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. Our results showed that osh1-osh7∆ deletions lead to autophagic dysfunction, with impaired GFP-Atg8 processing and the absence of autophagosomes and autophagic bodies in the cytosol and vacuole, respectively. Freeze-fracture electron microscopy (EM) revealed elevated phosphatidylinositol 4-phosphate (PtdIns(4)P) levels in cytoplasmic and luminal leaflets of autophagic bodies and vacuolar membranes in all deletion mutants. Phosphatidylserine (PtdSer) levels were significantly decreased in the autophagic bodies and vacuolar membranes in osh4∆ and osh5∆ mutants, whereas no significant changes were observed in other osh deletion mutants. Furthermore, we identified defects in autophagic processes in the osh4∆ and osh5∆ mutants, including rare autophagosome formation in the osh5∆ mutant and accumulation of autophagic bodies in the vacuole in the osh4∆ mutant, even in the absence of the proteinase inhibitor PMSF. These findings suggest that Osh4p and Osh5p play crucial roles in the transport of PtdSer to autophagic bodies and autophagosome membranes, respectively. The precise control of lipid composition in the membranes of autophagosomes and autophagic bodies by Osh4p and Osh5p represents an important regulatory mechanism in autophagy.

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. In this study, we developed an electron microscopy method that enabled us to determine the distributions of PtdSer and PtdEtn in individual leaflets of cellular membranes by using quick-freeze freeze-fracture replica labeling. Our findings show that PtdSer and PtdEtn are asymmetrically distributed, with substantial amounts localized at the luminal leaflet of the inner membrane complex (IMC), which comprises flattened vesicles located just underneath the plasma membrane (see Figs. 2B and 7). We also found that PtdSer was absent in the cytoplasmic leaflet of the inner IMC membrane, but was present in considerable amounts in the cytoplasmic leaflet of the middle IMC membrane, suggesting a barrier-like mechanism preventing the diffusion of PtdSer in the cytoplasmic leaflets of the two membranes. In addition, the expression levels of both PtdSer and PtdEtn in the luminal leaflet of the IMC membrane in the highly virulent RH strain were higher than those in the less virulent PLK strain. We also found that the amount of glycolipid GM3, a lipid raft component, was higher in the RH strain than in the PLK strain. These results suggest a correlation between lipid raft maintenance, virulence, and the expression levels of PtdSer and PtdEtn in T. gondii.

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. In this study, we analyzed the primary role of PtdIns(4)P phosphatases in yeast autophagy. The PtdIns(4)P labeling densities in the membranes of the vacuoles, mitochondria, nucleus, endoplasmic reticulum, and plasma membrane dramatically increased in the phosphatase deletion mutants sac1∆ and sjl3∆, and the temperature-sensitive mutant sac1ts/sjl3∆ at the restrictive temperature. GFP-Atg8 processing assay indicated defective autophagy in the sac1∆ and sac1ts/sjl3∆ mutants. In contrast to the localization of PtdIns(4)P in the luminal leaflet of autophagosomal membranes in the wild-type yeast, PtdIns(4)P was localized in both the luminal and cytoplasmic leaflets of the autophagosomal membranes in the sac1∆ strain. In addition, the number of autophagic bodies in the vacuole significantly decreased in the sac1∆ strain, although autophagosomes were present in the cytoplasm. In the sac1ts/sjl3∆ strain, the number of autophagosomes in the cytoplasm dramatically decreased at the restrictive temperature. Considering that the numbers of autophagosomes and autophagic bodies in the sjl3∆ strain were comparable to those in the wild-type yeast, we found that the autophagosome could not be formed when PtdIns(4)P phosphatase activities of both Sac1p and Sjl3p were diminished. Together, these results indicate that the turnover of PtdIns(4)P by phosphatases is essential for autophagosome biogenesis.

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. Using the electron microscopy method, distributions of four phospholipids were examined in the autophagosomal and autophagic body membranes upon autophagy induction. The labeling of phosphatidylserine (PtdSer) in the autophagic body membrane dramatically increased after it converted from the autophagosome, but remained low in the vacuolar membrane. PtdSer in the autophagic body membrane also increased in atg15∆ yeast. These results suggest that the selective increment of PtdSer in the autophagic body, but not the vacuolar, membrane, can explain the selective degradation of the autophagic membrane.

