Mapping Ceacam 1-4L's Nanoscale Spatial Distribution, Structure, Dynamics, and Association with Lipid Ordered Domains by Super-Resolution Microscopy

Abstract

Super-resolution microscopy has provided novel quantitative insights into the nanoscale spatial distribution of membrane proteins, including the observation that some systems form micro- and nano-sized clusters. However, details of the protein's self-association state (monomer / dimer / oligomer) within the different cluster types are often missing. Since it has been shown that a protein's oligomeric form might be a crucial determinant of its function, it is important to have a technique capable of measuring both spatial distribution and association state. To that end, we have applied a correlated dSTORM/homo-FRET approach to characterize CEACAM 1-4L, the progenitor isoform of the carcinoembryonic antigen-related cellular adhesion molecule (CEACAMs) family. CEACAMs are cell surface glycoproteins involved in homo- and hetero-philic intercellular interactions that control cellular growth, differentiation, tumourigenesis, inflammation and infection. While CEACAM1-4L is known to exist as a monomer or oligomer with the former being hypothesized to be involved in activatory intercellular interactions, the basis for its spatial distribution and association state as a function of distribution remain poorly understood. Our correlated approach involved live cell homo-FRET studies of eYFP-CEACAM1-4L in HeLa cells followed by fixed cell dSTORM imaging performed using nanobody labeling with Alexa Fluor 647 (AF647) conjugated to an anti-eYFP single domain antibody. Cluster analysis of the dSTORM datasets using Voronoi tessellation revealed that the CEACAM1-4L nanoclusters were preferentially comprised of monomeric CEACAM1-4L while microclusters contained a heterogeneous distribution of CEACAM1-4L monomers and oligomers. To further characterize these clusters, super-resolution imaging using Alexa-647 labelled cholera toxin was performed to examine the co-localization of lipid ordered domains, which are known to drive membrane protein lateral distribution and clustering, with CEACAM1-4L. Finally, live-cell single particle tracking (SPT) of CEACAM1-4L was performed to investigate the putative confined, obstructed, free diffusion, and directed motion dynamics of CEACAM1-4L.