title: Cholesterol-dependent energy transfer between fluorescent proteins-insights into protein proximity of APP and BACE1 in different membranes in Niemann-Pick type C disease cells.
abstract: Förster resonance energy transfer (FRET) -based techniques have recently been applied to study the interactions between β-site APP-cleaving enzyme-GFP (BACE1-GFP) and amyloid precursor protein-mRFP (APP-mRFP) in U373 glioblastoma cells. In this context, the role of APP-BACE1 proximity in Alzheimer's disease (AD) pathogenesis has been discussed. FRET was found to depend on intracellular cholesterol levels and associated alterations in membrane stiffness. Here, NPC1 null cells (CHO-NPC1-/-), exhibiting increased cholesterol levels and disturbed cholesterol transport similar to that observed in Niemann-Pick type C disease (NPC), were used to analyze the influence of altered cholesterol levels on APP-BACE1 proximity. Fluorescence lifetime measurements of whole CHO-wild type (WT) and CHO-NPC1-/- cells (EPI-illumination microscopy), as well as their plasma membranes (total internal reflection fluorescence microscopy, TIRFM), were performed. Additionally, generalized polarization (GP) measurements of CHO-WT and CHO-NPC1-/- cells incubated with the fluorescence marker laurdan were performed to determine membrane stiffness of plasma- and intracellular-membranes. CHO-NPC1-/- cells showed higher membrane stiffness at intracellular- but not plasma-membranes, equivalent to cholesterol accumulation in late endosomes/lysosomes. Along with higher membrane stiffness, the FRET efficiency between BACE1-GFP and APP-mRFP was reduced at intracellular membranes, but not within the plasma membrane of CHO-NPC1-/-. Our data show that FRET combined with TIRF is a powerful technique to determine protein proximity and membrane fluidity in cellular models of neurodegenerative diseases.
authors: Department of Experimental Neurology, Ulm University, Helmholtz Str. 8/1, 89081 Ulm, Germany. herbert.schneckenburger@htw-aalen.de.
authors: Bjoern
authors: B
authors: von Einem
authors: Petra
authors: P
authors: Weber
authors: Michael
authors: M
authors: Wagner
authors: Martina
authors: M
authors: Malnar
authors: Marko
authors: M
authors: Kosicek
authors: Silva
authors: S
authors: Hecimovic
authors: Christine A F von
authors: CA
authors: Arnim
authors: Herbert
authors: H
authors: Schneckenburger
mesh_terms: D053829:Amyloid Precursor Protein Secretases / Q000235:genetics / Q000378:metabolism*; D016564:Amyloid beta-Protein Precursor / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D002352:Carrier Proteins; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D002784:Cholesterol / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D017353:Gene Deletion; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; D000077296:Niemann-Pick C1 Protein; D052556:Niemann-Pick Disease, Type C / Q000235:genetics / Q000378:metabolism* / Q000473:pathology
chemical_list: C545487:APP protein, human; D016564:Amyloid beta-Protein Precursor; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; C106881:NPC1 protein, human; D000077296:Niemann-Pick C1 Protein; D049452:Green Fluorescent Proteins; D002784:Cholesterol; D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human