RESUMEN
Systemic Lupus Erythematosus (SLE) is an autoimmune disease, the pathophysiology and genetic basis of which are incompletely understood. Using a forward genetic screen in multiplex families with systemic lupus erythematosus (SLE) we identified an association between SLE and compound heterozygous deleterious variants in the non-receptor tyrosine kinases (NRTKs) ACK1 and BRK. Experimental blockade of ACK1 or BRK increased circulating autoantibodies in vivo in mice and exacerbated glomerular IgG deposits in an SLE mouse model. Mechanistically, non-receptor tyrosine kinases (NRTKs) regulate activation, migration, and proliferation of immune cells. We found that the patients' ACK1 and BRK variants impair efferocytosis, the MERTK-mediated anti-inflammatory response to apoptotic cells, in human induced Pluripotent Stem Cell (hiPSC)-derived macrophages, which may contribute to SLE pathogenesis. Overall, our data suggest that ACK1 and BRK deficiencies are associated with human SLE and impair efferocytosis in macrophages.
RESUMEN
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.
Asunto(s)
Proteínas Reguladoras de la Apoptosis , Basigina , Proteínas de la Membrana , Proteínas de Transferencia de Fosfolípidos , Humanos , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/genética , Basigina/química , Membrana Celular/metabolismo , Liposomas/química , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Nanopartículas/química , Proteínas de Transferencia de Fosfolípidos/química , Proteínas de Transferencia de Fosfolípidos/genética , Fosfolípidos , Conformación Proteica en Hélice alfa , Imagen Individual de MoléculaRESUMEN
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.
Asunto(s)
Interferón Tipo I , Humanos , Interferón Tipo I/genética , ADN Complementario/genética , Clonación Molecular , Proteínas Recombinantes/genética , Interferón-alfaRESUMEN
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.
Asunto(s)
Membrana Dobles de Lípidos , Fosfolípidos , Humanos , Membrana Dobles de Lípidos/metabolismo , Fosfolípidos/metabolismo , Membrana Celular/metabolismo , Glicerofosfolípidos/metabolismo , Fosfatidilserinas/metabolismoRESUMEN
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.
Asunto(s)
Transportador 1 de Casete de Unión a ATP , ATPasas Tipo P , Fosfatidilserinas , Proteínas de Transferencia de Fosfolípidos , Animales , Ratones , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Membrana Celular/metabolismo , ATPasas Tipo P/genética , ATPasas Tipo P/metabolismo , Fosfatidilserinas/metabolismo , Proteínas de Transferencia de Fosfolípidos/genética , Proteínas de Transferencia de Fosfolípidos/metabolismo , Fosfolípidos/metabolismo , Transportador 1 de Casete de Unión a ATP/genética , Transportador 1 de Casete de Unión a ATP/metabolismo , Técnicas de Inactivación de Genes , Linfocitos TRESUMEN
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.
Asunto(s)
Fosfatidilserinas , Receptores Purinérgicos P2X7 , Adenosina Trifosfato/metabolismo , Linfocitos T CD8-positivos/metabolismo , Muerte Celular , Membrana Celular/metabolismo , Fosfatidilserinas/metabolismo , Fosfolípidos/metabolismo , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismoRESUMEN
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.
Asunto(s)
Placenta , Trofoblastos , Transportador 1 de Casete de Unión a ATP , Adenosina Trifosfatasas/metabolismo , Animales , Membrana Celular/metabolismo , Femenino , Ratones , Fosfatidilserinas/metabolismo , Placenta/metabolismo , Embarazo , Trofoblastos/metabolismoRESUMEN
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.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Fosfolípidos/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Linfocitos T/fisiología , Proteínas de Transporte Vesicular/metabolismo , Adenosina Trifosfato , Sistemas de Transporte de Aminoácidos Neutros/genética , Animales , Sistemas CRISPR-Cas , Muerte Celular , Línea Celular , Eliminación de Gen , Regulación de la Expresión Génica/efectos de los fármacos , Estudio de Asociación del Genoma Completo , Células HEK293 , Humanos , Ratones , Ratones Transgénicos , Mutación , Fosfatidilserinas/farmacología , Receptores Purinérgicos P2X7/genética , Proteínas de Transporte Vesicular/genéticaRESUMEN
Xkr8-Basigin is a plasma membrane phospholipid scramblase activated by kinases or caspases. We combined cryo-EM and X-ray crystallography to investigate its structure at an overall resolution of 3.8 Å. Its membrane-spanning region carrying 22 charged amino acids adopts a cuboid-like structure stabilized by salt bridges between hydrophilic residues in transmembrane helices. Phosphatidylcholine binding was observed in a hydrophobic cleft on the surface exposed to the outer leaflet of the plasma membrane. Six charged residues placed from top to bottom inside the molecule were essential for scrambling phospholipids in inward and outward directions, apparently providing a pathway for their translocation. A tryptophan residue was present between the head group of phosphatidylcholine and the extracellular end of the path. Its mutation to alanine made the Xkr8-Basigin complex constitutively active, indicating that it plays a vital role in regulating its scramblase activity. The structure of Xkr8-Basigin provides insights into the molecular mechanisms underlying phospholipid scrambling.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/química , Basigina/química , Membrana Celular/metabolismo , Proteínas de la Membrana/química , Fosfolípidos/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Basigina/metabolismo , Microscopía por Crioelectrón , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/aislamiento & purificación , Complejos Multiproteicos/metabolismo , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Proteínas de Transferencia de Fosfolípidos/química , Proteínas de Transferencia de Fosfolípidos/metabolismo , Fosfolípidos/química , Estructura Terciaria de Proteína , Triptófano/químicaRESUMEN
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.
