Coordinated regulation of phosphatidylinositol 4-phosphate and phosphatidylserine levels by Osh4p and Osh5p is an essential regulatory mechanism in autophagy.

Macroautophagy (hereafter autophagy) is an intracellular degradative pathway in budding yeast cells. Certain lipid types play essential roles in autophagy; yet the precise mechanisms regulating lipid composition during autophagy remain unknown. Here, we explored the role of the Osh family proteins in the modulating lipid composition during autophagy in budding yeast.

Unique asymmetric distribution of phosphatidylserine and phosphatidylethanolamine in Toxoplasma gondii revealed by nanoscale analysis.

Toxoplasma gondii is a highly prevalent obligate apicomplexan parasite that is important in clinical and veterinary medicine. It is known that glycerophospholipids phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtn), especially their expression levels and flip-flops between cytoplasmic and exoplasmic leaflets, in the membrane of T. gondii play important roles in efficient growth in host mammalian cells, but their distributions have still not been determined because of technical difficulties in studying intracellular lipid distribution at the nanometer level.

Essential roles of phosphatidylinositol 4-phosphate phosphatases Sac1p and Sjl3p in yeast autophagosome formation.

Autophagy is regulated by phosphoinositides. We have previously shown that phosphatidylinositol 4-phosphate (PtdIns(4)P) is localized in the autophagosomal membrane. Additionally, in yeast cells, phosphatidylinositol 4-kinases Pik1p and Stt4p play important roles in the formation of the autophagosome and its fusion with the vacuole, respectively.

Selective increment of phosphatidylserine on the autophagic body membrane in the yeast vacuole.

In yeast cells, the autophagosome is a double-membrane structure; the inner membrane becomes the autophagic body membrane in the vacuole. Vacuolar enzymes degrade the autophagic body. There is no critical information regarding its selective degradation.

Glycosphingolipid GM3 is localized in both exoplasmic and cytoplasmic leaflets of Plasmodium falciparum malaria parasite plasma membrane.

Lipid rafts, sterol-rich and sphingolipid-rich microdomains on the plasma membrane are important in processes like cell signaling, adhesion, and protein and lipid transport. The virulence of many eukaryotic parasites is related to raft microdomains on the cell membrane. In the malaria parasite Plasmodium falciparum, glycosylphosphatidylinositol-anchored proteins, which are important for invasion and are possible targets for vaccine development, are localized in the raft.

The distribution of phosphatidylinositol 4,5-bisphosphate in the budding yeast plasma membrane.

Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P) is generated through phosphorylation of phosphatidylinositol 4-phosphate (PtdIns(4)P) by Mss4p, the only PtdIns phosphate 5-kinase in yeast cells. PtdIns(4,5)P is involved in various kinds of yeast functions. PtdIns(4)P is not only the immediate precursor of PtdIns(4,5)P, but also an essential signaling molecule in the plasma membrane, Golgi, and endosomal system.

Raft microdomain localized in the luminal leaflet of inner membrane complex of living Toxoplasma gondii.

Membrane microdomains or rafts, sterol- and sphingolipid-rich microdomains in the plasma membrane have been studied extensively in mammalian cells. Recently, rafts were found to mediate virulence in a variety of parasites, including Toxoplasma gondii. However, it has been difficult to examine a two-dimensional distribution of lipid molecules at a nanometer scale.

Microautophagy in the yeast vacuole depends on the activities of phosphatidylinositol 4-kinases, Stt4p and Pik1p.

Morphologically, the lipophagy in yeast cell mimics microautophagy, which includes a direct amendment of the vacuolar membrane that engulfs lipid droplets (LDs). The molecular mechanism of the membrane modifications that elicits microautophagy still remains elusive. In this study, an analysis of membrane lipid distribution at a nanoscale level showed that PtdIns(4)P is localized in the cytoplasmic leaflet of microautophagic vesicles, which are derived when the vacuole's membrane domains engulfed LDs both in the stationary phase and in acute nitrogen starvation.

Nanoscale analysis reveals no domain formation of glycosylphosphatidylinositol-anchored protein SAG1 in the plasma membrane of living Toxoplasma gondii.

Glycosylphosphatidylinositol (GPI)-anchored proteins typically localise to lipid rafts. GPI-anchored protein microdomains may be present in the plasma membrane; however, they have been studied using heterogeneously expressed GPI-anchored proteins, and the two-dimensional distributions of endogenous molecules in the plasma membrane are difficult to determine at the nanometre scale. Here, we used immunoelectron microscopy using a quick-freezing and freeze-fracture labelling (QF-FRL) method to examine the distribution of the endogenous GPI-anchored protein SAG1 in Toxoplasma gondii at the nanoscale.

