Two types of type IV P-type ATPases independently re-establish the asymmetrical distribution of phosphatidylserine in plasma membranes.

Phospholipids are asymmetrically distributed between the lipid bilayer of plasma membranes in which phosphatidylserine (PtdSer) is confined to the inner leaflet. ATP11A and ATP11C, type IV P-Type ATPases in plasma membranes, flip PtdSer from the outer to the inner leaflet, but involvement of other P4-ATPases is unclear. We herein demonstrated that once PtdSer was exposed on the cell surface of ATP11A-/-ATP11C-/- mouse T cell line (W3), its internalization to the inner leaflet of plasma membranes was negligible at 15 °C. However, ATP11A-/-ATP11C-/- cells internalized the exposed PtdSer at 37 °C, a temperature at which trafficking of intracellular membranes was active. In addition to ATP11A and 11C, W3 cells expressed ATP8A1, 8B2, 8B4, 9A, 9B, and 11B, with ATP8A1 and ATP11B being present at recycling endosomes. Cells deficient in four P4-ATPases (ATP8A1, 11A, 11B, and 11C) (QKO) did not constitutively expose PtdSer on the cell surface but lost the ability to re-establish PtdSer asymmetry within 1 hour, even at 37 °C. The expression of ATP11A or ATP11C conferred QKO cells with the ability to rapidly re-establish PtdSer asymmetry at 15 °C and 37 °C, while cells expressing ATP8A1 or ATP11B required a temperature of 37 °C to achieve this function, and a dynamin inhibitor blocked this process. These results revealed that mammalian cells are equipped with two independent mechanisms to re-establish its asymmetry: the first is a rapid process involving plasma membrane flippases, ATP11A and ATP11C, while the other is mediated by ATP8A1 and ATP11B, which require an endocytosis process.

Inefficient development of syncytiotrophoblasts in the Atp11a-deficient mouse placenta.

The P4-ATPases ATP11A and ATP11C function as flippases at the plasma membrane to translocate phosphatidylserine from the outer to the inner leaflet. We herein demonstrated that Atp11a-deficient mouse embryos died at approximately E14.5 with thin-walled heart ventricles. However, the cardiomyocyte- or epiblast-specific Atp11a deletion did not affect mouse development or mortality. ATP11C may have compensated for the function of ATP11A in most of the cell types in the embryo. On the other hand, Atp11a, but not Atp11c, was expressed in the mouse placenta, and the Atp11a-null mutation caused poor development of the labyrinthine layer with an increased number of TUNEL-positive foci. Immunohistochemistry and electron microscopy revealed a disorganized labyrinthine layer with unfused trophoblasts in the Atp11a-null placenta. Human placenta-derived choriocarcinoma BeWo cells expressed the ATP11A and ATP11C genes. A lack of ATP11A and ATP11C eliminated the ability of BeWo cells to flip phosphatidylserine and fuse when treated with forskolin. These results indicate that flippases at the plasma membrane play an important role in the formation of syncytiotrophoblasts in placental development.

Requirement of Xk and Vps13a for the P2X7-mediated phospholipid scrambling and cell lysis in mouse T cells.

A high extracellular adenosine triphosphate (ATP) concentration rapidly and reversibly exposes phosphatidylserine (PtdSer) in T cells by binding to the P2X7 receptor, which ultimately leads to necrosis. Using mouse T cell transformants expressing P2X7, we herein performed CRISPR/Cas9 screening for the molecules responsible for P2X7-mediated PtdSer exposure. In addition to Eros, which is required for the localization of P2X7 to the plasma membrane, this screening identified Xk and Vps13a as essential components for this process. Xk is present at the plasma membrane, and its paralogue, Xkr8, functions as a phospholipid scramblase. Vps13a is a lipid transporter in the cytoplasm. Blue-native polyacrylamide gel electrophoresis indicated that Xk and Vps13a interacted at the membrane. A null mutation in Xk or Vps13a blocked P2X7-mediated PtdSer exposure, the internalization of phosphatidylcholine, and cytolysis. Xk and Vps13a formed a complex in mouse splenic T cells, and Xk was crucial for ATP-induced PtdSer exposure and cytolysis in CD25+CD4+ T cells. XK and VPS13A are responsible for McLeod syndrome and chorea-acanthocytosis, both characterized by a progressive movement disorder and cognitive and behavior changes. Our results suggest that the phospholipid scrambling activity mediated by XK and VPS13A is essential for maintaining homeostasis in the immune and nerve systems.

