Abstract
The cytoplasm of bacterial cells is densely packed with highly polydisperse macromolecules that exhibit size-dependent glassy dynamics. Recent research has revealed that metabolic activities in living cells can counteract the glassy nature of these macromolecules, allowing the cell to maintain critical fluidity for its growth and function. While it has been proposed that the crowded cytoplasm is responsible for this glassy behavior, a detailed analysis of the size-dependent nature of the glassy dynamics and an explanation for how cellular activity induces its fluidization remains elusive. Here, we use a combination of computational models and targeted experiments to show that entropic segregation of the protein synthesis machinery from the chromosomal DNA causes size-dependent spatial organization of molecules within the cell, and the resultant crowding leads to size-dependent glassy dynamics. Furthermore, Brownian dynamics simulations of this in silico system supports a new hypothesis: protein synthesis in living cells contributes to the metabolism-dependent fluidization of the cytoplasm. The main protein synthesis machinery, ribosomes, frequently shift between fast and slow diffusive states. These states correspond to the independent movement of ribosomal subunits and the actively translating ribosome chains called polysomes, respectively. Our simulations demonstrate that the frequent transitions of the numerous ribosomes, which constitute a significant portion of the cell proteome, greatly enhance the mobility of other macromolecules within the bacterial cytoplasm. Considering that ribosomal protein synthesis is the largest consumer of ATP in growing bacterial cells, the translation process can serve as the primary mechanism for fluidizing the cytoplasm in metabolically active cells.