Scramblase TMEM16F Research Articles

Antiviral HIV-1 SERINC restriction factors disrupt virus membrane asymmetry

The host proteins SERINC3 and SERINC5 are HIV-1 restriction factors that reduce infectivity when incorporated into the viral envelope. The HIV-1 accessory protein Nef abrogates incorporation of SERINCs via binding to intracellular loop 4 (ICL4). Here, we determine cryoEM maps of full-length human SERINC3 and an ICL4 deletion construct, which reveal that hSERINC3 is comprised of two α-helical bundles connected by a ~ 40-residue, highly tilted, “crossmember” helix. The design resembles non-ATP-dependent lipid transporters. Consistently, purified hSERINCs reconstituted into proteoliposomes induce flipping of phosphatidylserine (PS), phosphatidylethanolamine and phosphatidylcholine. Furthermore, SERINC3, SERINC5 and the scramblase TMEM16F expose PS on the surface of HIV-1 and reduce infectivity, with similar results in MLV. SERINC effects in HIV-1 and MLV are counteracted by Nef and GlycoGag, respectively. Our results demonstrate that SERINCs are membrane transporters that flip lipids, resulting in a loss of membrane asymmetry that is strongly correlated with changes in Env conformation and loss of infectivity.

TMEM16A/F support exocytosis but do not inhibit Notch-mediated goblet cell metaplasia of BCi-NS1.1 human airway epithelium.

Cl- channels such as the Ca2+ activated Cl- channel TMEM16A and the Cl- permeable phospholipid scramblase TMEM16F may affect the intracellular Cl- concentration ([Cl-]i), which could act as an intracellular signal. Loss of airway expression of TMEM16A induced a massive expansion of the secretory cell population like goblet and club cells, causing differentiation into a secretory airway epithelium. Knockout of the Ca2+-activated Cl- channel TMEM16A or the phospholipid scramblase TMEM16F leads to mucus accumulation in intestinal goblet cells and airway secretory cells. We show that both TMEM16A and TMEM16F support exocytosis and release of exocytic vesicles, respectively. Lack of TMEM16A/F expression therefore causes inhibition of mucus secretion and leads to goblet cell metaplasia. The human basal epithelial cell line BCi-NS1.1 forms a highly differentiated mucociliated airway epithelium when grown in PneumaCult™ media under an air liquid interface. The present data suggest that mucociliary differentiation requires activation of Notch signaling, but not the function of TMEM16A. Taken together, TMEM16A/F are important for exocytosis, mucus secretion and formation of extracellular vesicles (exosomes or ectosomes) but the present data do no not support a functional role of TMEM16A/F in Notch-mediated differentiation of BCi-NS1.1 cells towards a secretory epithelium.

Lipid scrambling facilitates membrane vesiculation through decreasing membrane stiffness.

Membrane blebbing plays important roles in cell migration, cytokinesis, apoptosis, as well as extracellular vesicle formation for intercellular communication. During budding of the plasma membrane (PM) to form extracellular vesicles, phosphatidylserine is often translocated from the PM inner leaflet to the outer leaflet, indicative of lipid scrambling that results in loss of native membrane asymmetry. Using micrometer-sized giant plasma membrane vesicles (GPMV) as a readout of PM vesiculation, we found that knocking out (KO) the lipid scramblase TMEM16F reduced lipid scrambling and dramatically inhibited GPMV formation, in line with previous observations. TMEM16F repletion could rescue GPMV formation, indicating that lipid scrambling is necessary for membrane budding. We hypothesized three mechanisms to explain this surprising observation, namely that lipid scrambling: (1) depletes PI(4,5)P2 from the inner leaflet leading to detachment of the PM from the actin cytoskeleton, (2) makes PM bilayers softer and easier to bend, (3) externalizes lipids that stabilize highly curved bud necks. Depletion of PI(4,5)P2 was indeed observed during vesiculation, however this occurred in both wild type and KO cells, implying that detachment of PM from actin cytoskeleton due to PIP2 depletion is not sufficient to make large vesicles. Next, through fluorescence lifetime imaging microscopy of a lipid packing reporter (Di4), we found that lipid scrambling leads to a decrease of PM packing, potentially making the membrane softer for bending and thus facilitating membrane budding. Finally, we observed that lipid scrambling resulted in externalization of phosphatidylethanolamine (PE). This PE was preferentially located at the highly curved vesicle necks, suggesting that the intrinsic curvature of PE stabilizes this highly curved membrane structure. Thus, we conclude that lipid scrambling changes membrane biophysical properties, potentially explaining mechanisms underlying vesicle-budding in physiological contexts including apoptotic cell removal, bone mineralization, fertilization, and blood coagulation.

