Back and forth

Summary

That some membranes restrict certain lipid species to one side of the bilayer and others to the opposite side has been known for two decades. However, how this asymmetric transbilayer distribution is generated and controlled, how many and what type of membranes are so structured, and even the reason for its existence is just now beginning to be understood. It has been a decade since the discovery of an activity which transports in an ATP-dependent manner only the aminophospholipids from the outer to the inner leaflet of the plasma membrane. This aminophospholipid translocase has yet to be isolated, reconstituted, and identified molecularly. Elevating intracellular Ca²⁺ allows all the major classes of phospholipids to move freely across the bilayer, scrambling lipids and dissipating asymmetry. The nature of this pathway and its mode of activation by Ca²⁺ remain to be determined. Though loss of transbilayer asymmetry by blood cells clearly produces a procoagulant surface and increases interactions with the reticuloendothelial system, it remains to be elucidated whether maintenance of blood homeostasis is just one expression of a more general raison d'ětre for lipid asymmetry. It is these persisting uncertainties and gaps in our knowledge which make the field such an interesting and exciting challenge at the present time.     

Effect of transbilayer phospholipid distribution on erythrocyte fusion

Phospholipid packing has been suggested as a relevant variable in the control of membrane fusion events. To test this possibility in a model system, a comparison was made of the fusability of erythrocytes with a normal asymmetric transbilayer distribution of plasma membrane phospholipids (tightly packed exterior lipids) and erythrocytes with a symmetric transbilayer distribution of phospholipids (more loosely packed exterior lipids), using polyethylene glycol as fusogen. Not only were lipid-symmetric cells more readily fused, but fusions of mixtures of lipid-symmetric and lipid-asymmetric cells indicated that both fusing partners must have a symmetric distribution for fusion to be enhanced. Lipid-symmetric cells may fuse more readily because loose packing of the exterior lipids enhances hydrophobic interactions between cells. Alternatively, enhanced membrane fluidity may facilitate intramembranous particle clustering, previously implicated as a potentiator of fusion. Finally, exposure of phosphatidylserine on the surface of lipid-symmetric erythrocytes may be responsible for their enhanced fusion.     

Binding,Activation, and Solubilization of the Ca2+-ATPase from Sarcoplasmic Reticulum by Nonionic Detergents

Interactions between delipidated Ca²⁺-ATPase from sarcoplasmic reticulum and four nonionic detergents—dodecyl octaoxyethyleneglycol monoether (C₁₂E₈), Triton X-100, Brij 58, and Brij 35—were characterized with respect to activation of ATPase activity, binding, and solubilization. C₁₂E₈ and Triton X-100 activated the delipidated ATPase to at least 80% of the original activity at the critical micelle concentrations (CMCs), whereas Brij 58 and Brij 35 activated no more than 10% of the original activity. The inability of Brij 58 and Brij 35 to activate the delipidated enzyme was probably a result of reduced binding of these detergents below the CMCs; both detergents exhibited a sixteenfold reduction in binding at the CMC compared with C₁₂E₈. The two Brij detergents were also unable to solubilize the delipidated enzyme and form monomers, as determined by sedimentation experiments. Thus the reduced binding levels of these detergents may result from an inability to overcome protein/protein interactions in the delipidated preparation. However, the Brij detergents were capable of solubilizing active enzyme from membrane vesicles, although with lower efficiency than C₁₂E₈ and Triton X-100. These results suggest that Brij 58 and 35 may be useful for solubilization of membrane proteins without disrupting protein/protein interactions, while Triton X-100 and C₁₂E₈ are more useful when bulk solubilization is the goal.     

