Biological processes are sensitive to externally applied forces and internally generated forces that sculpt the cytoarchitecture of cells. Myogenesis and myofiber remodeling are critical to normal physiological processes such as development, injury repair and hypertrophy in response to chronic load changes in myofibers. These processes follow complex signaling pathways requiring mechanical cues. Dysfunction of myogenesis and remodeling is involved in many muscle pathologies. Ideally, one would want to monitor the stress on different structural proteins and mechanical domains directly during myogenesis and remodeling without perturbing the mechanics of the system.Our focus here is to study muscle differentiation in vitro and to measure the forces in different structural proteins using the genetically coded optical force probe cpstFRET. A major impediment to quantifying the sensitivity of FRET based stress probes are variable expression levels using transient transfections, sharing of stress with different levels of exogenous proteins, temporal expression patterns due to developmentally regulated expression, and large structural proteins that are difficult to manipulate genetically outside of its host.We will use CRISPR-Cas targeted gene cleavage and homologous recombination to introduce the probe into host proteins that include both constitutively expressed genes like laminA, actinin, filamin, and plectin, and developmentally regulated genes such as dystrophin. We will narrow our focus to the C2C12 myoblast cell line and monitor stress changes during development into myotubes on flexible micropatterned PDMS substrates. We will determine the stress changes on the proteins listed above at different developmental stages and during application of various uniaxial and isotropic forces.
Cells generate and respond to mechanical forces and some of these are familiar to us in our senses of touch and hearing and our beating hearts, but all cells generate and transduce forces. These are intimately involved in the development of tissues and pathologies such as cardiac arrhythmias, muscular dystrophy and metastatic cancer. We have developed a new family of tools to measure the forces in specific proteins by turning the force into an optical signal that can be imaged in real time in a microscope.
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