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. However, rafts are poorly understood. We used quick-freezing and freeze-fracture immuno-electron microscopy to examine the localization of monosialotetrahexosylganglioside (GM1) and monosialodihexosylganglioside (GM3), putative raft microdomain components in P. falciparum and infected erythrocytes. This method immobilizes molecules in situ, minimizing artifacts. GM3 was localized in the exoplasmic (EF) and cytoplasmic leaflets (PF) of the parasite and the parasitophorous vacuole (PV) membranes, but solely in the EF of the infected erythrocyte membrane, as in the case for uninfected erythrocytes. Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) was localized solely in the PF of erythrocyte, parasite, and PV membranes. This is the first time that GM3, the major component of raft microdomains, was found in the PF of a biological membrane. The unique localization of raft microdomains may be due to P. falciparum lipid metabolism and its unique biological processes, like protein transport from the parasite to infected erythrocytes.

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

Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) 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)P2 is involved in various kinds of yeast functions. PtdIns(4)P is not only the immediate precursor of PtdIns(4,5)P2, but also an essential signaling molecule in the plasma membrane, Golgi, and endosomal system. To analyze the distribution of PtdIns(4,5)P2 and PtdIns(4)P in the yeast plasma membrane at a nanoscale level, we employed a freeze-fracture electron microscopy (EM) method that physically immobilizes lipid molecules in situ. It has been reported that the plasma membrane of budding yeast can be divided into three distinct areas: furrowed, hexagonal, and undifferentiated flat. Previously, using the freeze-fracture EM method, we determined that PtdIns(4)P is localized in the undifferentiated flat area, avoiding the furrowed and hexagonal areas of the plasma membrane. In the present study, we found that PtdIns(4,5)P2 was localized in the cytoplasmic leaflet of the plasma membrane, and concentrated in the furrowed area. There are three types of PtdIns 4-kinases which are encoded by stt4, pik1, and lsb6. The labeling density of PtdIns(4)P in the plasma membrane significantly decreased in both pik1ts and stt4ts mutants. However, the labeling densities of PtdIns(4,5)P2 in the plasma membrane of both the pik1ts and stt4ts mutants were comparable to that of the wild type yeast. These results suggest that PtdIns(4)P produced by either Pik1p or Stt4p is immediately phosphorylated by Mss4p and converted to PtdIns(4,5)P2 at the plasma membrane.

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. We tried to determine the distribution of glycosphingolipids GM1 and GM3, putative raft components in the T. gondii cell membrane in this study, using a rapid-frozen and freeze-fractured immuno-electron microscopy method. This method physically stabilized molecules in situ, to minimize the probability of artefactual disruption. Labeling of GM3, but not GM1, was observed in the exoplasmic (or luminal), but not the cytoplasmic, leaflet of the inner membrane complex (IMC) in T. gondii infected in human foreskin fibroblast-1 (HFF-1). No labeling was detected in any leaflet of the T. gondii plasma membrane. In contrast to HFF-1, T. gondii infected in mouse fibroblast (MF), labelings of both GM1 and GM3 were detected in the IMC luminal leaflet, although GM1's gold labeling density was very low. The same freeze-fracture EM method showed that both GM1 and GM3 were expressed in the exoplasmic leaflet of the MF plasma membrane. However, labeling of only GM3, but not GM1, was detected in the exoplasmic leaflet of the HFF-1 plasma membrane. These results suggest that GM1 or GM3, localized in the IMC, is obtained from the plasma membranes of infected host mammalian cells. Furthermore, the localization of microdomains or rafts in the luminal leaflets of the intracellular confined space IMC organelle of T. gondii suggests a novel characteristic of rafts.

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. Furthermore, the PtdIns(4)P-positive raft-like domains engulf LDs through a microautophagic mechanism. When single temperature-conditional mutants of STT4 or PIK1 PtdIns 4-kinases were used, in the vacuole of STT4 and PIK1 mutant cells, microautophagic vesicles drastically decreased at restrictive temperatures, and the labeling density of PtdIns(4)P on the microautophagic vesicles and the sizes of the mutants' microautophagic vesicles also decreased. These results suggest that both Stt4p and Pik1p have important roles in the microautophagy of the vacuole in the stationary phase and under nitrogen starvation conditions.

