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1.
J Cell Sci ; 135(5)2022 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-34000034

RESUMO

Membrane phase separation to form micron-scale domains of lipids and proteins occurs in artificial membranes; however, a similar large-scale phase separation has not been reported in the plasma membrane of the living cells. We show here that a stable micron-scale protein-depleted region is generated in the plasma membrane of yeast mutants lacking phosphatidylserine at high temperatures. We named this region the 'void zone'. Transmembrane proteins and certain peripheral membrane proteins and phospholipids are excluded from the void zone. The void zone is rich in ergosterol, and requires ergosterol and sphingolipids for its formation. Such properties are also found in the cholesterol-enriched domains of phase-separated artificial membranes, but the void zone is a novel membrane domain that requires energy and various cellular functions for its formation. The formation of the void zone indicates that the plasma membrane in living cells has the potential to undergo phase separation with certain lipid compositions. We also found that void zones were frequently in contact with vacuoles, in which a membrane domain was also formed at the contact site.

2.
J Cell Biol ; 220(3)2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33439214

RESUMO

The mechanism of isolation membrane formation in autophagy is receiving intensive study. We recently found that Atg9 translocates phospholipids across liposomal membranes and proposed that this functionality plays an essential role in the expansion of isolation membranes. The distribution of phosphatidylinositol 3-phosphate in both leaflets of yeast autophagosomal membranes supports this proposal, but if Atg9-mediated lipid transport is crucial, symmetrical distribution in autophagosomes should be found broadly for other phospholipids. To test this idea, we analyzed the distributions of phosphatidylcholine, phosphatidylserine, and phosphatidylinositol 4-phosphate by freeze-fracture electron microscopy. We found that all these phospholipids are distributed with comparable densities in the two leaflets of autophagosomes and autophagic bodies. Moreover, de novo-synthesized phosphatidylcholine is incorporated into autophagosomes preferentially and shows symmetrical distribution in autophagosomes within 30 min after synthesis, whereas this symmetrical distribution is compromised in yeast expressing an Atg9 mutant. These results indicate that transbilayer phospholipid movement that is mediated by Atg9 is involved in the biogenesis of autophagosomes.

3.
J Cell Biol ; 220(1)2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33315072

RESUMO

Nuclear lipid droplets (LDs) in hepatocytes are derived from precursors of very-low-density lipoprotein in the ER lumen, but it is not known how cells lacking the lipoprotein secretory function form nuclear LDs. Here, we show that the inner nuclear membrane (INM) of U2OS cells harbors triglyceride synthesis enzymes, including ACSL3, AGPAT2, GPAT3/GPAT4, and DGAT1/DGAT2, and generates nuclear LDs in situ. mTOR inhibition increases nuclear LDs by inducing the nuclear translocation of lipin-1 phosphatidic acid (PA) phosphatase. Seipin, a protein essential for normal cytoplasmic LD formation in the ER, is absent in the INM. Knockdown of seipin increases nuclear LDs and PA in the nucleus, whereas seipin overexpression decreases these. Seipin knockdown also up-regulates lipin-1ß expression, and lipin-1 knockdown decreases the effect of seipin knockdown on nuclear LDs without affecting PA redistribution. These results indicate that seipin is not directly involved in nuclear LD formation but instead restrains it by affecting lipin-1 expression and intracellular PA distribution.


Assuntos
Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Gotículas Lipídicas/metabolismo , Membrana Nuclear/metabolismo , Linhagem Celular Tumoral , Proteínas de Fluorescência Verde/metabolismo , Humanos , Gotículas Lipídicas/ultraestrutura , Membrana Nuclear/ultraestrutura , Ácidos Fosfatídicos/metabolismo , Triglicerídeos/metabolismo
5.
Nat Struct Mol Biol ; 27(12): 1185-1193, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33106658

RESUMO

The molecular function of Atg9, the sole transmembrane protein in the autophagosome-forming machinery, remains unknown. Atg9 colocalizes with Atg2 at the expanding edge of the isolation membrane (IM), where Atg2 receives phospholipids from the endoplasmic reticulum (ER). Here we report that yeast and human Atg9 are lipid scramblases that translocate phospholipids between outer and inner leaflets of liposomes in vitro. Cryo-EM of fission yeast Atg9 reveals a homotrimer, with two connected pores forming a path between the two membrane leaflets: one pore, located at a protomer, opens laterally to the cytoplasmic leaflet; the other, at the trimer center, traverses the membrane vertically. Mutation of residues lining the pores impaired IM expansion and autophagy activity in yeast and abolished Atg9's ability to transport phospholipids between liposome leaflets. These results suggest that phospholipids delivered by Atg2 are translocated from the cytoplasmic to the luminal leaflet by Atg9, thereby driving autophagosomal membrane expansion.