Protocol to analyze lipid asymmetry in the plasma membrane.

The plasma membrane containing cholesterol exhibits phospholipid asymmetry, with phosphatidylcholine and sphingomyelin enriched in its outer leaflet and phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtn) on the cytoplasmic side. We herein describe steps for bacterial expression of recombinant proteins that bind to membrane lipids, followed by affinity purification. Using fluorescence-labeled phospholipid analogs, we further detail the assay to detect flippase activity, which maintains the single-sided distribution of PtdSer and PtdEtn, in mammalian cells. For complete details on the use and execution of this protocol, please refer to Segawa et al. (2021).1.

A sublethal ATP11A mutation associated with neurological deterioration causes aberrant phosphatidylcholine flipping in plasma membranes.

ATP11A translocates phosphatidylserine (PtdSer), but not phosphatidylcholine (PtdCho), from the outer to the inner leaflet of plasma membranes, thereby maintaining the asymmetric distribution of PtdSer. Here, we detected a de novo heterozygous point mutation of ATP11A in a patient with developmental delays and neurological deterioration. Mice carrying the corresponding mutation died perinatally of neurological disorders. This mutation caused an amino acid substitution (Q84E) in the first transmembrane segment of ATP11A, and mutant ATP11A flipped PtdCho. Molecular dynamics simulations revealed that the mutation allowed PtdCho binding at the substrate entry site. Aberrant PtdCho flipping markedly decreased the concentration of PtdCho in the outer leaflet of plasma membranes, whereas sphingomyelin (SM) concentrations in the outer leaflet increased. This change in the distribution of phospholipids altered cell characteristics, including cell growth, cholesterol homeostasis, and sensitivity to sphingomyelinase. Matrix-assisted laser desorption ionization-imaging mass spectrometry (MALDI-IMS) showed a marked increase of SM levels in the brains of Q84E-knockin mouse embryos. These results provide insights into the physiological importance of the substrate specificity of plasma membrane flippases for the proper distribution of PtdCho and SM.

Sensing and clearance of apoptotic cells.

Macrophages specifically engulf apoptotic cells but not healthy cells. Phosphatidylserine (PtdSer) is localized at the inner leaflet of plasma membranes as a result of the action of flippases (ATP11A and 11C). When cells undergo apoptosis, caspase 3 cleaves and inactivates the flippases, while simultaneously cleaving XKR8 to activate its phospholipid scramblase activity. PtdSer is thus swiftly and irreversibly exposed to the cell surface as an 'eat me' signal. Tissue resident macrophages recognize the apoptotic cells using a PtdSer-receptor TIM4 and engulf them with TAM tyrosine-kinase receptors, and integrins. The PtdSer 'eat me' signal appears to override 'don't eat me' signals in most cases.

Transport Cycle of Plasma Membrane Flippase ATP11C by Cryo-EM.

ATP11C, a plasma membrane phospholipid flippase, maintains the asymmetric distribution of phosphatidylserine accumulated in the inner leaflet. Caspase-dependent inactivation of ATP11C is essential for an apoptotic "eat me" signal, phosphatidylserine exposure, which prompts phagocytes to engulf cells. We show six cryo-EM structures of ATP11C at 3.0-4.0 Å resolution in five different states of the transport cycle. A structural comparison reveals phosphorylation-driven domain movements coupled with phospholipid binding. Three structures of phospholipid-bound states visualize phospholipid translocation accompanied by the rearrangement of transmembrane helices and an unwound portion at the occlusion site, and thus they detail the basis for head group recognition and the locality of the protein-bound acyl chains in transmembrane grooves. Invariant Lys880 and the surrounding hydrogen-bond network serve as a pivot point for helix bending and precise P domain inclination, which is crucial for dephosphorylation. The structures detail key features of phospholipid translocation by ATP11C, and a common basic mechanism for flippases is emerging.

