Cells can enter into a dormant state when faced with unfavorable conditions. However, how cells enter into and recover from this state is still poorly understood. Here, we study dormancy in different eukaryotic organisms and find it to be associated with a significant decrease in the mobility of organelles and foreign tracer particles. We show that this reduced mobility is caused by an influx of protons and a marked acidification of the cytoplasm, which leads to widespread macromolecular assembly of proteins and triggers a transition of the cytoplasm to a solid-like state with increased mechanical stability. We further demonstrate that this transition is required for cellular survival under conditions of starvation. Our findings have broad implications for understanding alternative physiological states, such as quiescence and dormancy, and create a new view of the cytoplasm as an adaptable fluid that can reversibly transition into a protective solid-like state.

One of the key questions in biology is how the metabolism of a cell responds to changes in the environment. In budding yeast, starvation causes a drop in intracellular pH, but the functional role of this pH change is not well understood. Here, we show that the enzyme glutamine synthetase (Gln1) forms filaments at low pH and that filament formation leads to enzymatic inactivation. Filament formation by Gln1 is a highly cooperative process, strongly dependent on macromolecular crowding, and involves back-to-back stacking of cylindrical homo-decamers into filaments that associate laterally to form higher order fibrils. Other metabolic enzymes also assemble into filaments at low pH. Hence, we propose that filament formation is a general mechanism to inactivate and store key metabolic enzymes during a state of advanced cellular starvation. These findings have broad implications for understanding the interplay between nutritional stress, the metabolism and the physical organization of a cell.

Acute stress causes a rapid redistribution of protein quality control components and aggregation-prone proteins to diverse subcellular compartments. How these remarkable changes come about is not well understood. Using a phenotypic reporter for a synthetic yeast prion, we identified two protein-sorting factors of the Hook family, termed Btn2 and Cur1, as key regulators of spatial protein quality control in Saccharomyces cerevisiae. Btn2 and Cur1 are undetectable under normal growth conditions but accumulate in stressed cells due to increased gene expression and reduced proteasomal turnover. Newly synthesized Btn2 can associate with the small heat shock protein Hsp42 to promote the sorting of misfolded proteins to a peripheral protein deposition site. Alternatively, Btn2 can bind to the chaperone Sis1 to facilitate the targeting of misfolded proteins to a juxtanuclear compartment. Protein redistribution by Btn2 is accompanied by a gradual depletion of Sis1 from the cytosol, which is mediated by the sorting factor Cur1. On the basis of these findings, we propose a dynamic model that explains the subcellular distribution of misfolded proteins as a function of the cytosolic concentrations of molecular chaperones and protein-sorting factors. Our model suggests that protein aggregation is not a haphazard process but rather an orchestrated cellular response that adjusts the flux of misfolded proteins to the capacities of the protein quality control system.

This protocol is associated to the publication “Non-invasive perturbations of intracellular flow reveal physical principles of cell organization” by Mittasch et al., published in Nature Cell Biology.In the following, we describe the procedure for maintenance and preparation of C. elegans zygotes for imaging and application of FLUCS. Preparation of S. cerevisiae cells for microscopy and intracellular flow perturbations. S. cerevisiae cells were grown into logarithmic stage. Subsequently the S. cerevisiae cells were either grown for 2 hours or alternatively energy depleted through addition of 2-Desoxy-D-Glucose (inhibition of glycolysis) and antimycin A (inhibition of mitochondrial ATP production) for 2 h prior to flow induction. This treatment causes a more than 95% reduction in cellular ATP 2. Potentially this protocol is suitable to energy deplete all S. cerevisiae strains and closely related yeast species. A specific marker such as µNS-GFP1is needed to observe subsequent flow perturbations by microscopy.