Visualizing clathrin-mediated IgE receptor internalization by electron and atomic force microscopy.
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.
Burns
Alan R
AR
Biomolecular Interfaces and Systems Department, Sandia National Laboratories, Albuquerque, NM, USA.
Oliver
Janet M
JM
Pfeiffer
Janet R
JR
Wilson
Bridget S
BS
D000818:Animals; D002478:Cells, Cultured; D022163:Clathrin-Coated Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism* / Q000648:ultrastructure; D004705:Endocytosis; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007150:Immunohistochemistry; D008407:Mast Cells / Q000378:metabolism* / Q000648:ultrastructure; D018625:Microscopy, Atomic Force; D046529:Microscopy, Electron, Transmission; D021381:Protein Transport; D017455:Receptors, IgE / Q000378:metabolism*
D017455:Receptors, IgE