Resolving Protein Interactions in Live Bacterial Cells through 3D Single‐Molecule Localization Microscopy

Abstract

In living cells, proteins form dynamic complexes essential for the functional regulation of cellular activities. While in vitro assays provide key insights into protein binding partners, interactions may be artificially reduced or enhanced once the proteins are removed from their native context. Single‐molecule localization microscopy is ideally suited to measure spatial locations of fluorescently‐labeled molecules in living cells and thus enables single‐particle tracking of the entire distribution of molecular behaviors. Here, we apply high‐throughput 3D single‐molecule localization microscopy to measure the diffusion behaviors of over 100,000 individual intracellular bacterial proteins. Quantitative analysis of the single‐molecule trajectories using Monte Carlo simulations of confined diffusion resolves the co‐existence of multiple diffusive states in the bacterial cytoplasm. Molecular and genetic perturbations show that these diffusive states consist of distinct hetero‐ and homo‐oligomeric complexes. These findings provide new molecular‐level insights into how dynamic molecular complexes function in their native environment.Support or Funding InformationResearch was supported by a Hartwell Postdoctoral Fellowship to CR and startup funds from the University of Virginia to AG.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.