Development of a Generalizable Assay for Probing the Effects of Mechanical Force on the Function of Fluorescent Proteins within Molecular Tension Sensors

The extracellular environment is a key regulator of cell behavior, providing both biochemical factors and mechanical signals to influence the form and function of cells. The process by which cells sense and respond to environmental mechanical signals is often mediated through force-dependent changes in protein structure and function through a poorly understood process known as mechanotransduction. Towards elucidating the molecular processes underlying mechanosensitive regulation, molecular tension sensors (MTSs) have been created to measure forces experienced by specific proteins inside cells. However, an incomplete understanding of the effects of intracellular forces on fluorescent protein (FP) function within the context of MTs limits sensor application and interpretation. To advance our understanding of the molecular events mediating mechanotransduction, it is necessary to improve on existing approaches as well as to develop new technologies for probing mechanical consequences inside cells. In this dissertation we aim to address this limitation by creating a generalizable assay for probing the effects of cell-generated forces on FP function towards improving the use and interpretation of MTSs. Additionally, we describe the development of a new “synthetic” actin crosslinking sensor which leverages FP mechanosensitivity to provide new insights into mechanical processes inside cells.In our initial efforts, we focused on investigating the effects of cell generated forces on FP function within vinculin-based MTSs. We chose vinculin as our model system as vinculin is a well-studied mechanosensitive protein, known to play a critical role in force transmission inside cells. Additionally, the vinculin tension sensor (VinTS) has been extensively characterized, validated, and utilized in a broad array of applications. Leveraging the relationship between FRET measurements and fluorophore stoichiometry with vinculin MTSs, we developed a generalizable assay for evaluating changes in ensemble MTS measurements in terms of fluorophore contributions. Furthermore, we validated this new method on an extensive MTS data set containing over 2000 cells expressing vinculin sensors. Our analysis revealed that FP stoichiometry within VinTS was modulated significantly within individual focal adhesions (FAs) in an actomyosin-dependent manner, and that both load magnitude and load duration likely play a role. Additionally, we found that this force-mediated loss of FP function, or “mechanical quenching,” is a reversible process, consistent with nonequilibrium transitions in protein structure. To investigate FP mechanosensitivity further, we developed an engineered FRET-based actin crosslinking (ABD) sensor to serve as an improved experimental platform, within which FPs would be subjected to higher loads in a manner free of endogenous biochemical regulation. Within this new system, higher tensile loading and FP mechanical quenching was observed at dynamic actin networks. Furthermore, we found that FP mechanical quenching within these sensors was mediated by non-muscle myosin II (NMII) activity and appears to be reversible. In addition, we found that FPs exhibit different sensitivities to intracellular mechanical loads. To probe the molecular origins of ABD sensors loading within cells, we manipulated the organization and dynamics of actin structures by tuning substrate stiffness within engineered in vitro culture systems. Using this approach, we found that ABD sensors reported increased loads and FP mechanical quenching at dynamic actin networks in response to softer substrates. By coupling FRET-based MTSs with the tunability of in vitro culture models, we demonstrated the application of ABD sensors to probe changes in tensile loading in response to environmental mechanical cues. In summary, this dissertation describes the development of novel tools for studying the effects of intracellular forces on FP function within the context of FRET-based tension sensors. Using these tools, we found that FPs, like mechanosensitive signaling proteins, can undergo nonequilibrium transitions in response to cell-generated forces. Based on these observations, we propose that FP mechanical quenching within MTSs could potentially serve as an entirely new way to visualize and probe mechanical consequences within force-sensitive proteins. By exploiting the mechanosensitivity of FPs as a mechanical consequence, new insights into molecular force-sensitive processes inside cells may be obtained.