Abstract C047: Phospholipid scramblase TMEM16F in cancer associated fibroblasts regulates trogocytosis to sustain critical dependency of pancreatic cancer cells on exogenous lipids

Abstract Pancreatic ductal adenocarcinoma (PDAC) cells derive their resistance to therapy and aggressive clinical course from the symbiotic signaling and metabolic interactions with cancer-associated fibroblasts (CAFs). Trogocytosis is a process of “nibbling” of plasma membranes between two living cells. Here, we demonstrate CAFs are the primary recipients of exogenous lipids which they transfer to metabolically “parasitic” PDAC cells via a contact-mediated trogocytosis. Whereas trogocytosis has been described in immune system and in normal development, the biochemical and signaling regulators of trogocytosis between CAFs and PDAC cells have not been defined. Results: We determine that trogocytosis occurs through heterotypic CAF-PDAC cell contacts: membrane blebbing (readily observed in activated CAFs and exaggerated by release of Ca2+ from the ER stores) results in the blebs uptake by the PDAC cells. The CAF blebs express externalized phosphatidylserine (PtdSer), and blockade of PtdSer in vitro transiently deters trogocytic uptake of CAF membranes. Probing a short list of candidate targets involved in regulation of cholesterol trafficking, membrane fusion and membrane protrusions using CRISPRi, has shown that CAFs deficient in CD81, ARF6 or TMEM16F exhibit markedly reduced ability to support the viability of cholesterol-auxotrophic PDAC cells in lipid-poor media. As a promising therapy target, TMEM16F is a Ca2+-regulated scramblase increasing PtdSer on the outer leaflet of the plasma membrane. TMEM16F protein is highly expressed in human PDAC CAFs compared to fibroblasts isolated from the adjacent non-malignant pancreatic tissues. The TMEM16F-null CAFs are unable to sustain of cholesterol-auxotrophic PDAC cells in lipid-poor co-cultures, and do not support the growth of PDAC cells in orthotopic co-implantation model. Furthermore, several clinically available antibiotics used to treat tapeworms are selective TMEM16F inhibitors. One of the widely available TMEM16F inhibitors, niclozamide, is effective in blocking cholesterol transfer from CAFs to PDAC cells in vivo. Conclusion: Our overall model is that activated CAFs initiate trogocytosis by expressing PtdSer as “eat me” signals on the surfaces of their membrane blebs. This process is regulated by Ca2+-dependent phospholipid scramblase TMEM16F which is an attractive drug-amenable target to dismantle the critical metabolic dependency in PDAC on the exogenous lipids. Re-purposing of clinically available TMEM16 inhibitors will make a tangible impact on treatment of PDAC patients in the near term. Citation Format: Charline Ogier, Alena Klochkova, Linara Gabitova-Cornell, Battuya Bayarmagnai, Diana Restifo, Aizhan Surumbayeva, Debora Barbosa Vendramini-Costa, Ralph Francescone, Janusz Franco-Barraza, Jaye Gardiner, Emmanuelle Nicolas, Andrei Efimov, Elizabeth A. Handorf, Kathy Q. Cai, Bojana Gligorijevic, Edna Cukierman, Igor Astsaturov. Phospholipid scramblase TMEM16F in cancer associated fibroblasts regulates trogocytosis to sustain critical dependency of pancreatic cancer cells on exogenous lipids [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C047.

Structural basis for the activation of the lipid scramblase TMEM16F

TMEM16F, a member of the conserved TMEM16 family, plays a central role in the initiation of blood coagulation and the fusion of trophoblasts. The protein mediates passive ion and lipid transport in response to an increase in intracellular Ca2+. However, the mechanism of how the protein facilitates both processes has remained elusive. Here we investigate the basis for TMEM16F activation. In a screen of residues lining the proposed site of conduction, we identify mutants with strongly activating phenotype. Structures of these mutants determined herein by cryo-electron microscopy show major rearrangements leading to the exposure of hydrophilic patches to the membrane, whose distortion facilitates lipid diffusion. The concomitant opening of a pore promotes ion conduction in the same protein conformation. Our work has revealed a mechanism that is distinct for this branch of the family and that will aid the development of a specific pharmacology for a promising drug target.