Crystal structure of a human plasma membrane phospholipid flippase.

ATP11C, a member of the P4-ATPase flippase, translocates phosphatidylserine from the outer to the inner plasma membrane leaflet, and maintains the asymmetric distribution of phosphatidylserine in the living cell. We present the crystal structures of a human plasma membrane flippase, ATP11C-CDC50A complex, in a stabilized E2P conformation. The structure revealed a deep longitudinal crevice along transmembrane helices continuing from the cell surface to the phospholipid occlusion site in the middle of the membrane. We observed that the extension of the crevice on the exoplasmic side is open, and the complex is therefore in an outward-open E2P state, similar to a recently reported cryo-EM structure of yeast flippase Drs2p-Cdc50p complex. We noted extra densities, most likely bound phosphatidylserines, in the crevice and in its extension to the extracellular side. One was close to the phosphatidylserine occlusion site as previously reported for the human ATP8A1-CDC50A complex, and the other in a cavity at the surface of the exoplasmic leaflet of the bilayer. Substitutions in either of the binding sites or along the path between them impaired specific ATPase and transport activities. These results provide evidence that the observed crevice is the conduit along that phosphatidylserine traverses from the outer leaflet to its occlusion site in the membrane and suggest that the exoplasmic cavity is important for phospholipid recognition. They also yield insights into how phosphatidylserine is incorporated from the outer leaflet of the plasma membrane into the transmembrane.

Flippase and scramblase for phosphatidylserine exposure.

In various biological processes, phosphatidylserine (PtdSer) that is normally sequestered to the inner leaflet of the plasma membrane (PM) is exposed to the cell surface. When platelets are activated, they expose PtdSer to activate the blood-clotting factors. Cells undergoing apoptosis and senescent neutrophils expose PtdSer that is recognized as an 'eat me' signal by phagocytes for clearance. The PtdSer-exposure and its internalization are mediated by phospholipid scramblases and flippases, respectively. Both have recently been molecularly identified, and their functional mechanism and physiological roles are being elucidated.

Functional Expression of the P2X7 ATP Receptor Requires Eros.

In response to extracellular ATP, the purinergic receptor P2X7 mediates various biological processes, including phosphatidylserine (PtdSer) exposure, phospholipid scrambling, dye uptake, ion transport, and IL-1ß production. A genome-wide CRISPR screen for molecules responsible for ATP-induced PtdSer exposure identified a transmembrane protein, essential for reactive oxygen species (Eros), as a necessary component for P2X7 expression. An Eros-null mouse T cell line lost the ability to expose PtdSer, to scramble phospholipids, and to internalize a dye YO-PRO-1 and Ca2+ ions. Eros-null mutation abolished the ability of an LPS-primed human THP-1 macrophage cell line and mouse bone marrow-derived macrophages to secrete IL-1ß in response to ATP. Eros is localized to the endoplasmic reticulum and functions as a chaperone for NADPH oxidase components. Similarly, Eros at the endoplasmic reticulum transiently associated with P2X7 to promote the formation of a stable homotrimeric complex of P2X7. These results indicated that Eros acts as a chaperone not only for NADPH oxidase, but also for P2X7, and contributes to the innate immune reaction.

Predominant localization of phosphatidylserine at the cytoplasmic leaflet of the ER, and its TMEM16K-dependent redistribution.