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. QF-FRL physically immobilised molecules in situ, minimising the possibility of artefactual perturbation. SAG1 labelling was observed in the exoplasmic, but not cytoplasmic, leaflets of T. gondii plasma membrane, whereas none was detected in any leaflet of the inner membrane complex. Point pattern analysis of SAG1 immunogold labelling revealed mostly random distribution in T. gondii plasma membrane. The present method obtains information on the molecular distribution of natively expressed GPI-anchored proteins and demonstrates that SAG1 in T. gondii does not form significant microdomains in the plasma membrane.

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.

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. PtdIns(4)P is confined to the cytoplasmic leaflet of autophagosomal inner and outer membranes in mammalian cells. Using temperature-conditional single PIK1 or STT4 PtdIns 4-kinase mutants, autophagic bodies in the vacuole of PIK1 and STT4 mutant cells dramatically decreased at restrictive temperatures, and the number of autophagosomes in the cytosol of PIK1 mutants cells was also decreased, whereas autophagosome levels of STT4 mutant cells were comparable to that of wild-type and STT4 mutant cells at permissive temperatures. Localization of PtdIns(4)P in the luminal leaflet in the biological membrane is a novel finding, and differences in PtdIns(4)P distribution suggest substantial differences between yeast and mammals. We also demonstrate in this study that Pik1p and Stt4p play essential roles in autophagosome formation and autophagosome-vacuole fusion in yeast cells, respectively.

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. To better understand autophagosome biogenesis, it is essential to determine the localization of PtdIns(4)P and to examine its relationship with LC3 and GABARAP subfamilies and Rab7. To analyze PtdIns(4)P distribution, we used an electron microscopy technique that labels PtdIns(4)P on the freeze-fracture replica of intracellular biological membranes, which minimizes the possibility of artificial perturbation because molecules in the membrane are physically immobilized in situ. Using this technique, we found that PtdIns(4)P is localized on the cytoplasmic, but not the luminal (exoplasmic), leaflet of the inner and outer membranes of autophagosomes. Double labeling revealed that PtdIns(4)P mostly colocalizes with Rab7, but not with LC3B, GABARAP, GABARAPL1 and GABARAPL2. Rab7 plays essential roles in autophagosome maturation and in autophagosome-lysosome fusion events. We suggest that PtdIns(4)P is localized to the cytoplasmic leaflet of the autophagosome at later stages, which may illuminate the importance of PtdIns(4)P at the later stages of autophagosome formation.

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. We observed that PtdIns(4)P is localised on the cytoplasmic leaflet, but not the exoplasmic leaflet, of the plasma membrane, Golgi body, vacuole, and vesicular structure membranes. PtdIns(4)P labelling was not observed in the membrane of the endoplasmic reticulum, and in the outer and inner membranes of the nuclear envelope or mitochondria. PtdIns(4)P forms clusters of <100 nm in diameter in the plasma membrane and vacuolar membrane according to point pattern analysis of immunogold labelling. There are three kinds of compartments in the cytoplasmic leaflet of the plasma membrane. In the present study, we showed that PtdIns(4)P is specifically localised in the flat undifferentiated plasma membrane compartment. In the vacuolar membrane, PtdIns(4)P was concentrated in intramembrane particle (IMP)-deficient raft-like domains, which are tightly bound to lipid droplets, but not surrounding IMP-rich non-raft domains in geometrical IMP-distributed patterns in the stationary phase. This is the first report showing microdomain formations of PtdIns(4)P in the plasma membrane and vacuolar membrane of budding yeast cells at a nanoscale level, which will illuminate the functionality of PtdIns(4)P in each membrane.

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)P2), 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)P2 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)P2 distributions, at a nanoscale, we employed an electron microscopy technique that specifically labels PtdIns(4)P and PtdIns(4,5)P2 on the freeze-fracture replica of intracellular biological membranes. This method minimizes the possibility of artificial perturbation, because molecules in the membrane are physically immobilized in situ. Using this technique, we found that PtdIns(4)P was localized to the cytoplasmic leaflet of Golgi apparatus and vesicular-shaped structures. The PtdIns(4,5)P2 labeling was observed in the cytoplasmic leaflet of the mitochondrial inner membrane and vesicular-shaped structures. Double labeling of PtdIns(4)P and PtdIns(4,5)P2 with endosome markers illustrated that PtdIns(4)P and PtdIns(4,5)P2 were mainly localized to the late endosome/lysosome and early endosome, respectively. PtdIns(4)P and PtdIns(4,5)P2 were colocalized in some endosomes, with the two phospholipids separated into distinct microdomains on the same endosomes. This is the first report showing, at a nanoscale, segregation of PtdIns(4)P- and PtdIns(4,5)P2-enriched microdomains in the endosome, of likely importance for endosome functionality.