Abstract 1350: The role of phosphatidylserine scramblases in tumor progression

Abstract Introduction: Phosphatidylserine (PS), an anionic phospholipid, is asymmetrically distributed in the inner leaflet of the phospholipid bilayer in normal cells under homeostasis. However, PS is constitutively externalized in the tumor microenvironment (TME); on viable cells that undergo stress, such as hypoxic tumor cells, stressed tumor vascular endothelial cells, as well as on cells undergoing apoptosis. This high PS harbors a pro-tumorigenic micro-environment, as myeloid cells expressing PS receptors (Mertk, Tyro3 and Axl) engage with externalized PS and secrete immunosuppressive cytokines. PS is externalized on viable cells by a calcium activated scramblase TMEM16F and on apoptotic cells by a caspase activated scramblase called Xkr8. Here, to study the mechanisms by which PS is externalized in the TME, we hypothesize that TMEM16F and Xkr8 are the major scramblases responsible for creating an immune suppressive milieu in the tumor microenvironment. Methods: To study the mechanisms of PS dysregulation in the tumor microenvironment, TMEM16F and Xkr8 were knocked out in E0771 murine breast cancer cell line using CRISPR/Cas9. The knockouts were validated by western blotting, q-RT-PCR and surveyor assays. Results: TMEM16F KO E0771 cells express lower basal levels of PS compared to WT E0771 cells when detected by Annexin V staining by flow cytometry. TMEM16F KO cells are also less responsive to activation by calcium ionophore. TMEM16F KO, Xkr8 KO and WT E0771 cells have similar viability and migration patterns in response to Gas6, indicating that the cells themselves do not have altered oncogenic potential. TMEM16F KO E0771 cells injected into the inguinal mammary gland fat pad of C57BL6 syngeneic mice showed modest but significantly reduced tumor growth compared to the E0771 WT tumors, suggesting that TMEM16F mediated PS externalization could play a role in exacerbating tumor progression. Conclusion: TMEM16F driven PS externalization contributes to tumor growth. The effect of TMEM16F KO on immune regulation of the tumor milieu will be discussed. These data corroborate previous studies targeting PS in tumors therapeutically using anti-PS antibodies and suggests a role for scramblases in pleiotropic immune-suppression in the tumor. Citation Format: Varsha Gadiyar, Viralkumar Davra, David Calianese, Kevin Lahey, Raymond B. Birge. The role of phosphatidylserine scramblases in tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1350.

Abstract 1571: Cancer associated fibroblasts sustain critical dependency of pancreatic cancer cells on exogenous lipids

Abstract Pancreatic ductal adenocarcinoma (PDAC) cells derive their resistance to therapy and aggressive clinical course from the symbiotic signaling and metabolic interactions with cancer-associated fibroblasts (CAFs). CAFs have been shown to provide nutrients to "parasitic" PDAC cells including water soluble glucose and amino acids. In hypoxic tumor microenvironments, aggressive PDAC are functional auxotroph for lipids, and scavenge exogenous lipids to build cellular membranes. However, the mechanism by which PDAC cells obtain these water-insoluble essential membrane building materials remains poorly understood. To gain insights into the mechanism of lipid scavenging, we generated cholesterol-auxotrophic human and mouse PDAC PDAC cells since cholesterol is the major constituent of lipid cellular membranes. Here, we discovered that PDAC cells utilize CAFs as a main source of lipids in vivo through direct heterotypic cellular contacts. In this process, PDAC cells directly acquire the CAF plasma membrane (PM) via trogocytosis. In response to yet unidentified paracrine “feed me” signals activating CAF-dependent trogocytosis, CAFs upregulate phosphatidylserine (PtdSer) on the outer leaflet of the PM. Consequently, blockade of PtdSer on CAFs partially deterred trogocytic transfer of CAF membranes to PDAC cells in vitro. Furthermore, Ca2+-dependent phospholipid scramblase TMEM16F is a critical regulator of PtdSer externalization. TMEM16F is highly expressed in human PDAC CAFs compared to fibroblasts isolated from the matching adjacent non-malignant pancreatic tissues CAFs deficient in TMEM16F scramblase exhibited markedly reduced ability to support the growth of cholesterol-auxotrophic PDAC cells cultured in lipid depleted media. We propose trogocytosis as a new mode of lipid scavenging by PDAC cells from CAFs involving activation of Ca2+-dependent phospholipid scramblase TMEM16F in CAFs and increased expression of PtdSer as “eat me” signals on CAF PM. To inactivate trogocytosis, we nominate TMEM16F as a plausible PDAC therapy target using clinically available TMEM16 inhibitors with a potential for impact on treatment of PDAC patients in the near term. Citation Format: Charline Ogier, Alena Klochkova, Battuya Bayarmagnai, Linara Gabitova, Diana Restifo, Aizhan Surumbayeva, Janusz Franco-Barraza, Debora Vendramini Costa, Ralph Francescone, Jaye Gardiner, Emmanuelle Nicolas, Elizabeth A. Handorf, Kathy Q. Cai, Edna Cukierman, Igor Astsaturov. Cancer associated fibroblasts sustain critical dependency of pancreatic cancer cells on exogenous lipids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1571.

