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Abstract

Living cells are densely populated with macromolecules (Fulton 1982). To understand how proteins and nucleic acids dynamically inhabit and function in cellular volumes, knowledge of molecular crowding is critical to appreciate their modes of interactions and their spatio-temporal distributions. However, none of the existing approaches to measure molecular crowding allow for label-free and spatially-resolved analyses at the molecular scale. In this thesis, I quantitatively and structurally described molecular crowding inside cells utilizing recent advances in cryo-electron tomography (cryo-ET) (Koning et al. 2018, Schaffer et al. 2017, Turk and Baumeister 2020). Specifically, I investigated intracellular crowding in yeast cells under varying nutritional conditions. As their cytosol undergoes a dramatic transition from a liquid- to a solid-like state upon starvation (Joyner et al. 2016, Munder et al. 2016), I mapped changes in local molecular concentrations of ribosomes and fatty acid synthase (FAS) complexes, as well as structural rearrangements of these macromolecules, and other meso-scale protein assemblies. For this purpose, I co-developed methods for automated, high-throughput cryo-sample preparations, in particular cryo-focused ion beam (FIB) milling, and automated data mining utilizing deep-learning algorithms. These workflows allow for the analysis of large datasets which take stochastic cell-to-cell variations into account and are also applicable to other cell types. The automated methods will increase throughput and enable exploration of new biological questions in the long term. In this thesis, I showed that energy-depletion leads to large-scale reorganization of the wild-type yeast cytosol, including variations in particle distributions, conformational changes of specific macromolecular species and the formation of various higher-order assemblies. Determination of their structures provided novel insights into local alterations of macromolecules within the cellular context under different physiologically-relevant conditions. In particular, for both ribosomes and FAS distinct structural conformations were observed upon energy depletion which hint at stationary states, possibly protecting these molecular machines during stress. Future structural analysis of all visualized macromolecular assemblies in combination with coarse-grain and molecular dynamics modeling, will ultimately enable a more holistic understanding of cytosolic phase transitions at a molecular level.