Characterization of P4 ATPase Phospholipid Translocases (Flippases) in Human and Rat Pancreatic Beta Cells: THEIR GENE SILENCING INHIBITS INSULIN SECRETION*

The negative charge of phosphatidylserine in lipid bilayers of secretory vesicles and plasma membranes couples the domains of positively charged amino acids of secretory vesicle SNARE proteins with similar domains of plasma membrane SNARE proteins enhancing fusion of the two membranes to promote exocytosis of the vesicle contents of secretory cells. Our recent study of insulin secretory granules (ISG) (MacDonald, M. J., Ade, L., Ntambi, J. M., Ansari, I. H., and Stoker, S. W. (2015) Characterization of phospholipids in insulin secretory granules in pancreatic beta cells and their changes with glucose stimulation. J. Biol. Chem. 290, 11075–11092) suggested that phosphatidylserine and other phospholipids, such as phosphatidylethanolamine, in ISG could play important roles in docking and fusion of ISG to the plasma membrane in the pancreatic beta cell during insulin exocytosis. P4 ATPase flippases translocate primarily phosphatidylserine and, to a lesser extent, phosphatidylethanolamine across the lipid bilayers of intracellular vesicles and plasma membranes to the cytosolic leaflets of these membranes. CDC50A is a protein that forms a heterodimer with P4 ATPases to enhance their translocase catalytic activity. We found that the predominant P4 ATPases in pure pancreatic beta cells and human and rat pancreatic islets were ATP8B1, ATP8B2, and ATP9A. ATP8B1 and CDC50A were highly concentrated in ISG. ATP9A was concentrated in plasma membrane. Gene silencing of individual P4 ATPases and CDC50A inhibited glucose-stimulated insulin release in pure beta cells and in human pancreatic islets. This is the first characterization of P4 ATPases in beta cells. The results support roles for P4 ATPases in translocating phosphatidylserine to the cytosolic leaflets of ISG and the plasma membrane to facilitate the docking and fusion of ISG to the plasma membrane during insulin exocytosis.     

AIF deficiency compromises oxidative phosphorylation

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that, after apoptosis induction, translocates to the nucleus where it participates in apoptotic chromatinolysis. Here, we show that human or mouse cells lacking AIF as a result of homologous recombination or small interfering RNA exhibit high lactate production and enhanced dependency on glycolytic ATP generation, due to severe reduction of respiratory chain complex I activity. Although AIF itself is not a part of complex I, AIF-deficient cells exhibit a reduced content of complex I and of its components, pointing to a role of AIF in the biogenesis and/or maintenance of this polyprotein complex. Harlequin mice with reduced AIF expression due to a retroviral insertion into the AIF gene also manifest a reduced oxidative phosphorylation (OXPHOS) in the retina and in the brain, correlating with reduced expression of complex I subunits, retinal degeneration, and neuronal defects. Altogether, these data point to a role of AIF in OXPHOS and emphasize the dual role of AIF in life and death.     

Autophagy genes coordinate with the class II PI/PtdIns 3-kinase PIKI-1 to regulate apoptotic cell clearance in C. elegans

Phagocytosis and autophagy are two lysosome-mediated cellular degradation pathways designed to eliminate extracellular and intracellular constituents, respectively. Recent studies suggest that these two processes intersect. Several autophagy proteins have been shown to participate in clearance of apoptotic cells, but whether and how the autophagy pathway is involved is unclear. Here we showed that loss of function mutations in 19 genes acting at overlapping or distinct stages of autophagy caused increased numbers of cell corpses in C. elegans embryos. In contrast, genes that mediate specific clearance of P granules or protein aggregates through autophagy are dispensable for cell corpse removal. We showed that defective autophagy impairs phagosome maturation and that autophagy genes act in parallel to the class II phosphoinositide (PI)/phosphatidylinositol (PtdIns) 3-kinase PIKI-1 to regulate phagosomal PtdIns3P in a similar manner as VPS-34. Our data indicate that autophagy may coordinate with PIKI-1 to promote phagosome maturation, thus ensuring efficient clearance of apoptotic cells.     

