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Influence of ABCA1 and ABCA7 on the Lipid Microenvironment of the Plasma Membrane

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Influence of ABCA1 and ABCA7 on the Lipid Microenvironment of the Plasma Membrane I Influence of ABCA1 and ABCA7 on the lipid microenvironment of the plasma membrane Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) im Fach Biologie eingereicht an der Mathematisch-Naturwissenschaftlichen Fakultät I der Humboldt-Universität zu Berlin von Dottore Magistrale in Biotecnologie Industriali Anna Pia Plazzo geb. 17.10.1981 in San Giovanni Rotondo, Italien Präsident der Humboldt-Universität zu Berlin Prof. Dr. Dr. h.c. Christoph Markschies Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I Prof. Dr. Lutz-Helmut Schön Gutachter: 1. Prof. Dr. Andreas Herrmann 2. Prof. Dr. Thomas Günther-Pomorski 3. Prof. Dr. Thomas Eitinger Tag der mündlichen Prüfung: 12.06.2009 II Alla mia famiglia III Zusammenfassung Der ABC-Transporter ABCA1 ist unmittelbar in die zelluläre Lipidhomeostasie einbezogen, in dem er die Freisetzung von Cholesterol an plasmatische Rezeptoren, wie ApoA-I, vermittelt. Trotz intensiver Untersuchungen ist dieser molekulare Mechanismus nicht verstanden. Verschiedene Studien deuten daraufhin, dass durch die Aktivität von ABCA1 bedingte Veränderungen in der Lipidphase der äußeren Hälfte der Plasmamembran (PM) wichtig für die Freisetzung des Cholesterols sind. In der vorliegenden Arbeit wird die Lipidumgebung von ABCA1 in der PM lebender Säugetierzellen unter Anwendung der Fluoreszenzlebenszeitmikroskopie von fluoreszierenden Lipidsonden untersucht. Es wurde eine breite Verteilung der Fluoreszenzlebenszeiten der Sonden gefunden, die sensitiv gegenüber Veränderungen der lateralen und transversalen Organisation der Lipide ist. Im Einklang mit Studien an riesengroßen unilamellaren Vesikeln und Plasmamembranvesikeln weisen unsere Ergebnisse die Existenz einer größeren Vielfalt submikroskopischer Lipiddomänen auf. Die FLIM-Untersuchungen an ABCA1 exprimierenden HeLa-Zellen weisen eine die Lipidphase destabilisierende Funktion des Transportes aus. Dieses wurde unterstützt durch die Lipidanalyse von Fraktionen der PM. Auf der Basis unserer Untersuchungen und früheren Daten stellen wir die Hypothese auf, dass die Exponierung von Phosphatidylserin (PS) auf der Zelloberfläche ein zentrales Ereignis der ABCA1 bedingten Veränderungen ist. Allerdings zeigen vergleichende Studien an ABCA7 exprimierenden Zellen, dass dies nicht ausreicht, um die ABCA1 verursachten Veränderungen in der Lipidpackung der PM zu erklären. Unsere Ergebnisse beweisen, dass die Fähigkeit von ABCA1, den Cholesterolefflux zu vermitteln, auf durch den Transporter bedingte Veränderungen in der LP der PM zurückzuführen sind, die unabhängig von der Bindung von ApoA-1 sind und dieser vorausgehen. Diese Veränderungen sind notwendig für die Lipidierung von ApoA-1 und der Generierung von HDL-Partikeln. Schlagwörter: ABCA1, Plasmamembran, Cholesterol, Lipiddomänen, FLIM IV Abstract The ABCA1 transporter organizes cellular lipid homeostasis by promoting the release of cholesterol to plasmatic acceptors such as ApoA-I. Despite intensive investigation, the molecular mechanism of such a process has not yet been clarified. In the present study we report on the analysis of the ABCA1 lipid microenvironment at the plasma membrane of living cells, by a novel approach based on fluorescence lifetime imaging microscopy (FLIM). In the plasma membrane of mammalian cells, a broad fluorescence lifetime distribution sensitive to treatments interfering with the membrane lateral and transbilayer organization was found. In agreement with investigations in giant unilamellar vesicles and giant plasma membrane vesicles, our results are consistent with the existence of a large variety of submicroscopic lipid domains. Based on that, FLIM in HeLa cells expressing ABCA1 revealed the destabilizing function of the transporter on the lipid arrangement at the membrane, indicating that lipid packing was a primary target of ABCA1 activity. This was corroborated by the analysis of plasma membrane fractions isolated by density fractionation. On the basis of our analysis and previous data, we speculate that the exposure of phosphatidylserine on the cell surface is a central event for ABCA1-dependent modifications. However, a comparative study of cells expressing ABCA7, the member of the ABCA subfamily with the highest homology to ABCA1, revealed that exposure of PS alone cannot account for the detected effects. Collectively, our data suggest that the ability of ABCA1 to promote cholesterol efflux is independent and precedes its actual binding to ApoA-I. Rather, ABCA1-induced plasma membrane modifications are necessary for the lipidation of ApoA-I and the generation of high density lipoprotein particles. Key Words: ABCA1, plasma membrane, cholesterol, lipid