Development of a novel spatiotemporal depletion system for cellular cholesterol

Ha Pham, Indira Singaram, Jiachen Sun, Arthur Ralko, Madalyn Puckett, Ashutosh Sharma, Alice Vrielink, Wonhwa Cho

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3 Citations (Scopus)


Cholesterol is an essential component of mammalian cell membranes whose subcellular concentration and function are tightly regulated by de novo biosynthesis, transport, and storage. Although recent reports have suggested diverse functions of cellular cholesterol in different subcellular membranes, systematic investigation of its site-specific roles has been hampered by the lack of a methodology for spatiotemporal manipulation of cellular cholesterol levels. Here we report development of a new cholesterol depletion system that allows for spatiotemporal manipulation of intracellular cholesterol levels. This system utilizes a genetically-encoded cholesterol oxidase whose intrinsic membrane binding activity is engineered in such a way that its membrane targeting can be controlled in a spatiotemporally specific manner via chemically-induced dimerization. In combination with in situ quantitative imaging of cholesterol and signaling activity measurements, this system allows for unambiguous determination of site-specific functions of cholesterol in different membranes, including the plasma membrane and the lysosomal membrane.

Original languageEnglish
Article number100178
JournalJournal of Lipid Research
Issue number3
Publication statusPublished - Mar 2022


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