Andrews Norma W
Department of Cell Biology and Molecular Genetics, University of Maryland at College Park, 2134 Bioscience Research Building, College Park, MD, 20742-5815, USA.
Methods Mol Biol. 2017;1594:205-211. doi: 10.1007/978-1-4939-6934-0_13.
Elevation in the cytosolic Ca concentration triggers exocytosis of lysosomes in many cell types. This chapter describes a method to detect lysosomal exocytosis in mammalian cells, which takes advantage of the presence of an abundant glycoprotein, Lamp1, on the membrane of lysosomes. Lamp1 is a transmembrane protein with a large, heavily glycosylated region that faces the lumen of lysosomes. When lysosomes fuse with the plasma membrane, epitopes present on the luminal domain of Lamp1 are exposed on the cell surface. The Lamp1 luminal epitopes can then be detected on the surface of live, unfixed cells using highly specific monoclonal antibodies and fluorescence microscopy. The main advantage of this method is its sensitivity, and the fact that it provides spatial information on lysosomal exocytosis at the single cell level.
胞质钙离子浓度升高会触发多种细胞类型中溶酶体的胞吐作用。本章介绍一种检测哺乳动物细胞中溶酶体胞吐作用的方法，该方法利用了溶酶体膜上存在的一种丰富的糖蛋白Lamp1。Lamp1是一种跨膜蛋白，其面向溶酶体腔的区域有一个大的、高度糖基化的区域。当溶酶体与质膜融合时，Lamp1腔域上的表位会暴露在细胞表面。然后，使用高度特异性的单克隆抗体和荧光显微镜，可以在活的、未固定的细胞表面检测到Lamp1腔表位。这种方法的主要优点是其灵敏度，以及它能在单细胞水平上提供溶酶体胞吐作用的空间信息。
