Owen Dylan M, Williamson David, Magenau Astrid, Gaus Katharina
Centre for Vascular Research, University of New South Wales, Sydney, Australia.
Methods Enzymol. 2012;504:221-35. doi: 10.1016/B978-0-12-391857-4.00011-2.
It is now recognized that the plasma membrane is not homogeneous but instead contains a variety of membrane microdomains. These include lipid microdomains (lipid rafts) and protein clusters which exist on a range of size and time scales but are often small and short-lived. The small size and dynamic nature of membrane domains has made them difficult to study by conventional fluorescence microscopy approaches. Photoactivated localization microscopy (PALM) is a super-resolution technique capable of localizing the positions of individual molecules with tens of nanometers precision. Here, we describe a method for imaging membrane proteins using PALM, including live-cell PALM, to detect the molecular clustering of plasma membrane proteins using a statistical cluster-analysis method based on Ripley's K-function. While the method is applicable to a wide variety of proteins in various biological systems, to illustrate the technique, we will image and analyze the clustering behavior of the adaptor protein Linker for activation of T cells (LAT) at the T cell immunological synapse [Williamson, D. J., Owen, D. M., Rossy, J., Magenau, A., Wehrmann, M., Gooding, J. J. , and Gaus, K. (2011). Pre-existing clusters of the adaptor Lat do not participate in early T cell signaling events. Nat. Immunol.12, 655-662.].
现在人们认识到，质膜并非均匀一致，而是包含多种膜微区。这些微区包括脂质微区（脂筏）和蛋白质簇，它们存在于一系列大小和时间尺度上，但通常较小且寿命短暂。膜结构域的小尺寸和动态性质使得用传统荧光显微镜方法研究它们变得困难。光激活定位显微镜（PALM）是一种超分辨率技术，能够以几十纳米的精度定位单个分子的位置。在这里，我们描述一种使用PALM对膜蛋白进行成像的方法，包括活细胞PALM，以使用基于Ripley's K函数的统计聚类分析方法检测质膜蛋白的分子簇集。虽然该方法适用于各种生物系统中的多种蛋白质，但为了说明该技术，我们将对T细胞免疫突触处的衔接蛋白T细胞活化连接蛋白（LAT）的簇集行为进行成像和分析[威廉姆森，D. J.，欧文，D. M.，罗西，J.，马根瑙，A.，韦尔曼，M.，古丁，J. J.，和高斯，K.（2011年）。衔接蛋白Lat的预先存在的簇不参与早期T细胞信号事件。《自然免疫学》12，655 - 662页。]
