Differential oligomeric nature of mEos2 and mEos3.2 fluorescent proteins is consequential to diffusion and confinement of membrane probes

Abstract

Lipid‐mediated membrane heterogeneity, often referred to as lipid rafts, is proposed to be an important organizing principle in mammalian cells. Using super‐resolution fluorescence localization imaging we track the diffusion of a large panel of fluorescent membrane probes ranging in size, mode of membrane anchoring, and putative ‘raft’ or ‘non‐raft’ phase‐association. Our expressed probes include fluorescently‐tagged palmitoylated or non‐palmitoylated versions of transmembrane domains including linker of activated T‐cell and hemagglutinin, and fluorescent protein anchored by GPI, palmitate and myristoyl moieties, or a geranyl‐geranyl moiety. The recent advent of photoconvertible fluorescent protein mEos3.2, a “true monomeric” mutant of its mEos2 predecessor1, has enabled us to compare directly monomeric and oligomeric versions of the same probes. Our results indicate that in some cases rates of diffusion are more than two fold lower in the mEos2 probes when compared to their mEos3.2 counterparts. Many mEos2‐labeled probes also differ from mEos3.2‐labeled probes in their exponents of anomalous diffusion. We attribute changes in mobility to the proteins’ differential propensity to oligomerize, and find that larger probes differentiate themselves more than smaller ones with respect to phase‐mediated mobility differences. We are using the clustering effect of mEos2, therefore, as a tool to elucidate phase heterogeneity. Funding for this project was provided by NIH: SV R00GM087810 and a start up grant from the University of Michigan.