Apoptosis and Clearance of Apoptotic Cells.

The human body generates 10-100 billion cells every day, and the same number of cells die to maintain homeostasis in our body. Cells infected by bacteria or viruses also die. The cell death that occurs under physiological conditions mainly proceeds by apoptosis, which is a noninflammatory, or silent, process, while pathogen infection induces necroptosis or pyroptosis, which activates the immune system and causes inflammation. Dead cells generated by apoptosis are quickly engulfed by macrophages for degradation. Caspases are a large family of cysteine proteases that act in cascades. A cascade that leads to caspase 3 activation mediates apoptosis and is responsible for killing cells, recruiting macrophages, and presenting an "eat me" signal(s). When apoptotic cells are not efficiently engulfed by macrophages, they undergo secondary necrosis and release intracellular materials that represent a damage-associated molecular pattern, which may lead to a systemic lupus-like autoimmune disease.

Regulation of phospholipid distribution in the lipid bilayer by flippases and scramblases.

Cellular membranes function as permeability barriers that separate cells from the external environment or partition cells into distinct compartments. These membranes are lipid bilayers composed of glycerophospholipids, sphingolipids and cholesterol, in which proteins are embedded. Glycerophospholipids and sphingolipids freely move laterally, whereas transverse movement between lipid bilayers is limited. Phospholipids are asymmetrically distributed between membrane leaflets but change their location in biological processes, serving as signalling molecules or enzyme activators. Designated proteins - flippases and scramblases - mediate this lipid movement between the bilayers. Flippases mediate the confined localization of specific phospholipids (phosphatidylserine (PtdSer) and phosphatidylethanolamine) to the cytoplasmic leaflet. Scramblases randomly scramble phospholipids between leaflets and facilitate the exposure of PtdSer on the cell surface, which serves as an important signalling molecule and as an 'eat me' signal for phagocytes. Defects in flippases and scramblases cause various human diseases. We herein review the recent research on the structure of flippases and scramblases and their physiological roles. Although still poorly understood, we address the mechanisms by which they translocate phospholipids between lipid bilayers and how defects cause human diseases.

The role of the C-terminal tail region as a plug to regulate XKR8 lipid scramblase.

XK-related 8 (XKR8), in complex with the transmembrane glycoprotein basigin, functions as a phospholipid scramblase activated by the caspase-mediated cleavage or phosphorylation of its C-terminal tail. It carries a putative phospholipid translocation path of multiple hydrophobic and charged residues in the transmembrane region. It also has a crucial tryptophan at the exoplasmic end of the path that regulates its scrambling activity. We herein investigated the tertiary structure of the human XKR8-basigin complex embedded in lipid nanodiscs at an overall resolution of 3.66 Å. We found that the C-terminal tail engaged in intricate polar and van der Waals interactions with a groove at the cytoplasmic surface of XKR8. These interactions maintained the inactive state of XKR8. Point mutations to disrupt these interactions strongly enhanced the scrambling activity of XKR8, suggesting that the activation of XKR8 is mediated by releasing the C-terminal tail from the cytoplasmic groove. We speculate that the cytoplasmic tail region of XKR8 functions as a plug to prevent the scrambling of phospholipids.

Autoimmunity and the clearance of dead cells.

To maintain organismal homeostasis, phagocytes engulf dead cells, which are recognized as dead by virtue of a characteristic "eat me" signal exposed on their surface. The dead cells are then transferred to lysosomes, where their cellular components are degraded for reuse. Inefficient engulfment of dead cells activates the immune system, causing disease such as systemic lupus erythematosus, and if the DNA of the dead cells is not properly degraded, the innate immune response becomes activated, leading to severe anemia and chronic arthritis. Here, we discuss how the endogenous components of dead cells activate the immune system through both extracellular and intracellular pathways.

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.

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.

The XK plasma membrane scramblase and the VPS13A cytosolic lipid transporter for ATP-induced cell death.

Extracellular ATP released from necrotic cells in inflamed tissues activates the P2X7 receptor, stimulates the exposure of phosphatidylserine, and causes cell lysis. Recent findings indicated that XK, a paralogue of XKR8 lipid scramblase, forms a complex with VPS13A at the plasma membrane of T cells. Upon engagement by ATP, an unidentified signal(s) from the P2X7 receptor activates the XK-VPS13A complex to scramble phospholipids, followed by necrotic cell death. P2X7 is expressed highly in CD25+ CD4+ T cells but weakly in CD8+ T cells, suggesting a role of this system in the activation of the immune system to prevent infection. On the other hand, a loss-of-function mutation in XK or VPS13A causes neuroacanthocytosis, indicating the crucial involvement of XK-VPS13A-mediated phospholipid scrambling at plasma membranes in the maintenance of homeostasis in the nervous and red blood cell systems.

Cloning of human Type I interferon cDNAs.