小线虫,大发现:Caenorhabditis elegans在生命科学研究中的重要贡献

自20世纪60年代开始,秀丽线虫作为重要的模式生物在生命科学的发展过程中发挥着举足轻重的作用。线虫中的许多重大发现为人们理解复杂的细胞生命活动做出了极大的贡献。本文对秀丽线虫的研究历史、重要成果及研究前景作一简要综述。     

Cocaine decreases expression of neurogranin via alterations in thyroid receptor/retinoid X receptor signaling

The molecule responsible for the enzyme activity plasma membrane (PM) aminophospholipid translocase (APLT), which catalyzes phosphatidylserine (PS) translocation from the outer to the inner leaflet of the plasma membrane, is unknown in mammals. A Caenorhabditis elegans study has shown that ablation of transbilayer amphipath transporter-1 (TAT-1), which is an ortholog of a mammalian P-type ATPase, Atp8a1, causes PS externalization in the germ cells. We demonstrate here that the hippocampal cells of the dentate gyrus, and Cornu Ammonis (CA1, CA3) in mice lacking Atp8a1 exhibit a dramatic increase in PS externalization. Although their hippocampi showed no abnormal morphology or heightened apoptosis, these mice displayed increased activity and a marked deficiency in hippocampus-dependent learning, but no hyper-anxiety. Such observations indicate that Atp8a1 plays a crucial role in PM-APLT activity in the neuronal cells. In corroboration, ectopic expression of Atp8a1 but not its close homolog, Atp8a2, caused an increase in the population (V(max) ) of PM-APLT without any change in its signature parameter K(m) in the neuronal N18 cells. Conversely, expression of a P-type phosphorylation-site mutant of Atp8a1 (Atp8a1*) caused a decrease in V(max) of PM-APLT without significantly altering its K(m) . The Atp8a1*-expressing N18 cells also exhibited PS externalization without apoptosis. Together, our data strongly indicate that Atp8a1 plays a central role in the PM-APLT activity of some mammalian cells, such as the neuronal N18 and hippocampal cells.     

Amphiphile-induced phosphatidylserine exposure in human erythrocytes

Nonionic and anionic water-soluble amphiphiles were shown to increase strongly the binding of fluorescein isothiocyanate-conjugated annexin V (FITC-annexin V) in human erythrocytes pretreated with the aminophospholipid translocase (APLT) inhibitor n-ethylmaleimide (NEM). At high sublytic amphiphile-concentrations the binding of FITC-annexin V, monitored in a flow cytometer, was time -and temperature-dependent and occurred heterogeneously in the cell population, with 43-81% of cells being stained above background following incubation for 60 minutes at 37°C. The increased FITC-annexin V binding apparently indicates an increased flop rate of phosphatidylserine (PS) to the outer membrane leaflet. When the NEM-pretreatment was omitted, the FITC-annexin V binding was markedly, but not completely, reduced. In erythrocytes incubated with a zwitter-ionic amphiphile, a small increase in FITC-annexin V binding was detected, while cationic amphiphiles did not induce an increased FITC-annexin V binding. The potency of amphiphiles to induce PS exposure was not related to the type of shape alteration or vesiculation induced. Our results indicate a significant role of the charge status of a membrane intercalated amphiphile for its capability to induce PS exposure.     

Involvement of phosphatidylserine externalization in the down-regulation of c-myb expression in differentiating C2C12 cells

Abstract Analysis of c-myb gene down-regulation in differentiating C212 cells revealed that in proliferating cells, c-myb expression is high and ceases as the proliferation rate decreases. However, a low level of c-myb mRNA was detected in confluent non-proliferating differentiating cells for an extended period of time before it declined to an undetectable level. The time course of c-myb gene silencing in differentiating cells correlated with exposition of phosphatidylserine (PS) on the cell surface. Moreover, the interaction of exposed PS with exogenously added annexin V perturbed PS-mediated cell signaling and transiently up-regulated the declining c-myb expression. We, therefore, suggest that cell surface-exposed PS, which plays a role in the process of myotube formation, is also involved in the down-regulation of c-myb expression.     

线虫发育模式的分子生物学研究概况

线虫是研究动物发育的理想模式系统.由其细胞谱系研究深入到细胞间相互作用及信号传递的研究、细胞程序性死亡的研究,乃至多基因相互作用调控发育的研究,这些都是今后这一领域的研究热点.     

