Elucidating the mechanisms of intracellular cholesterol homeostasis using a novel cholesterol biosensor

Abstract

Cholesterol plays a significant role in maintaining membrane integrity and cell signaling. Dysregulation of the distribution of cellular cholesterol is associated with numerous diseases, including cardiovascular and neurological diseases. While regulated transport of a specific pool of cholesterol, known as “accessible cholesterol” contributes to the maintenance of cellular cholesterol distribution and homeostasis, tools to visualize accessible cholesterol in live cells remain limited. Here, we engineered a highly sensitive accessible cholesterol biosensor by utilizing the cholesterol-sensing domain (the GRAM domain) of an evolutionarily conserved lipid transfer protein, GRAMD1b. Using this novel cholesterol biosensor, which we call GRAM-W, we successfully visualized the distribution of accessible cholesterol in many different cell types in real time, including human keratinocytes and iPSC-derived neurons, and showed differential dependences on cholesterol biosynthesis and uptake for maintaining levels of accessible cholesterol. Using GRAM-W, we found that keratinocytes maintain levels of accessible cholesterol in the plasma membrane largely through de novo cholesterol biosynthesis. They employ the evolutionarily conserved lipid transfer protein, namely oxysterol-binding protein (OSBP), for the maintenance of accessible cholesterol at the plasma membrane. Finally, we combined GRAM-W with a dimerization-dependent fluorescent protein (ddFP) and established a novel strategy for the ultrasensitive detection of accessible plasma membrane cholesterol. These new tools will allow us to gain important insights into the molecular mechanisms of cellular cholesterol transport mediated by various lipid transfer proteins at membrane contact sites, paving the way toward uncovering the pathogenesis of diseases related to dysregulation of cholesterol homeostasis