Visualization of single proteins from stripped native cell membranes: A protocol for high-resolution atomic force microscopy

Abstract

Atomic force microscopy (AFM) proved to be able to obtain high-resolution three-dimensional images of single-membrane proteins, isolated, crystallized, or included in reconstructed model membranes. The extension of this technique to native systems, such as the protein immersed in a cell membrane, needs a careful manipulation of the biological sample to meet the experimental constraints for high-resolution AFM imaging. In this article, a general protocol for sample preparation is presented, based on the mechanical stretch of the cell membrane. The effectiveness for AFM imaging has been tested on the basis of an integrated optical and AFM approach and the proposed method has been applied to cells expressing cystic fibrosis transmembrane conductance regulator, a channel involved in cystic fibrosis, showing the possibility to identify and analyze single proteins in the plasma membrane.

The structure and function of cell membranes examined by atomic force microscopy and single-molecule force spectroscopy

“…The apical cytoplasmic side of membranes can be prepared by stripping the membrane from the top of the cell. 96,97 Briefly, the cells are cultured on glass slides (Fig. 13).…”

Clustering of Major Histocompatibility Complex-Class I Molecules in Healthy and Cancer Colon Cells Revealed from Their Nanomechanical Properties

“…Nevertheless, the MHC-I clusters and the local mechanical properties of the cell have been kept disjointed so far: yet, the membrane nanoscale morphology encourages the use of AFM for assessing the correlation of proteins distribution, their functionality, and membrane mechanical behavior in a sub-micrometer spatial scale. The past two decades have seen enormous progress in the application of AFM to characterize biological molecules. − AFM has been widely applied in the study of membrane proteins directly on the cell membrane. − For example, Almqvist et al showed both the localization of VEGF receptor molecules on the cell surface and the changes in local elasticity of the plasma membrane upon their reorganization . The investigation of the interaction forces at play in the IS using AFM has been reported by Hosseini et al , where they investigated the interaction of T cells with MHC-II molecules on APCs.…”