{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,18]],"date-time":"2026-01-18T15:31:35Z","timestamp":1768750295275,"version":"3.49.0"},"reference-count":62,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2012,11,26]],"date-time":"2012-11-26T00:00:00Z","timestamp":1353888000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/3.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>F\u00f6rster resonance energy transfer (FRET) -based techniques have recently been applied to study the interactions between \u03b2-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\u2019s 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\u2212\/\u2212), 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\u2212\/\u2212 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\u2212\/\u2212 cells incubated with the fluorescence marker laurdan were performed to determine membrane stiffness of plasma- and intracellular-membranes. CHO-NPC1\u2212\/\u2212 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\u2212\/\u2212. 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.<\/jats:p>","DOI":"10.3390\/ijms131215801","type":"journal-article","created":{"date-parts":[[2012,11,26]],"date-time":"2012-11-26T11:24:24Z","timestamp":1353929064000},"page":"15801-15812","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Cholesterol-Dependent Energy Transfer between Fluorescent Proteins\u2014Insights into Protein Proximity of APP and BACE1 in Different Membranes in Niemann-Pick Type C Disease Cells"],"prefix":"10.3390","volume":"13","author":[{"given":"Bjoern","family":"Von Einem","sequence":"first","affiliation":[{"name":"Department of Experimental Neurology, Ulm University, Helmholtz Str. 8\/1, 89081 Ulm, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Petra","family":"Weber","sequence":"additional","affiliation":[{"name":"Institut f\u00fcr Angewandte Forschung, Hochschule Aalen, Anton-Huber Str. 21, 73430 Aalen, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Michael","family":"Wagner","sequence":"additional","affiliation":[{"name":"Institut f\u00fcr Angewandte Forschung, Hochschule Aalen, Anton-Huber Str. 21, 73430 Aalen, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Martina","family":"Malnar","sequence":"additional","affiliation":[{"name":"Division of Molecular Medicine, Rudjer Boskovic Institute, Bijenicka 54, 10000 Zagreb, Croatia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marko","family":"Kosicek","sequence":"additional","affiliation":[{"name":"Division of Molecular Medicine, Rudjer Boskovic Institute, Bijenicka 54, 10000 Zagreb, Croatia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Silva","family":"Hecimovic","sequence":"additional","affiliation":[{"name":"Division of Molecular Medicine, Rudjer Boskovic Institute, Bijenicka 54, 10000 Zagreb, Croatia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Christine","family":"VonArnim","sequence":"additional","affiliation":[{"name":"Department of Experimental Neurology, Ulm University, Helmholtz Str. 8\/1, 89081 Ulm, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Herbert","family":"Schneckenburger","sequence":"additional","affiliation":[{"name":"Institut f\u00fcr Angewandte Forschung, Hochschule Aalen, Anton-Huber Str. 21, 73430 Aalen, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2012,11,26]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"105","DOI":"10.1097\/00041433-200104000-00003","article-title":"Cholesterol metabolism in the brain","volume":"12","author":"Dietschy","year":"2001","journal-title":"Curr. 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