In the late 1970s, crude interferon samples were found to exhibit anti-tumour activity. This discovery led to the interferon as a "magic drug" for cancer patients. Many groups, including those in Tokyo, Zürich, and San Francisco, attempted to identify human interferon cDNAs. Tadatsugu Taniguchi was the first to announce the cloning of human interferon-ß cDNA in the December 1979 issue of Proc. Jpn. Acad. Ser. B. This was followed by the cloning of human interferon-α by a Zürich group and interferon-γ by a group in Genentech in San Francisco. Recombinant interferon proteins were produced on a large scale, and interferon-α was widely used to treat C-type hepatitis patients. The biological functions of interferons were quickly elucidated with the purified recombinant interferons. The molecular mechanisms underlying virus-induced interferon gene expression were also examined using cloned chromosomal genes. The background that led to interferon gene cloning and its impact on cytokine gene hunting is described herein.

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.

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.

Phosphorylation-mediated activation of mouse Xkr8 scramblase for phosphatidylserine exposure.

The exposure of phosphatidylserine (PtdSer) to the cell surface is regulated by the down-regulation of flippases and the activation of scramblases. Xkr8 has been identified as a scramblase that is activated during apoptosis, but its exogenous expression in the mouse Ba/F3 pro B cell line induces constitutive PtdSer exposure. Here we found that this Xkr8-mediated PtdSer exposure occurred at 4 °C, but not at 20 °C, although its scramblase activity was observed at 20 °C. The Xkr8-mediated PtdSer exposure was inhibited by a kinase inhibitor and enhanced by phosphatase inhibitors. Phosphorylated Xkr8 was detected by Phos-tag PAGE, and a mass spectrometric and mutational analysis identified three phosphorylation sites. Their phosphomimic mutation rendered Xkr8 resistant to the kinase inhibitor for PtdSer exposure at 4 °C, but unlike phosphatase inhibitors, it did not induce constitutive PtdSer exposure at 20 °C. On the other hand, when the flippase genes were deleted, the Xkr8 induced constitutive PtdSer exposure at high temperature, indicating that the flippase activity normally counteracted Xkr8's ability to expose PtdSer. These results indicate that PtdSer exposure can be increased by the phosphorylation-mediated activation of Xkr8 scramblase and flippase down-regulation.

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.

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.

Lupus-like autoimmune disease caused by a lack of Xkr8, a caspase-dependent phospholipid scramblase.

Apoptotic cells expose phosphatidylserine (PtdSer) on their cell surface and are recognized by macrophages for clearance. Xkr8 is a scramblase that exposes PtdSer in a caspase-dependent manner. Here, we found that among the three Xkr members with caspase-dependent scramblase activity, mouse hematopoietic cells express only Xkr8. The PtdSer exposure of apoptotic thymocytes, splenocytes, and neutrophils was strongly reduced when Xkr8 was absent. While wild-type apoptotic lymphocytes and neutrophils were efficiently engulfed in vitro by phagocytes expressing Tim4 and MerTK, Xkr8-deficient apoptotic cells were hardly engulfed by these phagocytes. Accordingly, the number of apoptotic thymocytes in the thymus and neutrophils in the peritoneal cavity of the zymosan-treated mice was significantly increased in Xkr8-deficient mice. The percentage of CD62Llo senescent neutrophils was increased in the spleen of Xkr8-null mice, especially after the treatment with granulocyte colony-stimulating factor. Xkr8-null mice on an MRL background showed high levels of autoantibodies, splenomegaly with high levels of effector CD4 T cells, and glomerulonephritis development with immune-complex deposition at glomeruli. These results indicate that the Xkr8-mediated PtdSer exposure in apoptotic lymphocytes and aged neutrophils is essential for their clearance, and its defect activates the immune system, leading to lupus-like autoimmune disease.

Single-molecule analysis of phospholipid scrambling by TMEM16F.

Transmembrane protein 16F (TMEM16F) is a Ca2+-dependent phospholipid scramblase that translocates phospholipids bidirectionally between the leaflets of the plasma membrane. Phospholipid scrambling of TMEM16F causes exposure of phosphatidylserine in activated platelets to induce blood clotting and in differentiated osteoblasts to promote bone mineralization. Despite the importance of TMEM16F-mediated phospholipid scrambling in various biological reactions, the fundamental features of the scrambling reaction remain elusive due to technical difficulties in the preparation of a platform for assaying scramblase activity in vitro. Here, we established a method to express and purify mouse TMEM16F as a dimeric molecule by constructing a stable cell line and developed a microarray containing membrane bilayers with asymmetrically distributed phospholipids as a platform for single-molecule scramblase assays. The purified TMEM16F was integrated into the microarray, and monitoring of phospholipid translocation showed that a single TMEM16F molecule transported phospholipids nonspecifically between the membrane bilayers in a Ca2+-dependent manner. Thermodynamic analysis of the reaction indicated that TMEM16F transported 4.5 × 104 lipids per second at 25 °C, with an activation free energy of 47 kJ/mol. These biophysical features were similar to those observed with channels, which transport substrates by facilitating diffusion, and supported the stepping-stone model for the TMEM16F phospholipid scramblase.

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.

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.

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.