RESUMO
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.
Assuntos
1-Fosfatidilinositol 4-Quinase/metabolismo , Autofagia , Mutação , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , 1-Fosfatidilinositol 4-Quinase/genética , Domínios Proteicos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Vacúolos/genéticaRESUMO
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.
Assuntos
1-Fosfatidilinositol 4-Quinase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , 1-Fosfatidilinositol 4-Quinase/análise , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Autofagia , Fosfatos de Fosfatidilinositol/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/análise , Vacúolos/metabolismo , Vacúolos/ultraestruturaRESUMO
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.
Assuntos
Autofagossomos/ultraestrutura , Técnica de Fratura por Congelamento/métodos , Fosfatidilinositóis/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose , Autofagossomos/metabolismo , Linhagem Celular Tumoral , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Proteínas Associadas aos Microtúbulos/metabolismo , proteínas de unión al GTP Rab7RESUMO
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 <100â¯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.
Assuntos
Microdomínios da Membrana/ultraestrutura , Fosfatos de Fosfatidilinositol/metabolismo , Saccharomyces cerevisiae/metabolismo , Microdomínios da Membrana/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Vacúolos/metabolismo , Vacúolos/ultraestruturaRESUMO
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.
Assuntos
Endossomos/metabolismo , Microdomínios da Membrana/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Linhagem Celular Tumoral , Citoplasma/metabolismo , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Lisossomos/metabolismo , Vesículas Transportadoras/metabolismoRESUMO
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.
Assuntos
Nanotecnologia , Fosfatos de Fosfatidilinositol/metabolismo , Angiotensina II/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células HeLa , Humanos , Microscopia de FluorescênciaRESUMO
Human hepatocellular carcinoma cell lines cultured in a monolayer show negligible activities of drug-metabolizing enzymes such as cytochrome P450s (CYPs) and UDP-glucuronosyltransferases (UGTs). Here, we show that culture of human hepatocellular carcinoma FLC-4 cells on 24-well plates arrayed with uniform micro-sized compartments on the bottom of the plates (micro-space cell culture plates) resulted in increased expression of drug-metabolizing enzymes (CYP1A2, CYP2C9, CYP3A4, UGT1A1, etc.) and nuclear receptors (pregnane X receptor, constitutive androstane receptor, etc.). When cells were treated with a typical CYP3A substrate (triazolam), CYP2C9 substrate (diclofenac) or UGT1A1 substrate (SN-38), large amounts of their metabolites were detected in the medium of cells cultured on micro-space cell culture plates. The formation of metabolites from triazolam, diclofenac and SN-38 was strongly inhibited by co-treatment with a CYP3A inhibitor (ketoconazole), CYP2C9 inhibitor (sulfaphenazole) and UGT1A1 inhibitor (ketoconazole), respectively. On the other hand, formation of metabolites was not observed in the medium of cells cultured in a monolayer. Finally, the cytotoxic effect of aflatoxin B1 was more potent in cells cultured on micro-space cell culture plates than in cells cultured in a monolayer. The results suggest that FLC-4 cells cultured on micro-space cell culture plates are useful for studying drug metabolism and drug-induced hepatotoxicity.