Macromolecular crowding between the natural and unnatural

Cells are densely packed with molecules of different sizes and chemistries that constantly move and interact with each other. This macromolecular crowding strongly influences how biological reactions occur, affecting processes such as diffusion, enzymatic activity, and molecular interactions. Despite its importance, macromolecular crowding is difficult to measure inside living cells, where many processes take place simultaneously and cannot be easily isolated or controlled. Understanding this crowded intracellular environment is therefore essential for explaining how cellular functions emerge at the molecular level. The work presented in this thesis investigates macromolecular crowding in both natural and artificial systems. To enable direct measurements in living cells, a mammalian cell line was generated that continuously reports its internal crowding state. This system allows the monitoring of cytosolic crowding under different conditions and shows that crowding is not static but responds to external perturbations, including osmotic stress, small-molecule treatments, and viral infection. In addition to these dynamic changes, crowding is not uniformly distributed within the cell. Measurements indicate that regions near the plasma membrane exhibit higher crowding than the cytosol, highlighting the role of subcellular organization in shaping the intracellular environment. To allow controlled experimentation, synthetic cell systems were developed that reproduce physicochemical conditions found inside real cells. Liposome-based artificial cells encapsulating bacterial cell extracts were used to mimic the heterogeneous composition of the cytoplasm. Under osmotic upshift, these systems reach crowding levels comparable to those of unstressed bacterial cells, while also revealing variability between individual compartments. These artificial systems make it possible to study crowding effects in isolation while maintaining key aspects of biological complexity, and show that crowding depends on both the identity and solubility of macromolecular components. In addition to macromolecules, small cosolutes were found to modulate macromolecular crowding in a molecule-specific manner. Disaccharides such as sucrose and trehalose reduce effective crowding despite constant macromolecular concentration, indicating changes in crowder organization. Other molecules, such as ATP and TMAO, exhibit context-dependent effects, demonstrating that crowding arises from interactions between macromolecules and small metabolites rather than from macromolecular concentration alone. By comparing bacterial cells, mammalian cells, and artificial cell systems, macromolecular crowding can be directly quantified across different biological contexts and environmental conditions. Together, this work helps bridge the gap between living and artificial cells and provides experimental platforms to better understand how the crowded intracellular environment shapes biological function and how it can be recreated in synthetic systems.