Supporting Cells of the Human Olfactory Epithelium Co-Express the Lipid Scramblase TMEM16F and ACE2 and May Cause Smell Loss by SARS-CoV-2 Spike-Induced Syncytia.

Quantitative and qualitative alterations in the sense of smell are well established symptoms of COVID-19. Some reports have shown that non-neuronal supporting (also named sustentacular) cells of the human olfactory epithelium co-express ACE2 and TMPRSS2 necessary for SARS-CoV-2 infection. In COVID-19, syncytia were found in many tissues but were not investigated in the olfactory epithelium. Some studies have shown that syncytia in some tissues are formed when SARS-CoV-2 Spike expressed at the surface of an infected cell binds to ACE2 on another cell, followed by activation of the scramblase TMEM16F (also named ANO6) which exposes phosphatidylserine to the external side of the membrane. Furthermore, niclosamide, an approved antihelminthic drug, inhibits Spike-induced syncytia by blocking TMEM16F activity. The aim of this study was to investigate if proteins involved in Spike-induced syncytia formation, i.e., ACE2 and TMEM16F, are expressed in the human olfactory epithelium. We analysed a publicly available single-cell RNA-seq dataset from human nasal epithelium and performed immunohistochemistry in human nasal tissues from biopsies. We found that ACE2 and TMEM16F are co-expressed both at RNA and protein levels in non-neuronal supporting cells of the human olfactory epithelium. Our results provide the first evidence that TMEM16F is expressed in human olfactory supporting cells and indicate that syncytia formation, that could be blocked by niclosamide, is one of the pathogenic mechanisms worth investigating in COVID-19 smell loss.

Paneth Cell Secretion invivo Requires Expression ofTmem16a and Tmem16f.

Paneth cells play a central role in intestinal innate immune response. These cells are localized at the base of small intestinal crypts of Lieberkuhn. The calcium-activated chloride channel TMEM16A and the phospholipid scramblase TMEM16F control intracellular Ca2+ signaling and exocytosis. We analyzed the role of TMEM16A and TMEM16F for Paneth cells secretion. Mice with intestinal epithelial knockout of Tmem16a (Tmem16a-/-) and Tmem16f (Tmem16f-/-) were generated. Tissue structures and Paneth cells were analyzed, and Paneth cell exocytosis was examined in small intestinal organoids invitro. Intracellular Ca2+ signals were measured and were compared between wild-type and Tmem16 knockout mice. Bacterial colonization and intestinal apoptosis were analyzed. Paneth cells in the crypts of Lieberkuhn from Tmem16a-/- and Tmem16f-/- mice demonstrated accumulation of lysozyme. Tmem16a and Tmem16f were localized in wild-type Paneth cells but were absent in cells from knockout animals. Paneth cell number and size were enhanced in the crypt base and mucus accumulated in intestinal goblet cells of knockout animals. Granule fusion and exocytosis on cholinergic and purinergic stimulation were examined online. Both were strongly compromised in the absence of Tmem16a or Tmem16f and were also blocked by inhibition of Tmem16a/f. Purinergic Ca2+ signaling was largely inhibited in Tmem16a knockout mice. Jejunal bacterial content was enhanced in knockout mice, whereas cellular apoptosis was inhibited. The present data demonstrate the role of Tmem16 for exocytosis in Paneth cells. Inhibition or activation of Tmem16a/f is likely to affect microbial content and immune functions present in the small intestine.

Abstract PR-015: Cancer-associated fibroblasts sustain critical dependency of pancreatic cancer cells on exogenous lipids