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. This method minimizes the possibility of artificial perturbation, because molecules in the membrane are physically immobilized in situ. Using this technique, we observed no PtdIns(4)P in the caveolae of normal cultured human fibroblasts, although PtdIns(4,5)P2 has been shown to be highly concentrated in them in our previous report. When cells were stimulated with angiotensin II, the level of PtdIns(4)P in the undifferentiated membrane transiently decreased to 64.3% at 10 s, began to increase at 30 s and largely increased to 341.9% at 40 s, and then returned to the initial level at 130 s after the stimulation. Interestingly, PtdIns(4)P localized at the caveolae at 70 and 130 s after the stimulation. These results suggest that the level of the PtdIns(4)P pool in the plasma membrane is sensitive and the distribution of PtdIns(4)P dramatically changes by agonist stimulation, and there are active sites of production or replenishment of PtdIns(4)P at undifferentiated membrane and caveolar areas.

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. Cells depleted of PtdIns4KIIα had an impaired ability to sort molecules destined for recycling from early endosomes. We further identify the Eps15 homology domain-containing protein 3 (EHD3) as a possible endosomal effector of PtdIns4KIIα. Tubular endosomes containing EHD3 were shortened and became more vesicular in PtdIns4KIIα-depleted cells. Endosomal PtdIns(4,5)P2 was also significantly reduced in PtdIns4KIIα-depleted cells. These results show that PtdIns4KIIα regulates receptor sorting at early endosomes through a PtdIns(4)P-dependent pathway and contributes substrate for the synthesis of endosomal PtdIns(4,5)P2.

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. Cell death and reactive oxygen species production were significantly suppressed by propolis in dose-dependent manner, compared with Hcy alone. For the in vivo assays, mice were fed a propolis-containing diet and Hcy thiolactone in water. Cognitive function was evaluated using the Morris water maze test. Propolis suppressed cognitive dysfunction caused by hyperhomocysteinemia. Accumulation of aggregated protein in brain was accelerated in hyperhomocysteinemia, and the accumulation was suppressed by propolis. Hyperhomocysteinemia, however, did not enhance the oxidative stress in brain. In vitro amyloid formation assay showed that Hcy accelerated lysozyme aggregation and propolis inhibited the aggregation.

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, Kir4.1, on the ependymal cell membrane suggesting involvement of the channel in this function. Immunohistochemical study with confocal microscopy identified Kir4.1 labeling on the lateral but not apical membrane of ependymal cells. Ultrastructural analysis revealed that Kir4.1-immunogold particles were specifically localized and clustered on adjacent membranes at puncta adherens type junctions, whereas an aquaporin water channel, AQP4, that was also detected on the lateral membrane only occurred at components other than adherens junctions. Therefore, in ependymal cells, Kir4.1 and AQP4 are partitioned into distinct membrane compartments that might respectively transport either K(+) or water. Kir4.1 was also expressed in a specialized form of ependymal cell, namely the tanycyte, being abundant in tanycyte processes wrapping neuropils and blood vessels. These specific localizations suggest that Kir4.1 mediates intercellular K(+) exchange between ependymal cells and also K(+)-buffering transport via tanycytes that can interconnect neurons and vessels/ventricles. We propose that ependymal cells and tanycytes differentially operate Kir4.1 and AQP4 actively to control the property of fluids at local areas in the brain.

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. In mutant yeast lacking two cytoplasmic phosphatases, ymr1Δ and sjl3Δ, PtdIns(3)P in the autophagosome is equally abundant in the two membrane leaflets, suggesting that the PtdIns(3)P asymmetry in wild-type yeast is generated by unilateral hydrolysis. The observed differences in PtdIns(3)P distribution suggest that autophagy in yeast and mammals may involve substantially different processes.