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

Bjoern von Einem³  Petra Weber²  Michael Wagner²  Martina Malnar¹  Marko Kosicek¹  Silva Hecimovic¹  Christine A. F. von Arnim³ 

[1]Division of Molecular Medicine, Rudjer Boskovic Institute, Bijenicka 54, 10000 Zagreb, Croatia

[2] E-Mails:

[3]Institut für Angewandte Forschung, Hochschule Aalen, Anton-Huber Str. 21, 73430 Aalen, Germany

[4] E-Mails:

[5]Department of Experimental Neurology, Ulm University, Helmholtz Str. 8/1, 89081 Ulm, Germany

[6] E-Mails:

关键词: FRET;    cholesterol;    APP;    BACE1;    NPC;    TIRFM;    neurodegeneration;    laurdan;    generalized polarization;   

DOI  :  10.3390/ijms131215801

来源: mdpi

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2012 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland.