Defects in mechanotransduction ? the cellular processes that convert mechanical stimuli into biochemical signals ? are implicated in the development of a wide range of diseases, including cardiomyopathies, muscular dystrophies, and cancer progression. Tension of the plasma membrane has been increasingly recognized to actively regulate many cellular processes, including cell migration and membrane trafficking. The conventional view that the plasma membrane is a two-dimensional fluid lipid bilayer with embedded proteins has led to the idea that membrane tension could transmit forces over long-range to regulate cell polarity and migration. However, recent work has pointed to local variation of membrane tension can mediate distinct sub-cellular processes. While there exist approaches to measure membrane tension, the techniques require specialized setup and expertise. This has severely limited research progress in key questions in cell mechanotransduction. To address this unmet need, the objective of the proposed work is to repurpose bacterial mechanosensitive channel MscL as a membrane tension biosensor. The large conformational changes predicted from structural and biophysical studies coupled with phenomenal successes in recent years on protein-based biosensors make MscL an ideal candidate for engineering a membrane tension sensor. Recent work in our lab has demonstrated functional reconstitution of MscL in mammalian cells. Further, we have accrued a range of innovative methodologies for reconstituting MscL in vitro and for manipulating and measuring cell mechanics properties.
In Aim 1, we will insert circular permutated GFP (cpGFP) in the periplasmic loop of MscL and systematically engineer it for increased responsiveness and sensitivity. We will characterize cpGFP-MscL reconstituted into lipid bilayer vesicles and select the most optimal sensor experiments in living cells.
In Aim 2, we will establish the connection between membrane tension and cell contractility, as this important relationship between the two has never been determined but assumed. We will measure membrane tension in cells with different spreading areas and also evaluate dynamic changes of membrane tension in cells subjected to hypo-osmotic shock. These experiments will resolve the spatiotemporal dynamics of membrane tension in cellular process known to have membrane tension changes. The high spatial and temporal resolution afforded by the fluorescence- based membrane tension biosensor is expected to have transformative impact in membrane biophysics, developmental biology (where dramatic morphological dynamics that takes place is expected to elevate membrane tension), and mechanobiology.
The proposed research is relevant to public health because defects in mechanotransduction, the conversion of mechanical cues to biochemical signals, underlie several debilitated diseases including cardiomyopathies, muscular dystrophies, and cancer. The proposed research is to develop a fluorescence-based biosensor for membrane tension and apply this new tool to fundamental questions in cell mechanics. Ultimately, it is envisioned that fundamental understanding of regulation of membrane tension will enable new approaches for treating diseases that arise from aberrant mechanotransduction. ! !
