A cell in motion
Bacterial cells display a highly organized internal architecture essential for their survival and adaptability. In Escherichia coli, a Gram-negative bacterium, spatial organization arises from both physical compartmentalization—cytoplasm, periplasm, inner and outer membranes—and dynamic processes such as liquid–liquid phase separation (LLPS) and protein redistribution. This thesis investigates the localization and mobility of diverse proteins in E. coli with sub-micrometer resolution, using advanced microscopy and functional assays.
Single-Molecule displacement Mapping (SMdM) was applied for the first time in bacteria to measure cytoplasmic protein diffusion, revealing location-dependent mobility and reduced diffusion at cell poles. Further analysis linked this asymmetry to protein aggregates, particularly at the old pole, rather than ribosome localization, highlighting the role of cellular aging.In the periplasm, protein diffusion exhibited both fast and slow components; osmotic upshift increased the fast fraction, indicating reduced crowding. Simulations suggested that large periplasmic structures hinder mobility beyond simple confinement effects.
We also explored LLPS in membranes by inducing condensate formation of the integral membrane protein LacY via PopTag fusion. Experimental and simulation data demonstrated 2D membrane condensates, with curvature influencing polar localization. Functional assays confirmed native LacY activity within condensates, suggesting applications in metabolic engineering.
Finally, SMdM was adapted for giant unilamellar vesicles (GUVs) to probe protein diffusion in controlled cell-mimicking environments, revealing effects of protein and membrane surface charge on mobility. Together, these findings provide new insights into the spatiotemporal organization of bacterial cells, with implications for understanding physiology, aging, and synthetic biology applications.