TMEM16K, a membrane protein carrying 10 transmembrane regions, has phospholipid scramblase activity. TMEM16K is localized to intracellular membranes, but whether it actually scrambles phospholipids inside cells has not been demonstrated, due to technical difficulties in studying intracellular lipid distributions. Here, we developed a freeze-fracture electron microscopy method that enabled us to determine the phosphatidylserine (PtdSer) distribution in the individual leaflets of cellular membranes. Using this method, we found that the endoplasmic reticulum (ER) of mammalian cells harbored abundant PtdSer in its cytoplasmic leaflet and much less in the luminal leaflet, whereas the outer and inner nuclear membranes (NMs) had equivalent amounts of PtdSer in both leaflets. The ER and NMs of budding yeast also harbored PtdSer in their cytoplasmic leaflet, but asymmetrical distribution in the ER was not observed. Treating mouse embryonic fibroblasts with the Ca2+ ionophore A23187 compromised the cytoplasmic leaflet-dominant PtdSer asymmetry in the ER and increased PtdSer in the NMs, especially in the nucleoplasmic leaflet of the inner NM. This Ca2+-induced PtdSer redistribution was not observed in TMEM16K-null fibroblasts, but was recovered in these cells by reexpressing TMEM16K. These results indicate that, similar to the plasma membrane, PtdSer in the ER of mammalian cells is predominantly localized to the cytoplasmic leaflet, and that TMEM16K directly or indirectly mediates Ca2+-dependent phospholipid scrambling in the ER.

MERTK tyrosine kinase receptor together with TIM4 phosphatidylserine receptor mediates distinct signal transduction pathways for efferocytosis and cell proliferation.

Apoptotic cells expose phosphatidylserine (PtdSer) on their surface, leading to efferocytosis, i.e. their engulfment by resident macrophages that express the PtdSer receptor T cell immunoglobulin mucin receptor 4 (TIM4) and TAM family receptor tyrosine kinase receptors (MERTK, AXL, and TYRO3). TAM family receptors stimulate cell proliferation, and the many aspects of the growth signaling pathway downstream of TAM family receptors have been elucidated previously. However, the signaling cascade for TAM receptor-mediated efferocytosis has been elusive. Here we observed that efferocytosis by mouse-resident peritoneal macrophages was blocked by inhibitors against the MERTK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK), AKT Ser/Thr kinase (AKT), focal adhesion kinase (FAK), or STAT6 pathway. Accordingly, apoptotic cells stimulated the phosphorylation of MERTK, ERK, AKT, FAK, and STAT6, but not of IκB or STAT5. A reconstituted efferocytosis system using MERTK- and TIM4-expressing NIH3T3-derived cells revealed that the juxtamembrane and C-terminal regions of MERTK have redundant roles in efferocytosis. The transformation of murine IL-3-dependent Ba/F3 cells (a pro-B cell line) with MERTK and TIM4 enabled them to proliferate in response to apoptotic cells in a PtdSer-dependent manner. This apoptotic cell-induced MERTK-mediated proliferation required both MERTK's juxtamembrane and C-terminal regions and was blocked by inhibitors of not only ERK, AKT, FAK, and STAT6 but also of NF-κB and STAT5 signaling. These results suggest that apoptotic cells stimulate distinct sets of signal transduction pathways via MERTK to induce either efferocytosis or proliferation.

Phospholipid flippases enable precursor B cells to flee engulfment by macrophages.

ATP11A and ATP11C, members of the P4-ATPases, are flippases that translocate phosphatidylserine (PtdSer) from the outer to inner leaflet of the plasma membrane. Using the W3 T lymphoma cell line, we found that Ca2+ ionophore-induced phospholipid scrambling caused prolonged PtdSer exposure in cells lacking both the ATP11A and ATP11C genes. ATP11C-null (ATP11C-/y ) mutant mice exhibit severe B-cell deficiency. In wild-type mice, ATP11C was expressed at all B-cell developmental stages, while ATP11A was not expressed after pro-B-cell stages, indicating that ATP11C-/y early B-cell progenitors lacked plasma membrane flippases. The receptor kinases MerTK and Axl are known to be essential for the PtdSer-mediated engulfment of apoptotic cells by macrophages. MerTK-/- and Axl-/- double deficiency fully rescued the lymphopenia in the ATP11C-/y bone marrow. Many of the rescued ATP11C-/y pre-B and immature B cells exposed PtdSer, and these cells were engulfed alive by wild-type peritoneal macrophages, in a PtdSer-dependent manner. These results indicate that ATP11A and ATP11C in precursor B cells are essential for rapidly internalizing PtdSer from the cell surface to prevent the cells' engulfment by macrophages.

