A DNA Nanoassembly-Based Approach to Map Membrane Protein Nanoenvironments

Abstract

Super-resolution imaging has revealed that most proteins at the plasma membrane are not uniformly distributed but localize to dynamic domains of nanoscale dimensions. To investigate their functional relevance, there is a need for methods that enable comprehensive analysis of the compositions and spatial organizations of membrane protein nanodomains in cell populations. However, super-resolution methods are limited to analysing small, preselected subsets of proteins, at very low sampling fractions, and multiplexing analysis remains challenging. Here we describe thedevelopment of a non-microscopy based method for ensemble analysis of membrane protein nanodomains. The method, termed NANOscale DEciphEring of membrane Protein nanodomains (NanoDeep), is based on the use of DNA nanoassemblies to translate membrane protein organization information into a DNA sequencing readout. This method allows for detection of the inventory of proteins that forms the nanoenvironment of any reference membrane protein in cell populations. Using NanoDeep, we characterised the protein nanoenvironments surrounding Her2, a membrane receptor of critical relevance in cancer. We found that the occupancies of Her2, Her3 and EGFR in the nanoenvironments surrounding Her2 were similar in two cell lines with vastly different expression levels of Her2. Further, we found that adding Heregulin-β1 to cancer cells led to increased occupancy of Her2 and Her3, and to a lesser extent EGFR, in Her2 nanoenvironments. Importantly, we were able to modulate by design the inventory of proteins analysed by NanoDeep, including in the analysis of Her2 nanoenvironment two membrane proteins that have been reported to interact with Her2 (integrin α5β1 and CD63) and a protein unrelated to Her2 signalling (CD71). NanoDeep has the potential to provide new insights into the roles of the composition and spatial organization of protein nanoenvironments in the regulation of membrane protein function.