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 |
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Authors: | Bjoern von Einem Petra Weber Michael Wagner Martina Malnar Marko Kosicek Silva Hecimovic Christine A F von Arnim Herbert Schneckenburger |
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Affiliation: | 1.Department of Experimental Neurology, Ulm University, Helmholtz Str. 8/1, 89081 Ulm, Germany; E-Mails: (B.E.); (C.A.F.A.);2.Institut für Angewandte Forschung, Hochschule Aalen, Anton-Huber Str. 21, 73430 Aalen, Germany; E-Mails: (P.W.); (M.W.);3.Division of Molecular Medicine, Rudjer Boskovic Institute, Bijenicka 54, 10000 Zagreb, Croatia; E-Mails: (M.M.); (M.K.); (S.H.) |
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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. |
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Keywords: | FRET cholesterol APP BACE1 NPC TIRFM neurodegeneration laurdan generalized polarization |
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