通过电子显微镜和原子力显微镜观察网格蛋白介导的IgE受体内吞作用

Visualizing clathrin-mediated IgE receptor internalization by electron and atomic force microscopy.

作者信息

Burns Alan R, Oliver Janet M, Pfeiffer Janet R, Wilson Bridget S

机构信息

Biomolecular Interfaces and Systems Department, Sandia National Laboratories, Albuquerque, NM, USA.

出版信息

Methods Mol Biol. 2008;440:235-45. doi: 10.1007/978-1-59745-178-9_18.

DOI:10.1007/978-1-59745-178-9_18

PMID:18369950

Abstract

A significant step in the immunoglobulin E (IgE) receptor signaling pathway in mast cell membranes is receptor internalization by clathrin-coated vesicles. Visualization in native membrane sheets of the emerging clathrin lattice structures containing the IgE receptor and associated signaling partners has been accomplished with high-resolution transmission electron microscopy (TEM). More recently, membrane sheets with labeled clathrin have also been characterized with atomic force microscopy (AFM) in combination with fluorescence imaging. We discuss here the procedure for creating fixed, native cell membrane sheets, labeling with immunogold or fluorescent labels, and utilization for TEM or AFM/fluorescence imaging of clathrin-mediated IgE internalization.

摘要

肥大细胞膜中免疫球蛋白E（IgE）受体信号通路的一个重要步骤是网格蛋白包被小泡介导的受体内化。利用高分辨率透射电子显微镜（TEM）已实现对含有IgE受体及相关信号伴侣的新生网格蛋白晶格结构在天然细胞膜片中的可视化观察。最近，结合荧光成像，用原子力显微镜（AFM）也对标记有网格蛋白的细胞膜片进行了表征。我们在此讨论制备固定的天然细胞膜片、用免疫金或荧光标记物进行标记以及将其用于TEM或AFM/荧光成像以观察网格蛋白介导的IgE内化的操作流程。