It is widely postulated that mechanotransduction is initiated at the local force-membrane interface by inducing local conformational changes of proteins, similar to soluble ligand-induced signal transduction. However, all published reports are limited in time scale to address this fundamental issue. Using a FRET-based cytosolic Src reporter in a living cell, we quantified changes of Src activities as a local stress via activated integrins was applied. The stress induced rapid (<0.3 s) activation of Src at remote cytoplasmic sites, which depends on the cytoskeletal prestress. In contrast, there was no Src activation within 12 s of soluble epidermal growth factor (EGF) stimulation. A 1.8-Pa stress over a focal adhesion activated Src to the same extent as 0.4 ng/ml EGF at long times (minutes), and the energy levels for mechanical stimulation and chemical stimulation were comparable. The effect of both stress and EGF was less than additive. Nanometer-scale cytoskeletal deformation analyses revealed that the strong activation sites of Src by stress colocalized with large deformation sites of microtubules, suggesting that microtubules are essential structures for transmitting stresses to activate cytoplasmic proteins. These results demonstrate that rapid signal transduction via the prestressed cytoskeleton is a unique feature of mechanotransduction.cytoskeleton ͉ growth factor ͉ mechanical force ͉ prestress ͉ microtubule T he sensing and response of living cells and tissues to mechanical forces and physical microenvironments are critical for their functions and survival (1-3). However, the underlying mechanisms remain largely elusive. Various models of mechanotransduction have been proposed (2, 4, 5); the most straightforward model involves force-induced local conformational changes of proteins (6). It is generally believed that like soluble ligand-induced signal transduction, mechanotransduction initiates at the local force-membrane interface (e.g., at focal adhesions) by inducing local conformational changes or unfolding of membrane-bound proteins, followed by a cascade of diffusion-based or translocation-based signaling in the cytoplasm. Recent reports demonstrate force-induced dynamic changes in Src activity (7), mechanical extension of the Src family kinase substrate p130Cas (8), and forced unfolding of proteins in living cells (9). However, all published reports, including past studies with the reporter-type of construct extended here (7), are limited in time scale. Therefore, it has not been possible to compare early dynamics of mechanotransduction with that of soluble ligand-induced signal transduction. Here, we applied a local stress of physiologic magnitude and simultaneously imaged changes in Src activity in living cells by using a CFP-YFP Src reporter and fluorescence resonance energy transfer (FRET) technology. We show that stress-induced Src activation occurs rapidly in the cytoplasm and depends on the integrity of the microfilaments and microtubules, substrate rigidity, and the cytoskeletal prestress, d...

The cytoskeletal adapter protein talin plays a prominent role in adhesive structures connecting integrins to the actin cytoskeleton. In this work, Kumar et al. use a novel talin sensor to measure talin tension and provide insights into focal adhesion force transmission and mechanosensitivity.

SummaryForces acting on cells govern many important regulatory events during development, normal physiology, and disease processes. Integrin-mediated adhesions, which transmit forces between the extracellular matrix and the actin cytoskeleton, play a central role in transducing effects of forces to regulate cell functions. Recent work has led to major insights into the molecular mechanisms by which these adhesions respond to forces to control cellular signaling pathways. We briefly summarize effects of forces on organs, tissues, and cells; and then discuss recent advances toward understanding molecular mechanisms.

Genetically encoded biosensors based on FRET have enabled the visualization of signaling events in live cells with high spatiotemporal resolution. However, the limited sensitivity of these biosensors has hindered their broad application in biological studies. We have paired enhanced CFP (ECFP) with YPet, a variant of YFP. This ECFP/YPet FRET pair markedly enhanced the sensitivity of biosensors (several folds enhancement without the need of tailored optimization for each individual biosensor) for a variety of signaling molecules, including tyrosine kinase Src, small GTPase Rac, calcium, and a membrane-bound matrix metalloproteinase MT1-MMP. The application of these improved biosensors revealed that the activations of Src and Rac by PDGF displayed distinct subcellular patterns during directional cell migration on micropatterned surface. The activity of Rac is highly polarized and concentrated at the leading edge, whereas Src activity is relatively uniform. These FRET biosensors also led to the discovery that Src and Rac mutually regulate each other. Our findings indicate that molecules within the same signaling feedback loop can be differentially regulated at different subcellular locations. In summary, ECFP/YPet may serve as a general FRET pair for the development of highly sensitive biosensors to allow the determination of molecular hierarchies at subcellular locations in live cells.RET is a phenomenon of quantum mechanics allowing the detection of distance/orientation changes between fluorophore pairs (1). Genetically encoded FRET biosensors consisting of a donor and an acceptor fluorescence protein (FP) can be conveniently introduced into cells and targeted to subcellular compartments. Hence, these biosensors have been widely developed and applied for the detection of various molecular activities in live cells with high spatiotemporal resolution (2). Early studies used enhanced blue FP (EBFP) and enhanced GFP (EGFP) as the FRET pair for biosensor development (1). Because enhanced CFP (ECFP) has better extinction coefficient, quantum yield, and photostability than EBFP (3), ECFP and variants of enhanced YFP (EYFP) have become the most popular FRET pairs. However, the sensitivity of many biosensors based on ECFP and EYFP is generally limited. Recently, a high-efficiency FRET pair, CyPet and YPet, has been developed to significantly enhance the dynamic range of a protease biosensor (4). However, CyPet folded poorly at 37°C and hence is not suitable for live cell imaging (5). Therefore, other FP pairs are desired for the development of highly sensitive FRET biosensors.Src kinase plays crucial roles in a variety of cellular functions, including angiogenesis and cancer development (6). For example, angiogenic VEGF has been shown to activate Src to regulate cell motility and migration during angiogenesis process (7). It has also been documented that Src contributes to cell migration by modulating Rac, a small GTPase controlling cell protrusion and lamellipodia formation (8). Src can phosphorylate p130cas, which recru...

Proper subcellular localization of focal adhesion kinase (FAK) is crucial for many cellular processes. It remains, however, unclear how FAK activity is regulated at subcellular compartments. To visualize the FAK activity at different membrane microdomains, we develop a fluorescence resonance energy transfer (FRET)-based FAK biosensor, and target it into or outside of detergent-resistant membrane (DRM) regions at the plasma membrane. Here we show that, on cell adhesion to extracellular matrix proteins or stimulation by platelet-derived growth factor (PDGF), the FRET responses of DRM-targeting FAK biosensor are stronger than that at non-DRM regions, suggesting that FAK activation can occur at DRM microdomains. Further experiments reveal that the PDGF-induced FAK activation is mediated and maintained by Src activity, whereas FAK activation on cell adhesion is independent of, and in fact essential for the Src activation. Therefore, FAK is activated at membrane microdomains with distinct activation mechanisms in response to different physiological stimuli.