Using GRAM Domain-Based Biosensors to Monitor Accessible Cholesterol Distribution in Live Cells

Cholesterol is a vital component of cellular membranes, playing a crucial role in membrane integrity and cell signaling. It is unevenly distributed among different cellular compartments, with the highest enrichment in the plasma membrane (PM). Dysregulation of cellular cholesterol distribution is linked to various disorders, including cardiovascular and neurological diseases. Regulated transport of a specific pool of cholesterol, known as accessible cholesterol, is essential for maintaining cellular cholesterol distribution, yet visualization tools for accessible cholesterol in live cells are limited. Here, we introduce methods for visualizing accessible cholesterol using biosensors based on the GRAM domain of GRAMD1s/Asters (GRAMD1a, GRAMD1b, and GRAMD1c). We present two variants of the GRAM domain of GRAMD1b, GRAM-H, and GRAM-W, with different affinities for cholesterol, enabling the detection of both increase and decrease of accessible cholesterol in the PM. Additionally, we demonstrate the successful coupling of dimerization-dependent fluorescent protein (ddFP) with these biosensors to monitor accessible PM cholesterol levels in cell populations using flow cytometry. These GRAM domain-based biosensors complement existing tools, providing new insights into the molecular mechanisms underlying accessible cholesterol distribution.