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

doi:10.1016/j.jlr.2022.100178

Ha Pham, Indira Singaram + Show 6 more

Open Access

https://doi.org/10.1016/j.jlr.2022.100178

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Abstract

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 the 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.

Highlights

Cholesterol is a major and essential component of the mammalian cell membranes [1,2,3,4]
Site-specific functions of cellular cholesterol probed with site-specific depletion systems We previously reported that stimulation of HEK293T cells with Wnt3a induced the increase in [Chol]i that leads to inner leaflet of the PM (IPM) recruitment of Dvl2 in a cholesterol-dependent manner, leading to the activation of the β-catenin-dependent canonical Wnt signaling pathway [19,20,21, 25]
The method has long served as a standard protocol for testing the involvement of cholesterol in various cellular processes for lack of alternatives, the method suffers from major shortcomings, including severe deformation of the cell membranes leading to cell death and non-specific alteration of cell physiology and signaling processes

Summary

Introduction

Cholesterol is a major and essential component of the mammalian cell membranes [1,2,3,4]. It has been reported that cholesterol mediates cell signaling events at lysosomes by activating the mechanistic target of rapamycin complex (mTORC1) via an SLC38A9-Niemann-Pick C1 (NPC1) signaling complex [22]. Together, these reports underscore the need to develop a robust method to precisely control the cholesterol level in a sitespecific manner to determine cellular functions of cholesterol. The cellular cholesterol levels have been commonly manipulated by two methods; chemical extraction and enrichment of cholesterol by methyl-β-cyclodextrin (MβCD) and MβCD-cholesterol adducts, respectively [23] and inhibition of cellular de novo cholesterol biosynthesis by statins [24] Despite their popularity due to experimental convenience, these methods offer no spatiotemporal control and cause nonspecific cholesterol depletion that could exert major deleterious effects on cell physiology

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