The LDL receptor is regulated by membrane cholesterol as revealed by fluorescence fluctuation analysis

Morales, Sebastian V., Mahmood, Ahmad, Pollard, Jacob, Mayne, Janice, Figeys, Daniel ORCID: https://orcid.org/0000-0002-5373-7546 and Wiseman, Paul W. (2023) The LDL receptor is regulated by membrane cholesterol as revealed by fluorescence fluctuation analysis. Biophysical Journal, 122 (18). pp. 3783-3797. ISSN 0006-3495

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Abstract

Membrane cholesterol-rich domains have been shown to be important for regulating a range of membrane protein activities. Low-density lipoprotein receptor (LDLR)-mediated internalization of cholesterol-rich LDL particles is tightly regulated by feedback mechanisms involving intracellular sterol sensors. Since LDLR plays a role in maintaining cellular cholesterol homeostasis, we explore the role that membrane domains may have in regulating LDLR activity. We expressed a fluorescent LDLR-mEGFP construct in HEK293T cells and imaged the unligated receptor or bound to an LDL/DiI fluorescent ligand using total internal reflection fluorescence microscopy. We studied the receptor's spatiotemporal dynamics using fluorescence fluctuation analysis methods. Image cross correlation spectroscopy reveals a lower LDL-to-LDLR binding fraction when membrane cholesterol concentrations are augmented using cholesterol esterase, and a higher binding fraction when the cells are treated with methyl-β-cyclodextrin) to lower membrane cholesterol. This suggests that LDLR's ability to metabolize LDL particles is negatively correlated to membrane cholesterol concentrations. We then tested if a change in activity is accompanied by a change in membrane localization. Image mean-square displacement analysis reveals that unligated LDLR-mEGFP and ligated LDLR-mEGFP/LDL-DiI constructs are transiently confined on the cell membrane, and the size of their confinement domains increases with augmented cholesterol concentrations. Receptor diffusion within the domains and their domain-escape probabilities decrease upon treatment with methyl-β-cyclodextrin, consistent with a change in receptor populations to more confined domains, likely clathrin-coated pits. We propose a feedback model to account for regulation of LDLR within the cell membrane: when membrane cholesterol concentrations are high, LDLR is sequestered in cholesterol-rich domains. These LDLR populations are attenuated in their efficacy to bind and internalize LDL. However, when membrane cholesterol levels drop, LDL has a higher binding affinity to its receptor and the LDLR transits to nascent clathrin-coated domains, where it diffuses at a slower rate while awaiting internalization.

Item Type:	Article
Additional Information:	Supporting material can be found online at https://doi.org/10.1016/j.bpj. 2023.08.005.
Uncontrolled Keywords:	biophysics ,/dk/atira/pure/subjectarea/asjc/1300/1304
Faculty \ School:	Faculty of Medicine and Health Sciences > Norwich Medical School
UEA Research Groups:	Faculty of Medicine and Health Sciences > Research Centres > Metabolic Health
Related URLs:	https://www.scopus.com/pages/publication...
Depositing User:	LivePure Connector
Date Deposited:	09 Mar 2026 11:30
Last Modified:	18 Jun 2026 20:55
URI:	https://ueaeprints.uea.ac.uk/id/eprint/102264
DOI:	10.1016/j.bpj.2023.08.005

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