Asunto(s)
Transportador 1 de Casete de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Fosfatidilcolinas/metabolismo , Mutación Puntual , Transportador 1 de Casete de Unión a ATP/deficiencia , Transportador 1 de Casete de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Adulto , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Encéfalo/diagnóstico por imagen , Membrana Celular/metabolismo , Femenino , Genes Letales , Heterocigoto , Humanos , Masculino , Lípidos de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Mutantes , Simulación de Dinámica Molecular , Enfermedades Neurodegenerativas/diagnóstico por imagen , Proteínas de Transferencia de Fosfolípidos/genética , Proteínas de Transferencia de Fosfolípidos/metabolismo , EmbarazoRESUMEN
Macrophage recognition and phagocytosis of crystals is critical for the associated fibrosis and cancer. Of note, multi-walled carbon nanotubes (MWCNTs), the highly representative products of nanotechnology, induce macrophage NLRP3 inflammasome activation and cause asbestosis-like pathogenesis. However, it remains largely unknown how macrophages efficiently recognize MWCNTs on their cell surfaces. Here, we identify by a targeted screening of phagocyte receptors the phosphatidylserine receptors T cell immunoglobulin mucin 4 (Tim4) and Tim1 as the pattern-recognition receptors for carbon crystals. Docking simulation studies reveal spatiotemporally stable interfaces between aromatic residues in the extracellular IgV domain of Tim4 and one-dimensional carbon crystals. Further, CRISPR-Cas9-mediated deletion of Tim4 and Tim1 reveals that Tim4, but not Tim1, critically contributes to the recognition of MWCNTs by peritoneal macrophages and to granuloma development in a mouse model of direct mesothelium exposure to MWCNTs. These results suggest that Tim4 recognizes MWCNTs through aromatic interactions and mediates phagocytosis leading to granulomas.
Asunto(s)
Granuloma/metabolismo , Macrófagos Peritoneales/metabolismo , Proteínas de la Membrana/metabolismo , Nanotubos de Carbono , Fagocitosis , Animales , Granuloma/genética , Humanos , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Células 3T3 NIH , Células THP-1RESUMEN
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.
Asunto(s)
Apoptosis/inmunología , Macrófagos/inmunología , Animales , HumanosRESUMEN
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.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Membrana Celular/metabolismo , Microscopía por Crioelectrón/métodos , Proteínas de Transporte de Membrana/metabolismo , Humanos , Modelos MolecularesRESUMEN
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.
Asunto(s)
Adenosina Trifosfatasas/química , Membrana Celular/química , Proteínas de la Membrana/química , Proteínas de Transporte de Membrana/química , Complejos Multiproteicos/química , Cristalografía por Rayos X , Humanos , Estructura Cuaternaria de ProteínaRESUMEN
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.
Asunto(s)
Macrófagos/inmunología , Proteínas de la Membrana/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Señalización del Calcio , Técnicas de Silenciamiento del Gen , Humanos , Interleucina-1beta/metabolismo , Proteínas de la Membrana/genética , Ratones , Mutación/genética , Fagocitosis/genética , Fosfatidilserinas/metabolismo , Receptores Purinérgicos P2X7/genética , Células THP-1RESUMEN
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.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Membrana Celular/metabolismo , Fosfatidilserinas/metabolismo , Adenosina Trifosfatasas/química , Animales , Membrana Celular/química , Humanos , Modelos Moleculares , Fosfatidilserinas/químicaRESUMEN
During spermatogenesis, up to 75% of germ cells in the testes undergo apoptosis and are cleared by Sertoli cells. X-linked XK blood group-related 8 (Xkr8) is a plasma membrane protein that scrambles phospholipids in response to apoptotic signals, exposing phosphatidylserine (PtdSer). Here, we found that Xkr8-/- male mice were infertile due to reduced sperm counts in their epididymides. Apoptotic stimuli could not induce PtdSer exposure in Xkr8-/- germ cells. Consistent with the hypothesis that PtdSer functions as an "eat-me" signal to phagocytes, cells expressing phosphatidylserine receptor TIM4 and MER tyrosine kinase receptor efficiently engulfed apoptotic wild-type male germ cells but not Xkr8-/- germ cells. Fluorescence and electron microscopy revealed Sertoli cells carrying engulfed and degenerated dead cells. However, many unengulfed apoptotic cells and residual bodies and much cell debris were present in Xkr8-/- testes and epididymides. These results indicate that Xkr8-mediated PtdSer exposure is essential for the clearance of apoptotic germ cells by Sertoli cells. There was no apparent inflammation in Xkr8-/- testes, suggesting that the unengulfed apoptotic cells may have undergone secondary necrosis, releasing noxious materials that affected the germ cells. Alternatively, failure to engulf the apoptotic germ cells may have caused the Sertoli cells to starve and lose their ability to support spermatogenesis.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Apoptosis , Células Germinativas/patología , Infertilidad Masculina/genética , Proteínas de la Membrana/genética , Animales , Eliminación de Gen , Células Germinativas/citología , Células Germinativas/metabolismo , Infertilidad Masculina/patología , Infertilidad Masculina/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Espermatogénesis , Testículo/citología , Testículo/metabolismo , Testículo/patologíaRESUMEN
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.