Studying membrane proteins in intact cells using nanoparticle labels and liquid‐phase electron microscopy

Abstract

Cells have receptor proteins in their plasma membranes ‘listening’ to chemical signals from the outside world. These signals consist of ligands, small molecules that bind specifically to a receptor. But how those signals are interpreted and lead to decisions is incompletely understood mainly on account of limitations of present analytical methods. It is typically extremely difficult to directly see how endogenously expressed individual proteins respond to ligand binding in the intact cell, which can lead, for example, to the formation of protein complexes triggering signaling processes. Much knowledge about cellular function has been obtained via biochemical methods but these analyze pooled material from many thousands of cells and the knowledge is thus based on population averages. But we need to look at the individual cell in order to understand the fundamentals of how a cell interprets a signal. Studying membrane proteins at the nanoscale in intact eukaryotic cells is now possible using liquid‐phase scanning transmission electron microscopy (STEM) [1, 2]. The key step is to specifically label the proteins of interest in a one‐to‐one ratio with small probes combined with nanoparticles, for example, gold nanoparticles or quantum dots. Cells in liquid are then placed in a microfluidic chamber enclosing the sample in the vacuum of the electron microscope, and are imaged with STEM. It is not always necessary to enclose the cells in the microfluidic chamber. For some studies, it is sufficient to obtain information from the thin outer regions of the cells, and those can be imaged with high resolution using environmental scanning electron microscopy (ESEM) with STEM detector [3]. Liquid‐phase STEM was used to explore the formation of the epidermal growth factor HER2 at the single‐molecule level in intact SKBR3 breast cancer cells in liquid state [4]. HER2 is a membrane protein and plays an important role in breast cancer aggressiveness and progression. Data analysis based on calculating the pair correlation function from individual HER2 positions revealed remarkable differences in functionality between different cellular regions, and between cells with possible relevance for studying cancer metastasis and drug response.