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 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:	http://www.scopus.com/inward/record.url?...
Depositing User:	LivePure Connector
Date Deposited:	09 Mar 2026 11:30
Last Modified:	15 Mar 2026 07:30
URI:	https://ueaeprints.uea.ac.uk/id/eprint/102264
DOI:	10.1016/j.bpj.2023.08.005

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