Shi Xiaojun, Kohram Maryam, Zhuang Xiaodong, Smith Adam W
Institute of Advanced Organic Materials, School of Chemistry and Chemical Engineering, Shanghai Jiao Tong University , 315 Jiangong Building, 800 Dongchuan Road, Shanghai 200240, China.
Langmuir. 2016 Feb 23;32(7):1732-41. doi: 10.1021/acs.langmuir.5b02814. Epub 2016 Feb 12.
Phosphatidylinositol phosphate (PIP) lipids are critical to many cell signaling pathways, in part by acting as molecular beacons that recruit peripheral membrane proteins to specific locations within the plasma membrane. Understanding the biophysics of PIP-protein interactions is critical to developing a chemically detailed model of cell communication. Resolving such interactions is challenging, even in model membrane systems, because of the difficulty in preparing PIP-containing membranes with high fluidity and integrity. Here we report on a simple, vesicle-based protocol for preparing asymmetric supported lipid bilayers in which fluorescent PIP lipid analogues are found only on the top leaflet of the supported membrane facing the bulk solution. With this asymmetric distribution of lipids between the leaflets, the fluorescent signal from the PIP lipid analogue reports directly on interactions between the peripheral molecules and the top leaflet of the membrane. Asymmetric PIP-containing bilayers are an ideal platform to investigate the interaction of PIP with peripheral membrane proteins using fluorescence-based imaging approaches. We demonstrate their usefulness here with a combined fluorescence correlation spectroscopy and single particle tracking study of the interaction between PIP2 lipids and a polycationic polymer, quaternized polyvinylpyridine (QPVP). With this approach we are able to quantify the microscopic features of the mobility coupling between PIP2 lipids and polybasic QPVP. With single particle tracking we observe individual PIP2 lipids switch from Brownian to intermittent motion as they become transiently trapped by QPVP.
磷脂酰肌醇磷酸（PIP）脂质对许多细胞信号通路至关重要，部分原因是它们作为分子信标，将外周膜蛋白招募到质膜内的特定位置。了解PIP-蛋白相互作用的生物物理学对于建立细胞通讯的化学详细模型至关重要。即使在模型膜系统中，解析这种相互作用也具有挑战性，因为制备具有高流动性和完整性的含PIP膜存在困难。在此，我们报告一种基于囊泡的简单方案，用于制备不对称支撑脂质双层，其中荧光PIP脂质类似物仅存在于面向本体溶液的支撑膜的顶层小叶上。通过小叶间脂质的这种不对称分布，PIP脂质类似物的荧光信号直接报告外周分子与膜顶层小叶之间的相互作用。含不对称PIP的双层是使用基于荧光的成像方法研究PIP与外周膜蛋白相互作用的理想平台。我们在此通过荧光相关光谱和单粒子追踪相结合的研究，证明了它们在PIP2脂质与聚阳离子聚合物季铵化聚乙烯吡啶（QPVP）相互作用研究中的有用性。通过这种方法，我们能够量化PIP2脂质与多碱性QPVP之间迁移率耦合的微观特征。通过单粒子追踪，我们观察到单个PIP2脂质在被QPVP短暂捕获时从布朗运动转变为间歇运动。