domain, fluorescence lifetime imaging V List of abbreviations ABC ATP binding cassette Lo Liquid ordered ACAT Acyl-CoA cholesteryl acyl transferase LXR Liver X receptor MALDI- Matrix assisted laser desorption ADP Adenosine diphosphate TOF ionization-time of flight ALD Adrenoleukodystrophy MβCD Methyl-β-cyclodextrin Apo Apolipoprotein MDR Multi drug resistance ATP Adenosine triphosphate Mg2+ Magnesium BSA Bovine serum albumine MLV Multilamellar vesicle BSEP Bile salt efflux pump MRP Multi drug resistance protein Ca2+ Calcium MS Mass spectrometry [N-(7-nitrobenz-2-oxa-1,3-diazol- cAMP Cyclic adenosine monophosphate NBD 4-yl)amino] CE Cholesterol esters NBF Nucleotide binding fold Cer Ceramide PA Phosphatidic acid Cystic fibrosis transmembrane CFTR PC Phosphatidylcholine conductance regulator CHO Chinese hamster ovary Pgp P glycoprotein CNX Calnexin PKA, C Protein kinase A, C ConA Concanavalin A PL Phospholipids CVD Cardiovascular disease PM Plasma membrane DHB 2,5-dihydroxybenzoic acid PO Palmitoyl-oleoyl Peroxisome proliferator-activated DO Di-oleoyl PPAR receptor DP Di-palmitoyl PS Phosphatidylserine DRM Detergent resistant membranes PSM N-palmitoyl-D-sphingomyelin ER Endoplasmic reticulum RCT Reverse cholesterol transport FL Flotillin Rho Rhodamine Fluorescence lifetime imaging FLIM RXR Retinoid X receptor microscopy Fluorescence recovery after FRAP SD Standard deviation photobleaching FRET Förster resonance energy transfer SEM Standard error of the mean GP Generalized polarization SM Sphingomyelin GPMV Giant plasma membrane vesicle SMase Sphingomyelinase GPI Glycosylphosphatidylinositol SSM N-stearoyl-D-sphingomyelin Transporter associated with GUV Giant unilamellar vesicle TAP antigen processing HDL High density lipoprotein TCR T cell receptor HEK Human embryonic kidney TD Tangier disease IDL Intermediate density lipoprotein TfR Transferrin receptor LacCer Lactosyl ceramide TMD Transmembrane domain Ld Liquid disordered VLDL Very low density lipoprotein LDL Low density lipoprotein WHAM Wisconsin hypoalpha mutant VI Index Zusammenfassung .............................................................................................................. III Abstract ................................................................................................................................IV List of abbreviations............................................................................................................. V 1 Introduction ...............................................................................................1 1.1 The plasma membrane .................................................................................................1 1.1.1 The repertoire of membrane lipids..................................................................................... 1 1.1.2 Phase separation in model membranes .............................................................................. 3 1.1.3 Lipid rafts in the plasma membrane................................................................................... 5 1.1.4 Lipid asymmetry and protein-mediated lipid translocation ............................................... 6 1.2 ATP-Binding Cassette (ABC) transporters ................................................................8 1.2.1 General features ................................................................................................................. 8 1.2.2 Domain organization and transport cycle........................................................................... 9 1.2.3 ABC transporters and the transport of lipids.................................................................... 10 1.2.4 The ABCA subfamily....................................................................................................... 12 1.2.4.1 ABCA1: the key controller of cholesterol efflux........................................................................14 1.2.4.2 ABCA7: a molecule with unknown function..............................................................................14 1.3 ABCA1 .........................................................................................................................16 1.3.1 General features ............................................................................................................... 16 1.3.1.1 Once upon a time…....................................................................................................................16 1.3.1.2 Topological model ......................................................................................................................17 1.3.1.3 Regulation of ABCA1 ................................................................................................................18 1.3.1.4 Tissue and intracellular distribution of ABCA1 .........................................................................19
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