Int. J. Mol. Sci. 2012, 13(12), 15801-15812; doi:10.3390/ijms131215801
Article

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

1 Department of Experimental Neurology, Ulm University, Helmholtz Str. 8/1, 89081 Ulm, Germany 2 Institut für Angewandte Forschung, Hochschule Aalen, Anton-Huber Str. 21, 73430 Aalen, Germany 3 Division of Molecular Medicine, Rudjer Boskovic Institute, Bijenicka 54, 10000 Zagreb, Croatia
* Author to whom correspondence should be addressed.
Received: 18 October 2012; in revised form: 14 November 2012 / Accepted: 15 November 2012 / Published: 26 November 2012
(This article belongs to the Special Issue Förster Resonance Energy Transfer (FRET))
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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.
Keywords: FRET; cholesterol; APP; BACE1; NPC; TIRFM; neurodegeneration; laurdan; generalized polarization

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MDPI and ACS Style

von Einem, B.; Weber, P.; Wagner, M.; Malnar, M.; Kosicek, M.; Hecimovic, S.; vonArnim, C.A.F.; Schneckenburger, H. 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. Int. J. Mol. Sci. 2012, 13, 15801-15812.

AMA Style

von Einem B, Weber P, Wagner M, Malnar M, Kosicek M, Hecimovic S, vonArnim CAF, Schneckenburger H. 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. International Journal of Molecular Sciences. 2012; 13(12):15801-15812.

Chicago/Turabian Style

von Einem, Bjoern; Weber, Petra; Wagner, Michael; Malnar, Martina; Kosicek, Marko; Hecimovic, Silva; vonArnim, Christine A.F.; Schneckenburger, Herbert. 2012. "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." Int. J. Mol. Sci. 13, no. 12: 15801-15812.

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