6.
Nat Commun ; 11(1): 4480, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32900992

RESUMO

Macroautophagy initiates by formation of isolation membranes, but the source of phospholipids for the membrane biogenesis remains elusive. Here, we show that autophagic membranes incorporate newly synthesized phosphatidylcholine, and that CTP:phosphocholine cytidylyltransferase ß3 (CCTß3), an isoform of the rate-limiting enzyme in the Kennedy pathway, plays an essential role. In starved mouse embryo fibroblasts, CCTß3 is initially recruited to autophagic membranes, but upon prolonged starvation, it concentrates on lipid droplets that are generated from autophagic degradation products. Omegasomes and isolation membranes emanate from around those lipid droplets. Autophagy in prolonged starvation is suppressed by knockdown of CCTß3 and is enhanced by its overexpression. This CCTß3-dependent mechanism is also present in U2OS, an osteosarcoma cell line, and autophagy and cell survival in starvation are decreased by CCTß3 depletion. The results demonstrate that phosphatidylcholine synthesis through CCTß3 activation on lipid droplets is crucial for sustaining autophagy and long-term cell survival.


Assuntos
Autofagia/fisiologia , Colina-Fosfato Citidililtransferase/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Animais , Autofagossomos/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Colina-Fosfato Citidililtransferase/antagonistas & inibidores , Colina-Fosfato Citidililtransferase/genética , Meios de Cultura , Ativação Enzimática , Técnicas de Silenciamento de Genes , Humanos , Gotículas Lipídicas/metabolismo , Camundongos , Modelos Biológicos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Fosfatidilcolinas/metabolismo
7.
Methods Mol Biol ; 2169: 43-52, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32548817

RESUMO

Fluorescence microscopy is currently one of the more powerful and versatile techniques available for biological studies. With conventional biological immunofluorescence microscopy, caveolin-1 (CAV1) is visualized as numerous small dots, which are often distributed as a linear array or along the edge of the cell. Although its presence, as well as that of other proteins, can be detected by conventional immunofluorescence microscopy, those results do not clarify whether two different proteins exist in the plasma membrane of a specimen or how they are distributed two-dimensionally. Here, we describe an unroofing procedure that clearly reveals CAV1 localization in a single plane of the plasma membrane and also demonstrate a super-resolution structured illumination microscopy technique for observation of CAV1 in the plasma membrane.

8.
Bioconjug Chem ; 31(6): 1611-1615, 2020 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-32378884

RESUMO

Membrane curvature plays a pivotal role in cellular life, including cellular uptake and membrane trafficking. The modulation of membrane curvature provides a novel means of manipulating cellular events. In this report, we show that a nine-residue amphiphilic peptide (R6W3) stimulates endocytic uptake by inducing membrane curvature. Curvature formation on cell membranes was confirmed by observing the cellular distribution of the curvature-sensing protein amphiphysin fused with a yellow fluorescent protein (Amp-YFP). Dot-like signals of Amp-YFP were visible following the addition of R6W3, suggesting curvature formation in cell membranes, leading to endocytic cup and vesicle formation. The promotion of endocytic uptake was confirmed using the endocytosis marker polydextran. Treatment of cells with R6W3 yielded a 4-fold dextran uptake compared with untreated cells. The amphiphilic helical structure of R6W3 was also crucial for R6W3-stimulated endocytic uptake.

9.
Semin Cell Dev Biol ; 2020 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-32169402

RESUMO

Lipid droplets (LDs) are not an inert storage of excessive lipids, but play various roles in cellular lipid metabolism. Autophagy involves several mechanisms for the degradation of cellular components, and is related to many aspects of lipid metabolism. LD and autophagic membranes often distribute in proximity, but their relationship is complex. LDs can be degraded by autophagy, but LDs are also generated as a result of autophagy or support the execution of autophagy. Moreover, several proteins crucial for autophagy were shown to affect different aspects of LD formation. This article aims to categorize this multifaceted and seemingly entangled LD-autophagy relationship and to discuss unresolved issues.