Abstract Pancreatic ductal adenocarcinoma (PDAC) cells derive their resistance to therapy and aggressive clinical course from the symbiotic signaling and metabolic interactions with cancer-associated fibroblasts (CAFs). CAFs have been shown to provide to metabolically “parasitic” PDAC cells with water-soluble metabolites such as glucose and amino acids, or bulk protein via micropinocytosis. To meet the elevated demands for cellular membrane lipids, cancer cells rely on uptake of the exogenous lipids. However, the mechanism by which PDAC cells obtain water-insoluble essential lipids, such as cholesterol, remains poorly understood. Here, we define CAFs as a main source of lipids for PDAC cells by direct “feeding” of the CAF plasma membrane (PM) to cancer cells via heterotypic cellular contacts, a phenomenon known as trogocytosis. To gain insights into the mechanisms of regulation of CAF trogocytosis, we engineered cholesterol-auxotrophic human and mouse PDAC cells. In the absence of exogenously provided cholesterol, these cancer cells undergo apoptosis, which is completely rescued in CAF co-cultures. Using CRISPRi-mediated depletion in CAFs of select genes involved in cholesterol trafficking, membrane fusion and membrane protrusions, we found that CAFs deficient in CD81, TMEM16F, or ARF6 exhibited markedly reduced ability to support the viability of cholesterol-auxotrophic PDAC cells in lipid-poor media. As a promising therapy target, TMEM16F is a Ca2+-regulated scramblase increasing phosphatidylserine (PtdSer) on the outer leaflet of the PM. TMEM16F protein is highly expressed in human PDAC CAFs compared to fibroblasts isolated from the adjacent non-malignant pancreatic tissues. The TMEM16F-null CAFs are unable to sustain of cholesterol-auxotrophic PDAC cells in lipid-poor co-cultures. Our overall model is that, to initiate trogocytosis, PDAC cells release paracrine “feed me” signals activating Ca2+-dependent phospholipid scramblase TMEM16F. As the result, increased outward expression of PtdSer on CAF PM is a hallmark “eat me” signal that is recognized by the trogocytic PDAC cells. We conclude that trogocytosis is the critical metabolic dependency in PDAC, and nominated TMEM16F as a plausible PDAC therapy target. Re-purposing of clinically available TMEM16 inhibitors will make a tangible impact on treatment of PDAC patients in the near term. Citation Format: Charline Ogier, Alena Klochkova, Linara Gabitova, Battuya Bayarmagnai, Diana Restifo, Aizhan Surumbayeva, Janusz Franco-Barraza, Ralph Francescone, Debora B. Barbosa Vendramini-Costa, Jaye Gardiner, Emmanuelle Nicolas, Elizabeth A. Handorf, Kathy Q. Cai, Edna Cukierman, Igor Astsaturov. Cancer-associated fibroblasts sustain critical dependency of pancreatic cancer cells on exogenous lipids [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-015.

SARS-CoV-2 Ion Channel ORF3a Enables TMEM16F-Dependent Phosphatidylserine Externalization to Augment Procoagulant Activity of the Tenase and Prothrombinase Complexes

Severe SARS-CoV-2 infection is complicated by dysregulation of the blood coagulation system and high rates of thrombosis, but virus-intrinsic mechanisms underlying this phenomenon are poorly understood. Increased intracellular calcium concentrations promote externalization of phosphatidylserine (PS), the membrane anionic phospholipid required for assembly and activation of the tenase and prothrombinase complexes to drive blood coagulation. TMEM16F is a ubiquitous phospholipid scramblase that mediates externalization of PS in a calcium-dependent manner. As SARS-CoV-2 ORF3a encodes a presumed cation channel with the ability to transport calcium, we hypothesized that ORF3a expression by infected host cells perturbs the cellular calcium rheostat, driving TMEM16F-dependent externalization of PS and enhancing procoagulant activity.Using a doxycycline-inducible system, synchronized expression of ORF3a in A549 pulmonary epithelial cells resulted in a time-dependent augmentation of tissue factor (TF) procoagulant activity exceeding 9-fold by 48 hours (p < 0.0001), with no change in TF cell-surface expression. This enhancement was dependent upon PS as determined by inhibition with the PS-binding protein lactadherin. Over 2-fold enhancement of prothrombinase activity (p < 0.0001) was also observed by 48 hours. ORF3a increased intracellular calcium levels by 18-fold at 48 hours (p < 0.0001), as determined by the intracellular calcium indicator fluo-4. After 16 hours of ORF3a expression, more than 60% of cells had externalized PS (p < 0.001) without increased cell death, as quantified by flow cytometry following annexin V binding. Immunofluorescence microscopy staining for ORF3a, annexin V, and nuclei confirmed ORF3a expression within internal and cell surface membranes and increased PS externalization. PS externalization was insensitive to the pan-caspase inhibitor z-VAD-FMK, and there was no evidence of apoptotic activation as determined by caspase-3 cleavage. By contrast, ORF3a expression did not augment coagulation in cells deficient in the calcium-dependent phospholipid scramblase TMEM16F. Similarly, ORF3a-enhanced TF procoagulant activity (p < 0.01) and prothrombinase activity (p<0.05) was completely abrogated using TMEM16 inhibitors, including the uricosuric agent benzbromarone that has been registered for human use in over 20 countries. Live SARS-CoV-2 infection of A549-ACE2 cells increased cell surface factor Xa generation at MOI 0.1 (p < 0.01) but not MOI 0.01 or following heat inactivation of the virus, and RNA sequencing confirmed ORF3a induction without increased F3 expression. RNA sequencing of human SARS-CoV-2 infected lung autopsy and control tissue (n= 53) confirmed these findings in vivo. Immunofluorescence staining for ORF3a and KRT8/18 and CD31 in SARS-CoV-2 infected human lung autopsy specimens demonstrated ORF3a expression in pulmonary epithelium and endothelial cells, highlighting the potential pathologic relevance of this mechanism.Here we demonstrate that expression of the SARS-CoV-2 accessory protein ORF3a increases the intracellular calcium concentration and TMEM16F-dependent PS scrambling to augment procoagulant activity of the tenase and prothrombinase complexes. Our studies of human cells and tissues infected with SARS-CoV-2 support the pathologic relevance of this mechanism. We highlight the therapeutic potential to target the ORF3a-TMEM16F axis as with benzbromarone to mitigate dysregulation of coagulation and thrombosis during severe SARS-CoV-2 infection. DisclosuresSchwartz: Miromatrix Inc: Membership on an entity's Board of Directors or advisory committees; Alnylam Inc.: Consultancy, Speakers Bureau. Schulman: CSL Behring: Consultancy, Research Funding.

