The Dynamic Structure of Escherichia coli Cytoplasm: Crowding and Protein Diffusion

Escherichia coli, which has led to a better understanding of the structure of the cell cytoplasm. Driven by the question whether the bacterial cytoplasm has a form of spatial organization, I embarked on a journey of innovative experimental designs and analytical methods. Central to this research is the use of cutting-edge fluorescence imaging techniques, which provide real-time insights into the dynamics of cellular processes. I investigated the phenomenon of macromolecular crowding and tools to determine this physicochemical parameter by Förster Resonance Energy Transfer (FRET) probes. Additionally, I used Single Molecule displacement Mapping (SMdM) to track protein diffusion with nanometer precision, revealing that protein movement scales with complex mass and is location dependent. Finally, I introduced a new method, Simulation-based Reconstructed Diffusion (SbRD), to accurately measure diffusion in confined spaces, such as the cell poles of bacteria. This work challenges the traditional view of the cytoplasm as a uniform environment, instead revealing a complex and varied landscape where diffusion significantly differs across the cell. The findings not only deepen our understanding of bacterial cells but also provide a solid foundation for future research into cellular processes. This research represents a step forward in our understanding of the intricate mechanics that drive life at the molecular level.