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

von Einem, Bjorn; Weber, Petra; Wagner, Michael; Malnar, Martina; Košiček, Marko; Hećimović, Silva; von Arnim, Christine; 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. International Journal of Molecular Sciences, 13 (12). pp. 15801-15812. ISSN 1661-6596

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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.

Item Type:	Article
Uncontrolled Keywords:	FRET; cholesterol; APP; BACE1; NPC; TIRFM; neurodegeneration; Laurdan; generalized polarization
Subjects:	NATURAL SCIENCES > Biology
Divisions:	Division of Molecular Medicine
Projects:	Project title Project leader Project code Project type Mehanizam djelovanja kolesterola u nastanku Alzheimerove bolesti- Silva Katušić Hećimović 098-0982522-2525 MZOS
Depositing User:	Dr. sc. Silva Katušić Hećimović
Date Deposited:	04 Mar 2020 09:06
URI:	http://fulir.irb.hr/id/eprint/5321
DOI:	10.3390/ijms131215801

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