10.
EMBO J ; 39(8): e104120, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32128853

RESUMO

Protein prenylation is essential for many cellular processes including signal transduction, cytoskeletal reorganization, and membrane trafficking. Here, we identify a novel type of protein prenyltransferase, which we named geranylgeranyltransferase type-III (GGTase-III). GGTase-III consists of prenyltransferase alpha subunit repeat containing 1 (PTAR1) and the ß subunit of RabGGTase. Using a biotinylated geranylgeranyl analogue, we identified the Golgi SNARE protein Ykt6 as a substrate of GGTase-III. GGTase-III transfers a geranylgeranyl group to mono-farnesylated Ykt6, generating doubly prenylated Ykt6. The crystal structure of GGTase-III in complex with Ykt6 provides structural basis for Ykt6 double prenylation. In GGTase-III-deficient cells, Ykt6 remained in a singly prenylated form, and the Golgi SNARE complex assembly was severely impaired. Consequently, the Golgi apparatus was structurally disorganized, and intra-Golgi protein trafficking was delayed. Our findings reveal a fourth type of protein prenyltransferase that generates geranylgeranyl-farnesyl Ykt6. Double prenylation of Ykt6 is essential for the structural and functional organization of the Golgi apparatus.

11.
EMBO J ; 39(2): e102586, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31802527

RESUMO

ER-phagy, the selective autophagy of endoplasmic reticulum (ER), safeguards organelle homeostasis by eliminating misfolded proteins and regulating ER size. ER-phagy can occur by macroautophagic and microautophagic mechanisms. While dedicated machinery for macro-ER-phagy has been discovered, the molecules and mechanisms mediating micro-ER-phagy remain unknown. Here, we first show that micro-ER-phagy in yeast involves the conversion of stacked cisternal ER into multilamellar ER whorls during microautophagic uptake into lysosomes. Second, we identify the conserved Nem1-Spo7 phosphatase complex and the ESCRT machinery as key components for micro-ER-phagy. Third, we demonstrate that macro- and micro-ER-phagy are parallel pathways with distinct molecular requirements. Finally, we provide evidence that the ESCRT machinery directly functions in scission of the lysosomal membrane to complete the microautophagic uptake of ER. These findings establish a framework for a mechanistic understanding of micro-ER-phagy and, thus, a comprehensive appreciation of the role of autophagy in ER homeostasis.


Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Retículo Endoplasmático/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte , Membranas Intracelulares/metabolismo , Microautofagia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Homeostase , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae/metabolismo
12.
Proc Natl Acad Sci U S A ; 116(51): 26020-26028, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31776261

RESUMO

The voltage-sensing phosphatase (VSP) is a unique protein that shows voltage-dependent phosphoinositide phosphatase activity. Here we report that VSP is activated in mice sperm flagellum and generates a unique subcellular distribution pattern of PtdIns(4,5)P2 Sperm from VSP-/- mice show more Ca2+ influx upon capacitation than VSP+/- mice and abnormal circular motion. VSP-deficient sperm showed enhanced activity of Slo3, a PtdIns(4,5)P2-sensitive K+ channel, which selectively localizes to the principal piece of the flagellum and indirectly enhances Ca2+ influx. Most interestingly, freeze-fracture electron microscopy analysis indicates that normal sperm have much less PtdIns(4,5)P2 in the principal piece than in the midpiece of the flagellum, and this polarized PtdIns(4,5)P2 distribution disappeared in VSP-deficient sperm. Thus, VSP appears to optimize PtdIns(4,5)P2 distribution of the principal piece. These results imply that flagellar PtdIns(4,5)P2 distribution plays important roles in ion channel regulation as well as sperm motility.


Assuntos
Canais Iônicos/metabolismo , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Motilidade Espermática/fisiologia , Animais , Canais de Cálcio/metabolismo , Flagelos/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Masculino , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Knockout , Monoéster Fosfórico Hidrolases/genética , Cauda do Espermatozoide/metabolismo , Espermatozoides/metabolismo
13.
Proc Natl Acad Sci U S A ; 116(27): 13368-13373, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31217287

RESUMO

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.


Assuntos
Anoctaminas/metabolismo , Retículo Endoplasmático/metabolismo , Fosfatidilserinas/metabolismo , Animais , Calcimicina/farmacologia , Cálcio/metabolismo , Ionóforos de Cálcio/farmacologia , Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Membranas Intracelulares/metabolismo , Camundongos , Membrana Nuclear/metabolismo
14.
Oncogene ; 38(26): 5142-5157, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30894682

RESUMO

The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a transcriptional target of the lineage-survival oncogene NKX2-1/TTF-1 in lung adenocarcinomas. In addition to its kinase-dependent role, ROR1 functions as a scaffold protein to facilitate interaction between caveolin-1 (CAV1) and CAVIN1, and consequently maintains caveolae formation, which in turn sustains pro-survival signaling toward AKT from multiple receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR), MET (proto-oncogene, receptor tyrosine kinase), and IGF-IR (insulin-like growth factor receptor 1). Therefore, ROR1 is an attractive target for overcoming EGFR-TKI resistance due to various mechanisms such as EGFR T790M double mutation and bypass signaling from other RTKs. Here, we report that ROR1 possesses a novel scaffold function indispensable for efficient caveolae-dependent endocytosis. CAVIN3 was found to bind with ROR1 at a site distinct from sites for CAV1 and CAVIN1, a novel function required for proper CAVIN3 subcellular localization and caveolae-dependent endocytosis, but not caveolae formation itself. Furthermore, evidence of a mechanistic link between ROR1-CAVIN3 interaction and consequential caveolae trafficking, which was found to utilize a binding site distinct from those for ROR1 interactions with CAV1 and CAVIN1, with RTK-mediated pro-survival signaling towards AKT in early endosomes in lung adenocarcinoma cells was also obtained. The present findings warrant future study to enable development of novel therapeutic strategies for inhibiting the multifaceted scaffold functions of ROR1 in order to reduce the intolerable death toll from this devastating cancer.


Assuntos
Adenocarcinoma de Pulmão/patologia , Cavéolas/fisiologia , Endocitose , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pulmonares/patologia , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Animais , Células COS , Cavéolas/metabolismo , Sobrevivência Celular/genética , Células Cultivadas , Chlorocebus aethiops , Endocitose/genética , Células HEK293 , Células HeLa , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ligação Proteica/fisiologia , Células Sf9 , Transdução de Sinais/genética , Spodoptera
15.
Nat Commun ; 10(1): 1230, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30862813

RESUMO

The original version of this Article contained errors in the Abstract and Introduction, whereby CCTα was incorrectly defined as an abbreviation of CDP-choline diacylglycerol phosphotransferase α, instead of CTP:phosphocholine cytidylyltransferase α. This has now been corrected in both the PDF and HTML versions of the Article.

16.
Nat Commun ; 10(1): 473, 2019 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-30692541

RESUMO

The origin and physiological significance of lipid droplets (LDs) in the nucleus is not clear. Here we show that nuclear LDs in hepatocytes are derived from apolipoprotein B (ApoB)-free lumenal LDs, a precursor to very low-density lipoproprotein (VLDL) generated in the ER lumen by microsomal triglyceride transfer protein. ApoB-free lumenal LDs accumulate under ER stress, grow within the lumen of the type I nucleoplasmic reticulum, and turn into nucleoplasmic LDs by disintegration of the surrounding inner nuclear membrane. Oleic acid with or without tunicamycin significantly increases the formation of nucleoplasmic LDs, to which CDP-choline diacylglycerol phosphotransferase α (CCTα) is recruited, resulting in activation of phosphatidylcholine (PC) synthesis. Perilipin-3 competes with CCTα in binding to nucleoplasmic LDs, and thus, knockdown and overexpression of perilipin-3 increases and decreases PC synthesis, respectively. The results indicate that nucleoplasmic LDs in hepatocytes constitute a feedback mechanism to regulate PC synthesis in accordance with ER stress.