P. aeruginosa Induced Lipid Peroxidation Causes Ferroptotic Cell Death in Airways.

Oxidative stress and infections by Pseudomonas aeruginosa (P. aeruginosa) are prominent in lungs of patients suffering from cystic fibrosis (CF). The present study examines effects of P. aeruginosa on lipid peroxidation in human and mouse lungs, and cell death induced by P. aeruginosa in human airway epithelial cells. The role of the Ca2+ activated Cl- channel TMEM16A, the phospholipid scramblase TMEM16F, and the CFTR Cl- channel for ferroptotic cell death is examined. Lipid peroxidation was detected in human CF lungs, which correlated with bacterial infection. In vivo inoculation with P. aeruginosa or Staphylococcus aureus (S. aureus) induced lipid peroxidation in lungs of mice lacking expression of CFTR, and in lungs of wild type animals. Incubation of CFBE human airway epithelial cells with P. aeruginosa induced an increase in reactive oxygen species (ROS), causing lipid peroxidation and cell death independent of expression of wt-CFTR or F508del-CFTR. Knockdown of TMEM16A attenuated P. aeruginosa induced cell death. Antioxidants such as coenzyme Q10 and idebenone as well as the inhibitor of ferroptosis, ferrostatin-1, inhibited P. aeruginosa-induced cell death. CFBE cells expressing wtCFTR, but not F508del-CFTR, activated a basal Cl- conductance upon exposure to P. aeruginosa, which was caused by an increase in intracellular basal Ca2+ concentrations and activation of Ca2+-dependent adenylate cyclase. The data suggest an intrinsic pro-inflammatory phenotype in CF epithelial cells, while ferroptosis is observed in both non-CF and CF epithelial cells upon infection with P. aeruginosa. CF cells fail to activate fluid secretion in response to infection with P. aeruginosa. The use of antioxidants and inhibitors of ferroptosis is proposed as a treatment of pneumonia caused by infection with P. aeruginosa.

Flipping the switch in T cells

Immunology The cytoplasmic domains of some CD3 subunits in the T cell receptor complex localize along the inner leaflet of the plasma membrane through electrostatic interactions, which inhibits their activation. Connolly et al. found that the scramblase TMEM16F, which redistributes phosphatidylserine from the inner to the outer leaflet of the plasma membrane, enabled the cytoplasmic domains of CD3 subunits in bystander T cell receptors to disengage from the plasma membrane, leading to enhanced T cell activation. These bystander TCRs were close to antigen-stimulated TCRs and amplified T cell signaling. Sci. Signal. 14 , eabb5146 (2021).

TMEM16F mediates bystander TCR-CD3 membrane dissociation at the immunological synapse and potentiates T cell activation

Electrostatic interactions regulate many aspects of T cell receptor (TCR) activity, including enabling the dynamic binding of the TCR-associated CD3ε and CD3ζ chains to anionic lipids in the plasma membrane to prevent spontaneous phosphorylation. Substantial changes in the electrostatic potential of the plasma membrane occur at the immunological synapse, the interface between a T cell and an antigen-presenting cell. Here, we investigated how the electrostatic interactions that promote dynamic membrane binding of the TCR-CD3 cytoplasmic domains are modulated during signaling and affect T cell activation. We found that Ca2+-dependent activation of the phosphatidylserine scramblase TMEM16F, which was previously implicated in T cell activation, reduced the electrostatic potential of the plasma membrane during immunological synapse formation by locally redistributing phosphatidylserine. This, in turn, increased the dissociation of bystander TCR-CD3 cytoplasmic domains from the plasma membrane and enhanced TCR-dependent signaling and consequently T cell activation. This study establishes the molecular basis for the role of TMEM16F in bystander TCR-induced signal amplification and identifies enhancement of TMEM16F function as a potential therapeutic strategy for promoting T cell activation.

