Characterization Of Nanoscale Her2-Containing Clusters At The Cell Membrane

The spatial distribution of proteins on the cell surface has emerged as a key regulator of membrane proteins function. Protein assemblies are of biological and therapeutic importance: the nanoscale spatial ordering and dynamic distribution of proteins at the cell membrane tune the complex molecular networks they regulate in normal physiology and in disease. Therefore, there is a need to develop high-throughput methods to understand how the organization of proteins on a nanoscale level regulates receptor signaling and cellular phenotypes. In this work, we present the development of a non-microscopy based super-resolution method for unbiased analysis of protein nanoclusters at the cell membrane, studying the frequency in which specific types of protein appear in the proximity of the protein of interest. We setup a DNA nanotechnology-based approach, using DNA nanostructures to decipher the position and the identity of proteins within the assembly. STORM-TIRF super-resolution microscopy analysis and advanced biochemical assays were combined to validate the methodology and to obtain exhaustive characterization of protein clusters. We focused here on clustering of the membrane receptor Her2. Although Her2 has well known roles in driving tumor progression, the molecular mechanisms by which Her2 can lead to diverse cellular outcomes are still unclear. Since Her2 forms homo- and hetero-dimers and oligomers, we aim to provide a precise portrait of the spatial organization of Her2-containing clusters and to understand how it correlates with downstream signaling and cellular outcomes. Our approach has the potential to contribute to developing a new paradigm in targeting receptor signaling at the nanoscale, focusing not on the membrane receptors per se, but on their spatial organization.