Cytoplasmic Microrheology in Bacteria and Archaea: Toward Integrative Models of Subcellular Organization

Abstract

In the absence of compartments separating and organizing their subcellular content, prokaryotic cells rely on the homeostasis of cytoplasm fluidity for proper spatiotemporal distribution and function of biomolecular complexes. In this thesis, I implemented a fluorescence single-particle tracking method to infer viscoelastic properties of the cytoplasm in live cells. Furthermore, I showed that these probes and analysis are robust to provide a direct comparison between eukaryotic, bacterial, and archaeal species. To validate and get biological insights, I aimed to elucidate a reported phenomenon from literature: how unchecked cytoplasmic protein accumulation affects cell division placement in the Gram-positive bacterium Bacillus subtilis. We show that a 42-fold cytoplasmic accumulation of Hag, the flagellin protein, results in impaired function of multiple cellular processes, such as decreased mesoscale diffusion, slow FtsZ treadmilling speeds and mislocalized FtsZ rings, and nucleoid condensation and de-centering. This results in a decreased fitness for cell lineages with higher division asymmetry, where cell sizes vary at death. The accumulation of mNeonGreen also produces asymmetric cell division, signifying that the altered biophysical properties of the cytoplasm can have nonspecific effects on cell biology. Altogether, these observations represent direct evidence that prokaryotic cells require careful tuning of their cytoplasmic material properties to orchestrate essential cellular processes.