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Focal adhesions (FAs) are integrin-based transmembrane assemblies that connect a cell to its extracellular matrix (ECM). They are mechanosensors through which cells exert actin cytoskeleton-mediated traction forces to sense the ECM stiffness. Interestingly, FAs themselves are dynamic structures that adapt their growth in response to mechanical force. It is unclear how the cell manages the plasticity of the FA structure and the associated traction force to accurately sense ECM stiffness. Strikingly, FA traction forces oscillate in time and space, and govern the cell mechanosensing of ECM stiffness. However, precisely how and why the FA traction oscillates is unknown. We developed a model of FA growth that integrates the contributions of the branched actin network and stress fibers (SFs). Using the model in combination with experimental tests, we show that the retrograde flux of the branched actin network promotes the proximal growth of the FA and contributes to a traction peak near the FA's distal tip. The resulting traction gradient within the growing FA favors SF formation near the FA's proximal end. The SF-mediated actomyosin contractility further stabilizes the FA and generates a second traction peak near the center of the FA. Formin-mediated SF elongation negatively feeds back with actomyosin contractility, resulting in central traction peak oscillation. This underpins the observed FA traction oscillation and, importantly, broadens the ECM stiffness range over which FAs can accurately adapt to traction force generation. Actin cytoskeleton-mediated FA growth and maturation thus culminate with FA traction oscillation to drive efficient FA mechanosensing.

G protein-coupled receptors (GPCRs) are crucial sensors of extracellular signals in eukaryotes, and direct measurement of GPCR-mediated signaling is useful for high-throughput mutational studies. However, this is particularly difficult for the light-activated GPCR rhodopsin... G protein-coupled receptors (GPCRs) are crucial sensors of extracellular signals in eukaryotes, with multiple GPCR mutations linked to human diseases. With the growing number of sequenced human genomes, determining the pathogenicity of a mutation is challenging, but can be aided by a direct measurement of GPCR-mediated signaling. This is particularly difficult for the visual pigment rhodopsin—a GPCR activated by light—for which hundreds of mutations have been linked to inherited degenerative retinal diseases such as retinitis pigmentosa. In this study, we successfully engineered, for the first time, activation by human rhodopsin of the yeast mating pathway, resulting in signaling via a fluorescent reporter. We combine this novel assay for rhodopsin light-dependent activation with studies of subcellular localization, and the upregulation of the unfolded protein response in response to misfolded rhodopsin protein. We use these assays to characterize a panel of rhodopsin mutations with known molecular phenotypes, finding that rhodopsin maintains a similar molecular phenotype in yeast, with some interesting differences. Furthermore, we compare our assays in yeast with clinical phenotypes from patients with novel disease-linked mutations. We demonstrate that our engineered yeast strain can be useful in rhodopsin mutant classification, and in helping to determine the molecular mechanisms underlying their pathogenicity. This approach may also be applied to better understand the clinical relevance of other human GPCR mutations, furthering the use of yeast as a tool for investigating molecular mechanisms relevant to human disease.