Understanding mechanisms of membrane tension loss and recovery using small-molecule tools
Maintenance of membrane tension at the plasma membrane (PM) is an important yet understudied aspect of cellular homeostasis. Dysregulated membrane tension homeostasis has roles in cancer, neurodegeneration, and metabolic syndrome but its study has been limited by a lack of tools to measure and manipulate it. The Target of Rapamycin Complex 2 (TORC2), an important signaling hub regulating cell growth, has an unexpected role in maintaining PM tension homeostasis. A screen for TORC2-specific inhibitors identified the lipophilic molecule palmitoylcarnitine (PalmC) which induces a reversible loss in PM tension. It is currently unknown how PalmC is able to induce this tension loss, but understanding both its mode-of-action and how cells are able to recover from its effects are important for its development as a potential membrane-targeting drug. In this proposal, we aim to reveal key proteins involved in the effects of PalmC (Aim 1). We expect that a specific plasma membrane-localized transporter will mediate its uptake. Both by identifying this protein and solving its structure in a native-like lipidic environment and/or in complex with PalmC, we hope to reveal new insights into how PalmC might be tailored for therapeutic potential.  We will also investigate the recovery process post-PalmC treatment, starting from the observation that the TORC2-activating protein Slm1 co-localizes with TORC2 and forms very large clusters at sites of PalmC-induced membrane invaginations (Aim 2). Previous observations of TORC1 regulation suggest that clustering is correlated with the formation of ordered polymers. To investigate whether this is a conserved mechanism of regulation, we will attempt to isolate a Slm1-TORC2 superstructure and determine its structure using cryoEM with the expectation that structural details about the interactions between these proteins within the superstructure will reveal mechanistic insights into the regulation of TORC2, as well as membrane tension.