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.

Essential and distinct roles of phosphatidylinositol 4-kinases, Pik1p and Stt4p, in yeast autophagy.

Autophagy is a degradative cellular pathway that protects eukaryotic cells from starvation/stress. Phosphatidylinositol 4-kinases, Pik1p and Stt4p, are indispensable for autophagy in budding yeast, but participation of PtdIns-4 kinases and their product, phosphatidylinositol 4-phosphate [PtdIns(4)P], is not understood. Nanoscale membrane lipid distribution analysis showed PtdIns(4)P is more abundant in yeast autophagosomes in the luminal leaflet than the cytoplasmic leaflet.

Phosphatidylinositol 4-phosphate on Rab7-positive autophagosomes revealed by the freeze-fracture replica labeling.

Phosphatidylinositol 4-phophate (PtdIns(4)P) is an essential signaling molecule in the Golgi body, endosomal system, and plasma membrane and functions in the regulation of membrane trafficking, cytoskeletal organization, lipid metabolism and signal transduction pathways, all mediated by direct interaction with PtdIns(4)P-binding proteins. PtdIns(4)P was recently reported to have functional roles in autophagosome biogenesis. LC3 and GABARAP subfamilies and a small GTP-binding protein, Rab7, are localized on autophagosomal membranes and participate at each stage of autophagosome formation and maturation.

Immunoelectron Microscopy of Gangliosides.

Because chemical fixatives like aldehydes do not work on most lipid molecules in the membrane, small-scale lipid distribution cannot be identified by immunoelectron microscopy in cells fixed by conventional methods. Here we describe a method for physically stabilizing membranes through quick-freezing and freeze-fracture replica formation and for specifically labeling gangliosides for electron microscopy. This method enables the ultrahigh-resolution mapping of membrane lipids including gangliosides within the two-dimensional plane of membranes.

Nanoscale domain formation of phosphatidylinositol 4-phosphate in the plasma and vacuolar membranes of living yeast cells.

In budding yeast Saccharomyces cerevisiae, PtdIns(4)P serves as an essential signalling molecule in the Golgi complex, endosomal system, and plasma membrane, where it is involved in the control of multiple cellular functions via direct interactions with PtdIns(4)P-binding proteins. To analyse the distribution of PtdIns(4)P in yeast cells at a nanoscale level, we employed an electron microscopy technique that specifically labels PtdIns(4)P on the freeze-fracture replica of the yeast membrane. This method minimizes the possibility of artificial perturbation, because molecules in the membrane are physically immobilised in situ.

Segregation of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate into distinct microdomains on the endosome membrane.

Phosphatidylinositol 4-phosphate (PtdIns(4)P) is the immediate precursor of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P), which is located on the cytoplasmic leaflet of the plasma membrane and has been reported to possess multiple cellular functions. Although PtdIns(4)P and PtdIns(4,5)P have been reported to localize to multiple intracellular compartments and to each function as regulatory molecules, their generation, regulation and functions in most intracellular compartments are not well-defined. To analyze PtdIns(4)P and PtdIns(4,5)P distributions, at a nanoscale, we employed an electron microscopy technique that specifically labels PtdIns(4)P and PtdIns(4,5)P on the freeze-fracture replica of intracellular biological membranes.

Nanoscale analysis reveals agonist-sensitive and heterogeneous pools of phosphatidylinositol 4-phosphate in the plasma membrane.

Phosphatidylinositol 4-phosphate [PtdIns(4)P] is the immediate precursor of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], which is localized to the cytoplasmic leaflet of the plasma membrane and has been reported to possess multiple cell biological functions. Direct evidence showing the distribution of PtdIns(4)P pools at a nanoscale when the plasma membrane PtdIns(4,5)P2 is hydrolyzed by agonist stimulation is lacking. To analyze the distribution of PtdIns(4)P at a nanoscale, we employed an electron microscopy technique that specifically labels PtdIns(4)P on the freeze-fracture replica of the plasma membrane.

PtdIns4KIIα generates endosomal PtdIns(4)P and is required for receptor sorting at early endosomes.

