The plasma membrane is the outer border of a cell and physically separates its interior from the surrounding environment. However, the plasma membrane is not an inert shell. Rather, it is utilized in many cellular processes, and therefore its composition and structure are of great interest to many researchers. One of the early attempts to describe the plasma membrane was the “fluid mosaic” model by Singer and Nicholson (1
). In this model, the plasma membrane is a homogeneous two-dimensional lipid bilayer in which proteins can diffuse freely. Over the last decades, this view has been revised, as it became clear that diffusion in the plasma membrane is slower than expected, molecules are not evenly distributed over the cell surface, and molecules are confined to membrane domains and/or surface areas with dimensions of less than one micrometer. These findings led to more complex models, including the “lipid raft” (2
), “picket fence” (3
), and “protein island” models (4
). However, the actual organization of the plasma membrane and its associated molecules remains controversial.
Many biological processes in immune cells utilize and reorganize the plasma membrane. Most immune cells are activated via cell surface receptors, which in some cases is accompanied by the reorganizations of the plasma membrane, most dramatically seen in the formation of the immunological synapse (5
). The systematic functions of immune cells often involve the plasma membrane, e.g., endocytosis, phagocytosis, and secretion. Here, we describe a method based on Sanan et al. (6
) that has been successfully used in the analyses of the two-dimensional architecture of the plasma membrane in T cells (4
), B cells (8
), mast cells (9
), and other cell types (6
). It is based on the generation of plasma membrane sheets attached to EM grids (), by breaking (“ripping”) cells open through forces applied by separating two opposing surfaces sandwiching the cells of interest (). This procedure has been used with a variety of modifications, each optimized for the study of a particular question. In this chapter, the method is divided into four steps: (1) Surface choices and preparations, (2) Cell binding to primary surface, (3) Generation of plasma membrane sheets, and (4) Labeling of plasma membrane sheets. For each step, up to three possible protocols are shown, which can be “mixed and matched,” and further adapted, to suit any specific study aim.
Fig. 1 (a) Whole plasma membrane sheet from an activated T cell bound to an EM grid coated with stimulatory ligands (peptide-MHC II and co-stimulatory B7.1 molecules). (b) Magnified area of a plasma membrane sheet from a quiescent T cell bound to an EM grid (more ...)
Fig. 2 Simplified three-step illustrations of the “ripping” procedures to obtain plasma membrane sheets. (For simplification, working surface, filter papers and acetate disks are not included in the illustration.) (a) Example for the generation (more ...)