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.

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.

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.

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.

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.

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.

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.

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.