Calcium ions trigger the exposure of phosphatidylserine on the surface of necrotic cells.

Intracellular Ca2+ level is under strict regulation through calcium channels and storage pools including the endoplasmic reticulum (ER). Mutations in certain ion channel subunits, which cause mis-regulated Ca2+ influx, induce the excitotoxic necrosis of neurons. In the nematode Caenorhabditis elegans, dominant mutations in the DEG/ENaC sodium channel subunit MEC-4 induce six mechanosensory (touch) neurons to undergo excitotoxic necrosis. These necrotic neurons are subsequently engulfed and digested by neighboring hypodermal cells. We previously reported that necrotic touch neurons actively expose phosphatidylserine (PS), an “eat-me” signal, to attract engulfing cells. However, the upstream signal that triggers PS externalization remained elusive. Here we report that a robust and transient increase of cytoplasmic Ca2+ level occurs prior to the exposure of PS on necrotic touch neurons. Inhibiting the release of Ca2+ from the ER, either pharmacologically or genetically, specifically impairs PS exposure on necrotic but not apoptotic cells. On the contrary, inhibiting the reuptake of cytoplasmic Ca2+ into the ER induces ectopic necrosis and PS exposure. Remarkably, PS exposure occurs independently of other necrosis events. Furthermore, unlike in mutants of DEG/ENaC channels, in dominant mutants of deg-3 and trp-4, which encode Ca2+ channels, PS exposure on necrotic neurons does not rely on the ER Ca2+ pool. Our findings indicate that high levels of cytoplasmic Ca2+ are necessary and sufficient for PS exposure. They further reveal two Ca2+-dependent, necrosis-specific pathways that promote PS exposure, a “two-step” pathway initiated by a modest influx of Ca2+ and further boosted by the release of Ca2+ from the ER, and another, ER-independent, pathway. Moreover, we found that ANOH-1, the worm homolog of mammalian phospholipid scramblase TMEM16F, is necessary for efficient PS exposure in thapsgargin-treated worms and trp-4 mutants, like in mec-4 mutants. We propose that both the ER-mediated and ER-independent Ca2+ pathways promote PS externalization through activating ANOH-1.

Evidence that polyphenols do not inhibit the phospholipid scramblase TMEM16F

TMEM16 Ca2+-activated phospholipid scramblases (CaPLSases) mediate rapid transmembrane phospholipid flip-flop and as such play essential roles in various physiological and pathological processes such as blood coagulation, skeletal development, viral infection, cell-cell fusion, and ataxia. Pharmacological tools specifically targeting TMEM16 CaPLSases are urgently needed to understand these novel membrane transporters and their contributions to health and disease. Tannic acid (TA) and epigallocatechin gallate (EGCG) were recently reported as promising TMEM16F CaPLSase inhibitors. However, our present study shows that TA and EGCG do not inhibit the phospholipid-scrambling or ion conduction activities of the dual-functional TMEM16F. Instead, we found that TA and EGCG mainly acted as fluorescence quenchers that rapidly suppress the fluorophores conjugated to annexin V, a phosphatidylserine-binding probe commonly used to report on TMEM16 CaPLSase activity. These data demonstrate the false positive effects of TA and EGCG on inhibiting TMEM16F phospholipid scrambling and discourage the use of these polyphenols as CaPLSase inhibitors. Appropriate controls as well as a combination of both fluorescence imaging and electrophysiological validation are necessary in future endeavors to develop TMEM16 CaPLSase inhibitors.