An essential role for a membrane lipid in cytokinesis. Regulation of contractile ring disassembly by redistribution of phosphatidylethanolamine

Phosphatidylethanolamine (PE) is a major membrane phospholipid that is mainly localized in the inner leaflet of the plasma membrane. We previously demonstrated that PE was exposed on the cell surface of the cleavage furrow during cytokinesis. Immobilization of cell surface PE by a PE-binding peptide inhibited disassembly of the contractile ring components, including myosin II and radixin, resulting in formation of a long cytoplasmic bridge between the daughter cells. This blockade of contractile ring disassembly was reversed by removal of the surface-bound peptide, suggesting that the PE exposure plays a crucial role in cytokinesis. To further examine the role of PE in cytokinesis, we established a mutant cell line with a specific decrease in the cellular PE level. On the culture condition in which the cell surface PE level was significantly reduced, the mutant ceased cell growth in cytokinesis, and the contractile ring remained in the cleavage furrow. Addition of PE or ethanolamine, a precursor of PE synthesis, restored the cell surface PE on the cleavage furrow and normal cytokinesis. These findings provide the first evidence that PE is required for completion of cytokinesis in mammalian cells, and suggest that redistribution of PE on the cleavage furrow may contribute to regulation of contractile ring disassembly.     

p62/SQSTM1: A Missing Link between Protein Aggregates and the Autophagy Machinery

In eukaryotic cells short-lived proteins are degraded in a specific process by the ubiquitin-proteasome system (UPS), whereas long-lived proteins and damaged organelles are degraded by macroautophagy (hereafter referred to as autophagy). A growing body of evidence now suggests that autophagy is important for clearance of protein aggregates that form in cells as a consequence of ageing, oxidative stress, alterations that elevate the amounts of certain aggregation-prone proteins or expression of aggregating mutant variants of specific proteins. Autophagy is generally considered to be a non-specific, bulk degradation process. However, a recent study suggests that p62/SQSTM1 may link the recognition of polyubiquitinated protein aggregates to the autophagy machinery.1 This protein is able to polymerize via its N-terminal PB1 domain and to recognize polyubiquitin via its C-terminal UBA domain. It can also recruit the autophagosomal protein LC3 and co-localizes with many types of polyubiquitinated protein aggregates.1 Here we discuss possible implications of these findings and raise some questions for further investigation.     

Autophagy in embryonic erythroid cells: its role in maturation

Yolk sac-derived embryonic erythroid cells differentiate synchronously in the peripheral blood of Syrian hamster. The stage of differentiation on day 10 of gestation is equivalent to polychromatophilic erythroblast stage and that on day 13 is equivalent to the reticulocyte stage in adult animals. The cytoplasm of embryonic erythroid cells became scant and devoid of most organelles on day 12 of gestation. In addition, there were very few non-erythroid cells in circulation before day 13. Thus the embryonic erythroid cells serve a pure and synchronous system to study the mechanisms of terminal differentiation. The number of mitochondria in the embryonic erythroid cells decreased to about 10% of the initial number during the period between day 10 and day 12 of gestation. In contrast, the frequency of autophagy of mitochondria increased 4.6-fold in the same period. The cytochrome c content of the cell decreased as the mitochondria became extinct. However, release of cytochrome c into the cytoplasm was not detectable through day 10-13 of gestation, suggesting that the mitochondria were digested within a closed compartment. Decomposed mitochondria and ferritin particles were detected in lysosomes by electron microscopy on and after day 12 of gestation, which also suggested digestion in a closed compartment. Mitochondrial ATP synthase subunit c, which is known to be a protease-refractory protein, was retained in the cells even after the disappearance of mitochondria, indicating that most of the mitochondria were not extruded from the cells. The digestion of mitochondria in autolysosomes may allow the cells to escape from rapid apoptotic cell death through concomitant removal of mitochondrial death-promoting factors such as cytochrome c.