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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 (CROSBI ID 187785)

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von Einem, Bjorn ; Weber, Petra ; Wagner, Michael ; Malnar, Martina ; Košiček, Marko ; Hećimović, Silva ; von Arnim, Christine ; Schneckenburger, Herbert 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 (2012), 12; 15801-15812. doi: 10.3390/ijms131215801

Podaci o odgovornosti

von Einem, Bjorn ; Weber, Petra ; Wagner, Michael ; Malnar, Martina ; Košiček, Marko ; Hećimović, Silva ; von Arnim, Christine ; Schneckenburger, Herbert

engleski

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

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.

FRET ; cholesterol ; APP ; BACE1 ; NPC ; TIRFM ; neurodegeneration ; Laurdan ; generalized polarization

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Podaci o izdanju

13 (12)

2012.

15801-15812

objavljeno

1422-0067

10.3390/ijms131215801

Povezanost rada

Biologija

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