Lysophosphatidic acid-induced pro-thrombotic phosphatidylserine exposure and ionophore-induced microvesiculation is mediated by the scramblase TMEM16F in erythrocytes

Recent studies indicate that erythrocytes actively modulate blood clotting and thrombus formation. The lipid mediator lysophosphatidic acid (LPA) is produced by activated platelets, and triggers a signaling process in erythrocytes. This results in cellular calcium uptake and exposure of phosphatidylserine (PS) at the cell surface, thereby generating activated membrane binding sites for factors of the clotting cascade. Moreover, erythrocytes of patients with a bleeding disorder and mutations in the scramblase TMEM16F show impaired PS exposure and microvesiculation upon treatment with calcium ionophore. We report that TMEM16F inhibitors tannic acid (TA) and epigallocatechin-3-gallate (EGCG) inhibit LPA-induced PS exposure and calcium uptake at low micromolar concentrations; fluoxetine, an antidepressant and a known activator of TMEM16F, enhances these processes. These effectors likewise modulate erythrocyte PS exposure and microvesicle shedding induced by calcium ionophore treatment. Further, LPA-treated erythrocytes triggered thrombin generation in platelet-free plasma which was partially impaired in the presence of TA and EGCG. Thus, this study suggests that LPA activates the scramblase TMEM16F in erythrocytes, thereby possibly mediating a pro-thrombotic function in these cells. EGCG as well as fluoxetine, substances with potentially high plasma concentrations due to alimentation or medical treatment, should be considered as potential effectors of systemic hemostatic regulation.

Critical Role of Lipid Scramblase TMEM16F in Phosphatidylserine Exposure and Repair of Plasma Membrane after Pore Formation

Plasma membrane damage and cell death during processes such as necroptosis and apoptosis result from cues originating intracellularly. However, death caused by pore-forming agents, like bacterial toxins or complement, is due to direct external injury to the plasma membrane. To prevent death, the plasma membrane has an intrinsic repair ability. Here, we found that repair triggered by pore-forming agents involved TMEM16F, a calcium-activated lipid scramblase also mutated in Scott's syndrome. Upon pore formation and the subsequent influx of intracellular calcium, TMEM16F induced rapid "lipid scrambling" in the plasma membrane. This response was accompanied by membrane blebbing, extracellular vesicle release, preserved membrane integrity, and increased cell viability. TMEM16F-deficient mice exhibited compromised control of infection by Listeria monocytogenes associated with a greater sensitivity of neutrophils to the pore-forming Listeria toxin listeriolysin O (LLO). Thus, the lipid scramblase TMEM16F is critical for plasma membrane repair after injury by pore-forming agents.

Cryo-EM structures and functional characterization of the murine lipid scramblase TMEM16F.

The lipid scramblase TMEM16F initiates blood coagulation by catalyzing the exposure of phosphatidylserine in platelets. The protein is part of a family of membrane proteins, which encompasses calcium-activated channels for ions and lipids. Here, we reveal features of murine TMEM16F (mTMEM16F) that underlie its function as a lipid scramblase and an ion channel. The cryo-EM data of mTMEM16F in absence and presence of Ca2+ define the ligand-free closed conformation of the protein and the structure of a Ca2+-bound intermediate. Both conformations resemble their counterparts of the scrambling-incompetent anion channel mTMEM16A, yet with distinct differences in the region of ion and lipid permeation. In conjunction with functional data, we demonstrate the relationship between ion conduction and lipid scrambling. Although activated by a common mechanism, both functions appear to be mediated by alternate protein conformations that are at equilibrium in the ligand-bound state.

Activation of the phospholipid scramblase TMEM16F by nanosecond pulsed electric fields (nsPEF) facilitates its diverse cytophysiological effects

Nanosecond pulsed electric fields (nsPEF) are emerging as a novel modality for cell stimulation and tissue ablation. However, the downstream protein effectors responsible for nsPEF bioeffects remain to be established. Here we demonstrate that nsPEF activate TMEM16F (or Anoctamin 6), a protein functioning as a Ca2+-dependent phospholipid scramblase and Ca2+-activated chloride channel. Using confocal microscopy and patch clamp recordings, we investigated the relevance of TMEM16F activation for several bioeffects triggered by nsPEF, including phosphatidylserine (PS) externalization, nanopore-conducted currents, membrane blebbing, and cell death. In HEK 293 cells treated with a single 300-ns pulse of 25.5 kV/cm, Tmem16f expression knockdown and TMEM16F-specific inhibition decreased nsPEF-induced PS exposure by 49 and 42%, respectively. Moreover, the Tmem16f silencing significantly decreased Ca2+-dependent chloride channel currents activated in response to the nanoporation. Tmem16f expression also affected nsPEF-induced cell blebbing, with only 20% of the silenced cells developing blebs compared with 53% of the control cells. This inhibition of cellular blebbing correlated with a 25% decrease in cytosolic free Ca2+ transient at 30 s after nanoporation. Finally, in TMEM16F-overexpressing cells, a train of 120 pulses (300 ns, 20 Hz, 6 kV/cm) decreased cell survival to 34% compared with 51% in control cells (*, p < 0.01). Taken together, these results indicate that TMEM16F activation by nanoporation mediates and enhances the diverse cellular effects of nsPEF.