The CDC50A extracellular domain is required for forming a functional complex with and chaperoning phospholipid flippases to the plasma membrane.

Flippases are enzymes that translocate phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtn) from the outer to the inner leaflet in the lipid bilayer of the plasma membrane, leading to the asymmetric distribution of aminophospholipids in the membrane. One mammalian phospholipid flippase at the plasma membrane is ATP11C, a type IV P-type ATPase (P4-ATPase) that forms a heterocomplex with the transmembrane protein CDC50A. However, the structural features in CDC50A that support the function of ATP11C and other P4-ATPases have not been characterized. Here, using error-prone PCR-mediated mutagenesis of human CDC50A cDNA followed by functional screening and deep sequencing, we identified 14 amino acid residues that affect ATP11C's flippase activity. These residues were all located in CDC50A's extracellular domain and were evolutionarily well-conserved. Most of the mutations decreased CDC50A's ability to chaperone ATP11C and other P4-ATPases to their destinations. The CDC50A mutants failed to form a stable complex with ATP11C and could not induce ATP11C's PtdSer-dependent ATPase activity. Notably, one mutant variant could form a stable complex with ATP11C and transfer ATP11C to the plasma membrane, yet the ATP11C complexed with this CDC50A variant had very weak or little PtdSer- or PtdEtn-dependent ATPase activity. These results indicated that the extracellular domain of CDC50A has important roles both in CDC50A's ability to chaperone ATP11C to the plasma membrane and in inducing ATP11C's ATP hydrolysis-coupled flippase activity.

Mouse macrophages show different requirements for phosphatidylserine receptor Tim4 in efferocytosis.

Protein S (ProS) and growth arrest-specific 6 (Gas6) bind to phosphatidylserine (PtdSer) and induce efferocytosis upon binding TAM-family receptors (Tyro3, Axl, and Mer). Here, we produced mouse ProS, Gas6, and TAM-receptor extracellular region fused to IgG fragment crystallizable region in HEK293T cells. ProS and Gas6 bound Ca2+ dependently to PtdSer (Kd 20-40 nM), Mer, and Tyro3 (Kd 15-50 nM). Gas6 bound Axl strongly (Kd < 1.0 nM), but ProS did not bind Axl. Using NIH 3T3-based cell lines expressing a single TAM receptor, we showed that TAM-mediated efferocytosis was determined by the receptor-binding ability of ProS and Gas6. Tim4 is a membrane protein that strongly binds PtdSer. Tim4 alone did not support efferocytosis, but enhanced TAM-dependent efferocytosis. Resident peritoneal macrophages, Kupffer cells, and CD169+ skin macrophages required Tim4 for TAM-stimulated efferocytosis, whereas efferocytosis by thioglycollate-elicited peritoneal macrophages or primary cultured microglia was TAM dependent, but not Tim4 dependent. These results indicate that TAM and Tim4 collaborate for efficient efferocytosis in certain macrophage populations.

Characterization of the scrambling domain of the TMEM16 family.

The TMEM16 protein family has 10 members, each of which carries 10 transmembrane segments. TMEM16A and 16B are Ca2+-activated Cl- channels. Several other members, including TMEM16F, promote phospholipid scrambling between the inner and outer leaflets of a cell membrane in response to intracellular Ca2+ However, the mechanism by which TMEM16 proteins translocate phospholipids in plasma membranes remains elusive. Here we show that Ca2+-activated, TMEM16F-supported phospholipid scrambling proceeds at 4 °C. Similar to TMEM16F and 16E, seven TMEM16 family members were found to carry a domain (SCRD; scrambling domain) spanning the fourth and fifth transmembrane segments that conferred scrambling ability to TMEM16A. By introducing point mutations into TMEM16F, we found that a lysine in the fourth transmembrane segment of the SCRD as well as an arginine in the third and a glutamic acid in the sixth transmembrane segment were important for exposing phosphatidylserine from the inner to the outer leaflet. However, their role in internalizing phospholipids was limited. Our results suggest that TMEM16 provides a cleft containing hydrophilic "stepping stones" for the outward translocation of phospholipids.

