Dynamics and Co-Localization of the Electron Transport Chain of Escherichia Coli: Investigations Through Fluorescence Microscopy

Abstract

Total Internal Reflection Fluorescence Microscopy (TIRFM) is a key tool in probing the dynamics and distribution of fluorescently tagged fusion proteins. This non-invasive in vivo technique is suited to the investigation of membrane-bound proteins due to the low cytoplasmic response, sometimes allowing for ∼millisecond trajectory sampling of membrane bound fusion proteins. In addition, multi-wavelength TIRFM set-ups allow for investigations into co-localization of multiple fluorescently labeled protein species.Presented here are simultaneous dual-color TIRFM investigations of key proteins involved in the branched Electron Transport Chain (ETC) in Escherichia coli, using a series of single and dual-labeled mutant strains. Of interest was the comparative degree to which these proteins reflected a ‘free-diffusion-collision’ model of protein interactions, to that of a ‘solid state’ model in which reacting proteins are confined and co-localized.This builds upon previous work that identified a key protein in the ETC as having a highly non-random heterogeneous distribution, being located into functional mobile membrane patches.We investigated the spatial distribution and the degree of co-localization of these protein pairs at the cellular level. We quantified the diffusion behavior of the observed trajectories comparing the relative populations which displayed freely diffusing characteristics (through standard Brownian motion), with the populations that exhibited confined or anomalous modes. We discuss the implications of the observed dynamics at the systems level, and how this relates to the overall functioning of the branched ETC.