Naito Tomoki, Na Minyoung, Koh Dylan Hong Zheng, Saheki Yasunori
Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore.
Institute of Resource Development and Analysis, Kumamoto University, Kumamoto, Japan.
Methods Mol Biol. 2025;2888:67-82. doi: 10.1007/978-1-0716-4318-1_6.
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.
胆固醇是细胞膜的重要组成部分，在膜完整性和细胞信号传导中发挥着关键作用。它在不同细胞区室中分布不均，在质膜（PM）中富集程度最高。细胞胆固醇分布失调与包括心血管和神经疾病在内的各种疾病有关。特定胆固醇池（称为可及胆固醇）的调节转运对于维持细胞胆固醇分布至关重要，但活细胞中可及胆固醇的可视化工具有限。在这里，我们介绍了使用基于GRAMD1s/Asters（GRAMD1a、GRAMD1b和GRAMD1c）的GRAM结构域的生物传感器来可视化可及胆固醇的方法。我们展示了GRAMD1b的GRAM结构域的两种变体，GRAM-H和GRAM-W，它们对胆固醇具有不同的亲和力，能够检测质膜中可及胆固醇的增加和减少。此外，我们证明了二聚化依赖性荧光蛋白（ddFP）与这些生物传感器的成功偶联，以使用流式细胞术监测细胞群体中质膜可及胆固醇水平。这些基于GRAM结构域的生物传感器补充了现有工具，为可及胆固醇分布的分子机制提供了新的见解。
