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

1email, 2email, 2email, 3email, 3email, 3email, 1email and 2,* email
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))
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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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