Phosphatidylinositol 4-kinase IIα (PtdIns4KIIα) localizes to the trans-Golgi network and endosomal compartments and has been implicated in the regulation of endosomal traffic, but the roles of both its enzymatic activity and the site of its action have not been elucidated. This study shows that PtdIns4KIIα is required for production of endosomal phosphatidylinositol 4-phosphate (PtdIns(4)P) on early endosomes and for the sorting of transferrin and epidermal growth factor receptor into recycling and degradative pathways. Depletion of PtdIns4KIIα with small interfering RNA significantly reduced the amount of vesicular PtdIns(4)P on early endosomes but not on Golgi membranes.

Ethanol extract of Brazilian propolis ameliorates cognitive dysfunction and suppressed protein aggregations caused by hyperhomocysteinemia.

Homocysteine (Hcy) has been proposed to be a risk factor for cognitive dysfunction. We investigated the effects and the underlying mechanisms of action of propolis, which has antioxidant activity on Hcy-induced oxidative stress in vitro and in vivo. For the in vitro assays, neuroblastoma SH-SY5Y and glioblastoma U-251MG cells were cultured with Hcy and various concentrations of propolis.

Clustering of Kir4.1 at specialized compartments of the lateral membrane in ependymal cells of rat brain.

Brain ependymal cells, which form an epithelial layer covering the cerebral ventricles, have been shown to play a role in the regulation of cerebrospinal and interstitial fluids. The machinery underlying this, however, remains largely unknown. Here, we report the specific localization of an inwardly rectifying K(+) channel, Kir4.

Yeast and mammalian autophagosomes exhibit distinct phosphatidylinositol 3-phosphate asymmetries.

Phosphatidylinositol 3-kinase is indispensable for autophagy but it is not well understood how its product, phosphatidylinositol 3-phosphate (PtdIns(3)P), participates in the biogenesis of autophagic membranes. Here, by using quick-freezing and freeze-fracture replica labelling, which enables determination of the nanoscale distributions of membrane lipids, we show that PtdIns(3)P in yeast autophagosomes is more abundant in the luminal leaflet (the leaflet facing the closed space between the outer and inner autophagosomal membranes) than in the cytoplasmic leaflet. This distribution is drastically different from that of the mammalian autophagosome in which PtdIns(3)P is confined to the cytoplasmic leaflet.

A method for efficient observation of intracellular membranes of monolayer culture cells by quick-freeze and freeze-fracture electron microscopy.

We previously developed a method of analyzing the two-dimensional distribution of membrane lipids by combining quick-freezing and freeze-fracture replica labeling (QF-FRL). In principle, this method can be applied to any membrane, but in practice it is not easy to observe cytoplasmic organelles efficiently without ice crystal damage. In this paper, we report a modification of our method that circumvents this problem.

The distribution of phosphatidylinositol 4,5-bisphosphate in acinar cells of rat pancreas revealed with the freeze-fracture replica labeling method.

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] is a phospholipid that has been implicated in multiple cellular activities. The distribution of PI(4,5)P(2) has been analyzed extensively using live imaging of the GFP-coupled phospholipase C-δ1 pleckstrin homology domain in cultured cell lines. However, technical difficulties have prevented the study of PI(4,5)P(2) in cells of in vivo tissues.

Claudin-4 induction by E-protein activity in later stages of CD4/8 double-positive thymocytes to increase positive selection efficiency.

Claudins (Clds) are crucial constituents of tight-junction strands in epithelial cells and have a central role in barrier functions. We show that Cld4 is unexpectedly expressed in normal thymic lymphocytes independently of tight junctions. The Cld4 expression was mostly confined to a portion of the CD4/CD8 double-positive (DP) cells.

High-resolution molecular localization by freeze-fracture replica labeling.

The freeze-fracture technique splits the frozen lipid bilayer membrane into two halves and immobilizes membrane proteins and lipids by the vacuum evaporation of platinum and carbon. After a treatment by SDS to remove extramembrane materials, the specimen is subjected to immunogold labeling, which gives information on the two-dimensional distribution of membrane molecules and their relationship to various differentiated structures. In combination with rapid freezing, the freeze-fracture technique has an advantage over other methods using conventional chemical fixation because the distribution of lipids as well as proteins can be observed at the mesoscale in a wide area of the membrane.

Quantitative electron microscopy for the nanoscale analysis of membrane lipid distribution.

An important goal of membrane biology is to define the local heterogeneity of membrane lipid composition. Here we describe a quantitative electron microscopic method that enables the localization of specific membrane lipids at the nanometer scale. The method involves freezing cells rapidly to halt the molecular motion, physically stabilizing membrane molecules in the freeze-fracture replica by the deposition of evaporated platinum and carbon layers and labeling with specific probes for electron microscopic observation.