Elucidation of Unknown Signaling Roles Mediated by Lipid Scramblase TMEM16F

Abstract

TMEM16F is a calcium-activated phospholipid scramblase that rapidly disrupts plasma membrane phospholipid asymmetry. It is traditionally recognized for its role in exposing phosphatidylserine (PS) on the cell surface, thereby initiating extracellular signaling events critical for processes such as blood coagulation, phagocytosis, and cell fusion. Despite its well-established extracellular signaling functions, its potential roles in intracellular signaling remain poorly understood, and the physiological calcium sources that activate TMEM16F are still unclear.The potential impact of TMEM16F on the inner leaflet of the plasma membrane is often overlooked. TMEM16F-mediated PS exposure has the potential to alter the electrostatic landscape of the inner leaflet and disrupt the binding of membrane proteins that rely on PS for membrane association. To elucidate the intracellular signaling role of TMEM16F, we used endothelial cells as a model system and found that TMEM16F regulates angiogenesis by modulating VE-cadherin–Src kinase signaling, revealing a previously unrecognized intracellular signaling function (Chapter 2). TMEM16F is traditionally believed to require sustained, high intracellular calcium concentrations for activation. As a result, most studies have used non-physiological stimuli, such as calcium ionophores, to artificially activate TMEM16F. However, endogenous calcium signals rarely reach these levels, raising questions about how TMEM16F is activated under native physiological and pathological conditions. To address this, we used placental trophoblasts as a model to examine the upstream regulation of TMEM16F-mediated fusion. We discovered that the mechanosensitive, calcium-permeable channel PIEZO1 functions as a physiological upstream activator of TMEM16F and that its expression is essential for trophoblast fusion and placental development (Chapter 3). TMEM16F has also been implicated in regulated cell death, including ferroptosis, an iron-dependent form of cell death marked by lipid peroxidation. However, its mechanistic contribution to ferroptosis execution remains unclear. Using placental trophoblasts, which highly express TMEM16F and exhibit distinct morphological features during ferroptosis, we investigated its role in this process. We show that ferroptosis is a calcium-dependent process and that TMEM16F promotes oxidative membrane damage by facilitating ROS accumulation and phospholipid peroxidation. These findings reveal a previously unrecognized role for TMEM16F in modulating redox imbalance and membrane remodeling during ferroptosis (Chapter 4). In summary, this thesis establishes TMEM16F as a multifunctional membrane regulator at the intersection of lipid scrambling, calcium signaling, and membrane dynamics. By dissecting its roles in endothelial signaling, trophoblast fusion, and ferroptotic cell death, we uncover previously unknown intracellular functions, identify PIEZO1 as a physiological activator, and elucidate a sequence of ferroptotic membrane remodeling events orchestrated by TMEM16F. Together, these findings position TMEM16F as a central transducer linking calcium influx to membrane remodeling and intracellular signaling in both physiological and pathological contexts.