Membrane protein stoichiometry studied in intact mammalian cells using liquid‐phase electron microscopy

Abstract

Receptor membrane proteins in the plasma membranes of cells respond to extracellular chemical signals by conformational changes, spatial redistribution, and (re-)assembly into protein complexes, for example, into homodimers (pairs of the same protein type). The functional state of the proteins can be determined from information about how subunits are assembled into protein complexes. Stoichiometric information about the protein complex subunits, however, is generally not obtained from intact cells but from pooled material extracted from many cells, resulting in a lack of fundamental knowledge about the functioning of membrane proteins. First, functional states may dramatically differ from cell to cell on account of cell heterogeneity. Second, extracting the membrane proteins from the plasma membrane may lead to many artefacts. Liquid-phase scanning transmission electron microscopy (STEM), in short liquid STEM, is a new technique capable of determining the locations of individual membrane proteins within the intact plasma membranes of cells in liquid. Many tens of whole cells can readily be imaged. It is possible to analyse the stoichiometry of membrane proteins in single cells while accounting for heterogenic cell populations. Liquid STEM was used to image epidermal growth factor receptors in whole COS7 cells. A study of the dimerisation of the HER2 protein in breast cancer cells revealed the presence of rare cancer cells in which HER2 was in a different functional state than in the bulk cells. Stoichiometric information about receptors is essential not only for basic science but also for biomedical application because they present many important pharmaceutical targets.

Linear Chains of HER2 Receptors Found in the Plasma Membrane Using Liquid-Phase Electron Microscopy

“…The study of the spatial arrangement of membrane proteins at the nanoscale was possible because of the 2-nm precision of the ESEM-STEM method in combination with the capability to analyze large areas approaching 1000 mm 2 of intact plasma membrane. Several types of liquid-phase electron microscopy systems exist for studying membrane proteins in cells (3,(27)(28)(29)(30). Most other methods for analyzing membrane proteins in cells do not achieve sufficient spatial resolution to resolve individual protein positions for endogenous membrane proteins, as is the case for super-resolution fluorescence microscopy (31,32), or are incapable of handling a series of whole cells, such as in cryo transmission electron microscopy, which typically focuses on analyzing a subsection of a cell (33).…”

Liquid-phase electron microscopy of molecular drug response in breast cancer cells reveals irresponsive cell subpopulations related to lack of HER2 homodimers

“…It is known that the problem of drug resistance development should be tackled by taking cancer cell heterogeneity into account ( Hanahan and Weinberg, 2011 ). However, this has been challenging until now due to limitations of the available analytical methods ( de Jonge, 2017 ). Biochemical methods and x-ray crystallography generally used to study the cellular responses to drugs use pooled cellular material so that information is obtained from average responses across a cell population only.…”

Liquid cell transmission electron microscopy and its applications

“…For example, from the inter-label distances, singlelabelled protein monomer and double-labelled protein pairs can be distinguished, and from their distribution, Peckys et al [144] revealed the dimeric conformation of the TMEM16A protein. This technique is an efficient way to elucidate the stoichiometry distribution and any possible change of various proteins on the cell membrane [47,145,146]. And not only proteins, it has been shown that nanoparticle labelling can also be done on DNA [49].…”