Cell-cell fusion or syncytialization is fundamental to the reproduction, development, and homeostasis of multicellular organisms. In addition to various cell type–specific fusogenic proteins, cell surface externalization of phosphatidylserine (PS), a universal eat-me signal in apoptotic cells, has been observed in different cell fusion events. Nevertheless, the molecular underpinnings of PS externalization and cellular mechanisms of PS-facilitated cell-cell fusion are unclear. Here, we report that TMEM16F, a Ca²⁺-activated phospholipid scramblase (CaPLSase), plays an essential role in placental trophoblast fusion by translocating PS to cell surface independent of apoptosis. The placentas from the TMEM16F knockout mice exhibit deficiency in trophoblast syncytialization and placental development, which lead to perinatal lethality. We thus identified a new biological function of TMEM16F CaPLSase in trophoblast fusion and placental development. Our findings provide insight into understanding cell-cell fusion mechanism of other cell types and on mitigating pregnancy complications such as miscarriage, intrauterine growth restriction, and preeclampsia.

TMEM16F, a Ca²⁺-activated phospholipid scramblase (CaPLSase), is critical for placental trophoblast syncytialization, HIV infection, and SARS-CoV2-mediated syncytialization, however, how TMEM16F is activated during cell fusion is unclear. Here, using trophoblasts as a model for cell fusion, we demonstrate that Ca²⁺ influx through the Ca²⁺ permeable transient receptor potential vanilloid channel TRPV4 is critical for TMEM16F activation and plays a role in subsequent human trophoblast fusion. GSK1016790A, a TRPV4 specific agonist, robustly activates TMEM16F in trophoblasts. We also show that TRPV4 and TMEM16F are functionally coupled within Ca²⁺ microdomains in a human trophoblast cell line using patch-clamp electrophysiology. Pharmacological inhibition or gene silencing of TRPV4 hinders TMEM16F activation and subsequent trophoblast syncytialization. Our study uncovers the functional expression of TRPV4 and one of the physiological activation mechanisms of TMEM16F in human trophoblasts, thus providing us with novel strategies to regulate CaPLSase activity as a critical checkpoint of physiologically and disease-relevant cell fusion events.

Cell-surface exposure of phosphatidylserine (PS) is essential for phagocytic clearance and blood clotting. Although a calcium-activated phospholipid scramblase (CaPLSase) has long been proposed to mediate PS exposure in red blood cells (RBCs), its identity, activation mechanism, and role in RBC biology and disease remain elusive. Here, we demonstrate that TMEM16F, the long-sought-after RBC CaPLSase, is activated by calcium influx through the mechanosensitive channel PIEZO1 in RBCs. PIEZO1-TMEM16F functional coupling is enhanced in RBCs from individuals with hereditary xerocytosis (HX), an RBC disorder caused by PIEZO1 gain-of-function channelopathy. Enhanced PIEZO1-TMEM16F coupling leads to an increased propensity to expose PS, which may serve as a key risk factor for HX clinical manifestations including anemia, splenomegaly, and postsplenectomy thrombosis. Spider toxin GsMTx-4 and antigout medication benzbromarone inhibit PIEZO1, preventing force-induced echinocytosis, hemolysis, and PS exposure in HX RBCs. Our study thus reveals an activation mechanism of TMEM16F CaPLSase and its pathophysiological function in HX, providing insights into potential treatment.

The immunosuppressive tumor microenvironment enables immune evasion through mechanisms beyond canonical immune checkpoints. While phosphatidylserine (PS) externalization coordinates apoptotic clearance under physiological conditions, tumors hijack this mechanism through apoptotic mimicry to subvert antitumor immunity. Here, we identify TMEM16F, a calcium-activated phospholipid scramblase, as a driver of tumor-intrinsic PS externalization. TMEM16F-mediated PS scrambling polarized macrophages to an immunosuppressive M2 phenotype, which promotes TGF-β1 secretion and regulatory T cell expansion to suppress cytotoxic lymphocytes. Genetic ablation of TMEM16F abolished PS exposure, systemically reprogrammed the tumor microenvironment and primary immune organs toward immune activation, and suppressed tumor growth across cancer models. Pharmacological scramblase inhibition produced these effects, demonstrating therapeutic potential. Our findings establish TMEM16F-dependent phospholipid scrambling as a critical immune evasion axis and propose targeting this pathway for cancer treatment.

The calcium-activated TMEM16 proteins and the mechanosensitive/osmolarity-activated OSCA/TMEM63 proteins belong to the Transmembrane Channel/Scramblase (TCS) superfamily. Within the superfamily, OSCA/TMEM63 proteins, as well as TMEM16A and TMEM16B, are thought to function solely as ion channels. However, most TMEM16 members, including TMEM16F, maintain an additional function as scramblases, rapidly exchanging phospholipids between leaflets of the membrane. Although recent studies have advanced our understanding of TCS structure–function relationships, the molecular determinants of TCS ion and lipid permeation remain unclear. Here, we show that single mutations along the transmembrane helix (TM) 4/6 interface allow non-scrambling TCS members to permeate phospholipids. In particular, this study highlights the key role of TM 4 in controlling TCS ion and lipid permeation and offers novel insights into the evolution of the TCS superfamily, suggesting that, like TMEM16s, the OSCA/TMEM63 family maintains a conserved potential to permeate ions and phospholipids.

The calcium-activated TMEM16 proteins and the mechanosensitive/osmolarity-activated OSCA/TMEM63 proteins belong to the Transmembrane Channel/Scramblase (TCS) superfamily. Within the superfamily, OSCA/TMEM63 proteins, as well as TMEM16A and TMEM16B, are thought to function solely as ion channels. However, most TMEM16 members, including TMEM16F, maintain an additional function as scramblases, rapidly exchanging phospholipids between leaflets of the membrane. Although recent studies have advanced our understanding of TCS structure-function relationships, the molecular determinants of TCS ion and lipid permeation remain unclear. Here we show that single mutations along the transmembrane helix (TM) 4/6 interface allow non-scrambling TCS members to permeate phospholipids. In particular, this study highlights the key role of TM 4 in controlling TCS ion and lipid permeation and offers novel insights into the evolution of the TCS superfamily, suggesting that, like TMEM16s, the OSCA/TMEM63 family maintains a conserved potential to permeate ions and phospholipids.