell migration, establishing key cellular functions for Vcn directional catch bonding. This study provides insights into the directional catch bonding of the linker protein vinculin by elucidating the molecular basis of mechanical reinforcement as well as unveiling its key role in subcellular organization and cellular processes.

Although understanding cells' responses to mechanical stimuli is seen as increasingly important for understanding cell biology, how to best measure, interpret, and model cells' mechanical properties remains unclear. We determine the frequency-dependent shear modulus of cultured mammalian cells by using four different methods, both unique and well established. This approach clarifies the effects of cytoskeletal heterogeneity, ATP-dependent processes, and cell regional variations on the interpretation of such measurements. Our results clearly indicate two qualitatively similar, but distinct, mechanical responses, corresponding to the cortical and intracellular networks, each having an unusual, weak power-law form at low frequency. The two frequency-dependent responses we observe are remarkably similar to those reported for a variety of cultured mammalian cells measured with different techniques, suggesting it is a useful consensus description. Finally, we discuss possible physical explanations for the observed mechanical response.

Cellular responses to mechanical forces are crucial in embryonic development and adult physiology, and are involved in numerous diseases, including atherosclerosis, hypertension, osteoporosis, muscular dystrophy, myopathies and cancer. These responses are mediated by load-bearing subcellular structures, such as the plasma membrane, cell-adhesion complexes and the cytoskeleton. Recent work has demonstrated that these structures are dynamic, undergoing assembly, disassembly and movement, even when ostensibly stable. An emerging insight is that transduction of forces into biochemical signals occurs within the context of these processes. This framework helps to explain how forces of varying strengths or dynamic characteristics regulate distinct signalling pathways.

Meniscus injuries are highly prevalent, and both meniscus injury and subsequent surgery are linked to the development of post-traumatic osteoarthritis (PTOA). Although the pathogenesis of PTOA remains poorly understood, the inflammatory cytokine IL-1 is elevated in synovial fluid following acute knee injuries and causes degradation of meniscus tissue and inhibits meniscus repair. Dynamic mechanical compression of meniscus tissue improves integrative meniscus repair in the presence of IL-1 and dynamic tensile strain modulates the response of meniscus cells to IL-1. Despite the promising observed effects of physiologic mechanical loading on suppressing inflammatory responses of meniscus cells, there is a lack of knowledge on the global effects of loading on meniscus transcriptomic profiles. In this study, we compared two established models of physiologic mechanical stimulation, dynamic compression of tissue explants and cyclic tensile stretch of isolated meniscus cells, to identify conserved responses to mechanical loading. RNA sequencing was performed on loaded and unloaded meniscus tissue or isolated cells from inner and outer zones, with and without IL-1. Overall, results from both models showed significant modulation of inflammation-related pathways with mechanical stimulation. Anti-inflammatory effects of loading were well-conserved between the tissue compression and cell stretch models for inner zone; however, the cell stretch model resulted in a larger number of differentially regulated genes. Our findings on the global transcriptomic profiles of two models of mechanical stimulation lay the groundwork for future mechanistic studies of meniscus mechanotransduction, which may lead to the discovery of novel therapeutic targets for the treatment of meniscus injuries.

Although actin and myosin are important contributors to cell-force generation, shape change, and motility, their contributions to cell stiffness and frequency-dependent rheology have not been conclusively determined. We apply several pharmacological interventions to cultured epithelial cells to elucidate the roles of actin and myosin in the mechanical response of cells and intracellular fluctuations. A suite of different methods is used to separately examine the mechanics of the deep cell interior and cortex, in response to depletion of intracellular ATP, depolymerization of F-actin, and inhibition of myosin II. Comparison of these results shows that F-actin plays a significant role in the mechanics of the cortical region of epithelial cells, but its disruption has no discernable effect on the rheology of the deeper interior. Moreover, we find that myosins do not contribute significantly to the rheology or ATP-dependent, non-Brownian motion in the cell interior. Finally, we investigate the broad distribution of apparent stiffness values reported by some microrheology methods, which are not observed with two-point microrheology. Based on our findings and a simple model, we conclude that heterogeneity of the tracer-cytoskeleton contacts, rather than the network itself, can explain the broad distribution of apparent stiffnesses.

Therapeutic ultrasound or shockwave has shown its great potential to stimulate neural and muscle tissue, where cavitation microbubble induced Ca signaling is believed to play an important role. However, the pertinent mechanisms are unknown, especially at the single-cell level. Particularly, it is still a major challenge to get a comprehensive understanding of the effect of potential mechanosensitive molecular players on the cellular responses, including mechanosensitive ion channels, purinergic signaling and integrin ligation by extracellular matrix. Here, laser-induced cavitation microbubble was used to stimulate individual HEK293T cells either genetically knocked out or expressing Piezo1 ion channels with different normalized bubble-cell distance. Ca signaling and potential membrane poration were evaluated with a real-time fluorescence imaging system. Integrin-binding microbeads were attached to the apical surface of the cells at mild cavitation conditions, where the effect of Piezo1, P2X receptors and integrin ligation on single cell intracellular Ca signaling was assessed. Ca responses were rare at normalized cell-bubble distances that avoided membrane poration, even with overexpression of Piezo1, but could be increased in frequency to 42% of cells by attaching integrin-binding beads. We identified key molecular players in the bead-enhanced Ca response: increased integrin ligation by substrate ECM triggered ATP release and activation of P2X-but not Piezo1-ion channels. The resultant Ca influx caused dynamic changes in cell spread area. This approach to safely eliciting a Ca response with cavitation microbubbles and the uncovered mechanism by which increased integrin-ligation mediates ATP release and Ca signaling will inform new strategies to stimulate tissues with ultrasound and shockwaves.

Electrotransfection is a widely used method for delivering genes into cells with electric pulses. Although different hypotheses have been proposed, the mechanism of electrotransfection remains controversial. Previous studies have indicated that uptake and intracellular trafficking of plasmid DNA (pDNA) are mediated by endocytic pathways, but it is still unclear which pathways are directly involved in the delivery. To this end, the present study investigated the dependence of electrotransfection on macropinocytosis. Data from the study demonstrated that electric pulses induced cell membrane ruffling and actin cytoskeleton remodeling. Using fluorescently labeled pDNA and a macropinocytosis marker (i.e., dextran), the study showed that electrotransfected pDNA co-localized with dextran in intracellular vesicles. Furthermore, electrotransfection efficiency could be decreased significantly by reducing temperature or treatment of cells with a pharmacological inhibitor of Rac1 and could be altered by changing Rac1 activity. Taken together, the findings suggested that electrotransfection of pDNA involved Rac1-dependent macropinocytosis. Mao et al. demonstrated that macropinocytosis of plasmid DNA was important for electrotransfection of cells in vitro. They further showed that the macropinocytosis was Rac1 dependent.