Assuntos
Núcleo Celular/metabolismo , Gotículas Lipídicas/metabolismo , Lipoproteínas/metabolismo , Fosfatidilcolinas/biossíntese , Precursores de Proteínas/metabolismo , Células A549 , Animais , Linhagem Celular Tumoral , Colina-Fosfato Citidililtransferase/metabolismo , Células HEK293 , Células HeLa , Hepatócitos/metabolismo , Humanos , Ácido Oleico/metabolismo , Perilipina-3/metabolismo , Ratos
17.
Curr Opin Cell Biol ; 57: 33-39, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30423517

RESUMO

New functionalities of phosphoinositides (PIs) are being revealed continuously, and the scale of the membrane area studied is becoming smaller, from the micrometer range like the entire surface of organelles to the nanometer range as in subdomains of organelles. Concurrently, function of less abundant PIs, such as PI(3,4)P2 and PI(3,5)P2, attracts increasing attention. In accordance with the progress, finer and more accurate information on PI distribution is required. The fluorescence biosensor method utilizing PI-binding domains and/or immunolabeling with anti-PI antibodies are used for this purpose in most studies but both methods are known to have caveats. In this article, we examined how PI distribution was defined in recent studies and discussed whether methodological uncertainty has any bearing on the results.


Assuntos
Organelas/química , Fosfatidilinositóis/análise , Animais , Transporte Biológico , Técnicas Biossensoriais , Membrana Celular/química , Cílios/química , Fluorescência , Imuno-Histoquímica
18.
EMBO J ; 37(21)2018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-30237312

RESUMO

PGAM5, a mitochondrial protein phosphatase that is genetically and biochemically linked to PINK1, facilitates mitochondrial division by dephosphorylating the mitochondrial fission factor Drp1. At the onset of mitophagy, PGAM5 is cleaved by PARL, a rhomboid protease that degrades PINK1 in healthy cells, and the cleaved form facilitates the engulfment of damaged mitochondria by autophagosomes by dephosphorylating the mitophagy receptor FUNDC1. Here, we show that the function and localization of PGAM5 are regulated by syntaxin 17 (Stx17), a mitochondria-associated membrane/mitochondria protein implicated in mitochondrial dynamics in fed cells and autophagy in starved cells. In healthy cells, loss of Stx17 causes PGAM5 aggregation within mitochondria and thereby failure of the dephosphorylation of Drp1, leading to mitochondrial elongation. In Parkin-mediated mitophagy, Stx17 is prerequisite for PGAM5 to interact with FUNDC1. Our results reveal that the Stx17-PGAM5 axis plays pivotal roles in mitochondrial division and PINK1/Parkin-mediated mitophagy.


Assuntos
Dinâmica Mitocondrial , Proteínas Mitocondriais/metabolismo , Mitofagia , Fosfoproteínas Fosfatases/metabolismo , Proteínas Qa-SNARE/metabolismo , Transdução de Sinais , Autofagossomos/metabolismo , Dinaminas , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Células HeLa , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Metaloproteases/genética , Metaloproteases/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Mitocondriais/genética , Fosfoproteínas Fosfatases/genética , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteólise , Proteínas Qa-SNARE/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
19.
Biochem Soc Trans ; 46(5): 1047-1054, 2018 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-30242116

RESUMO

The membrane raft has been a focus of intensive research for the past two decades. Liquid-ordered domains form in artificial liposomes containing sterol and saturated lipids, but their presence in living cell membranes has been controversial. The yeast vacuole is exceptional in that micron-sized raft-like domains form in the stationary phase and under several other conditions. The sterol content of the vacuole in the log phase is much lower than that of liposomes showing liquid-ordered domains, suggesting that sterols may need to be supplied to the vacuole for the raft-like domain formation. We will discuss how lipids and lipid domains are organized in the vacuolar membrane and examine whether evidence is strong enough to conclude that the observed micron-sized domains are rafts.


Assuntos
Lipídeos de Membrana/química , Microdomínios da Membrana/química , Saccharomyces cerevisiae/química , Esteróis/química , Vacúolos/química , Transporte Biológico , Membrana Celular/química , Colesterol/química , Lipossomos/química , Osmose , Domínios Proteicos , Estresse Fisiológico
20.
Methods Mol Biol ; 1804: 231-239, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29926412

RESUMO

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.


Assuntos
Gangliosídeos/química , Microscopia Imunoeletrônica/métodos , Animais , Técnicas de Cultura de Células , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Fibroblastos/metabolismo , Técnica de Fratura por Congelamento , Congelamento , Camundongos , Coloração e Rotulagem
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