Human Type IV P-type ATPases That Work as Plasma Membrane Phospholipid Flippases and Their Regulation by Caspase and Calcium.

In plasma membranes, flippases translocate aminophospholipids such as phosphatidylserine and phosphatidylethanolamine from the extracellular to the cytoplasmic leaflet. Mammalian ATP11C, a type IV P-type ATPase, acts as a flippase at the plasma membrane. Here, by expressing 12 human type IV P-type ATPases in ATP11C-deficient cells, we determined that ATP8A2 and ATP11A can also act as plasma membrane flippases. As with ATP11C, ATP8A2 and ATP11A localized to the plasma membrane in a CDC50A-dependent manner. ATP11A was cleaved by caspases during apoptosis, and a caspase-resistant ATP11A blocked apoptotic PtdSer exposure. In contrast, ATP8A2 was not cleaved by caspase, and cells expressing ATP8A2 did not expose PtdSer during apoptosis. Similarly, high Ca(2+) concentrations inhibited the ATP11A and ATP11C PtdSer flippase activity, but ATP8A2 flippase activity was relatively resistant to Ca(2+). ATP11A and ATP11C were ubiquitously expressed in human and mouse adult tissues. In contrast, ATP8A2 was expressed in specific tissues, such as the brain and testis. Thus, ATP8A2 may play a specific role in translocating PtdSer in these tissues.

An Apoptotic 'Eat Me' Signal: Phosphatidylserine Exposure.

Apoptosis and the clearance of apoptotic cells are essential processes in animal development and homeostasis. For apoptotic cells to be cleared, they must display an 'eat me' signal, most likely phosphatidylserine (PtdSer) exposure, which prompts phagocytes to engulf the cells. PtdSer, which is recognized by several different systems, is normally confined to the cytoplasmic leaflet of the plasma membrane by a 'flippase'; apoptosis activates a 'scramblase' that quickly exposes PtdSer on the cell surface. The molecules that flip and scramble phospholipids at the plasma membrane have recently been identified. Here we discuss recent findings regarding the molecular mechanisms of apoptotic PtdSer exposure and the clearance of apoptotic cells.

Clearance of Apoptotic Cells and Pyrenocytes.

Apoptotic cells are engulfed and digested by macrophages to maintain homeostasis in animals. If dead cells are not engulfed swiftly, they undergo secondary necrosis and release intracellular components that activate the immune system. Apoptotic cells are efficiently cleared due to phosphatidylserine (PtdSer) exposed on the cell surface that acts as an "eat me" signal. PtdSer is exposed through the activation of phospholipid scramblase and the inactivation of phospholipid flippase, which are both caspase-mediated events. Macrophages express a variety of molecules to recognize PtdSer, and use a sophisticated mechanism to engulf apoptotic cells. In red blood cells, the nucleus is lost when it is extruded as a pyrenocyte during definitive erythropoiesis. These pyrenocytes (nuclei surrounded by plasma membrane) also expose PtdSer on their surface and are efficiently engulfed by macrophages in a PtdSer-dependent manner. Macrophages transfer the engulfed apoptotic cell or pyrenocyte into lysosomes, where the components of the dead cell or pyrenocyte are degraded. If lysosomes cannot digest the DNA from apoptotic cells or pyrenocytes, the undigested DNA accumulates in the lysosome and activates macrophages to produce type I interferon (IFN) via a STING-dependent pathway; in embryos, this causes severe anemia. Here, we discuss how macrophages clear apoptotic cells and pyrenocytes.