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How cells adapt to varying environmental conditions is largely unknown. Here, we show that, in budding yeast, the RNA-binding and stress granule protein Pub1 has an intrinsic property to form condensates upon starvation or heat stress and that condensate formation is associated with cell-cycle arrest. Release from arrest coincides with condensate dissolution, which takes minutes (starvation) or hours (heat shock). In vitro reconstitution reveals that the different dissolution rates of starvation- and heat-induced condensates are due to their different material properties: starvation-induced Pub1 condensates form by liquid-liquid demixing and subsequently convert into reversible gel-like particles; heat-induced condensates are more solid-like and require chaperones for disaggregation. Our data suggest that different physiological stresses, as well as stress durations and intensities, induce condensates with distinct physical properties and thereby define different modes of stress adaptation and rates of recovery., Cell Reports, 23 (11), ISSN:2666-3864, ISSN:2211-1247

Recent advances in cell biology enable precise molecular perturbations. The spatiotemporal organization of cells and organisms, however, also depends on physical processes such as diffusion or cytoplasmic flows, and strategies to perturb physical transport inside cells are not yet available. Here, we demonstrate focused-light-induced cytoplasmic streaming (FLUCS). FLUCS is local, directional, dynamic, probe-free, physiological, and is even applicable through rigid egg shells or cell walls. We explain FLUCS via time-dependent modelling of thermoviscous flows. Using FLUCS, we demonstrate that cytoplasmic flows drive partitioning-defective protein (PAR) polarization in Caenorhabditis elegans zygotes, and that cortical flows are sufficient to transport PAR domains and invert PAR polarity. In addition, we find that asymmetric cell division is a binary decision based on gradually varying PAR polarization states. Furthermore, the use of FLUCS for active microrheology revealed a metabolically induced fluid-to-solid transition of the yeast cytoplasm. Our findings establish how a wide range of transport-dependent models of cellular organization become testable by FLUCS.

© 2013 International Society for Neurochemistry, Several neurodegenerative diseases, such as Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), or prion diseases, are known for their intimate association with protein misfolding and aggregation. These disorders are characterized by the loss of specific neuronal populations in the brain and are highly associated with aging, suggesting a decline in proteostasis capacity may contribute to pathogenesis. Nevertheless, the precise molecular mechanisms that lead to the selective demise of neurons remain poorly understood. As a consequence, appropriate therapeutic approaches and effective treatments are largely lacking. The development of cellular and animal models that faithfully reproduce central aspects of neurodegeneration has been crucial for advancing our understanding of these diseases. Approaches involving the sequential use of different model systems, starting with simpler cellular models and ending with validation in more complex animal models, resulted in the discovery of promising therapeutic targets and small molecules with therapeutic potential. Within this framework, the simple and well-characterized eukaryote Saccharomyces cerevisiae, also known as budding yeast, is being increasingly used to study the molecular basis of several neurodegenerative disorders. Yeast provides an unprecedented toolbox for the dissection of complex biological processes and pathways. Here, we summarize how yeast models are adding to our current understanding of several neurodegenerative disorders., This study was supported by Fundação para a Ciência e Tecnologia projects PTDC/SAU-NEU/105215/2008 and PTDC/BIA-BCM/117975/2010, and post-doctoral fellowship SFRH/BPD/35767/2007 (ST). TFO was supported by an EMBO Installation Grant and by the DFG Cluster of Excellence Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB). SA is supported by the Max Planck Society and MCM by a fellowship from the Dresden International Graduate School for Biomedicine and Bioengineering (DIGS-BB).

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. DOI: http://dx.doi.org/10.7554/eLife.09347.001, eLife digest Most organisms live in unpredictable environments, which can often lead to nutrient shortages and other conditions that limit their ability to grow. To survive in these harsh conditions, many organisms adopt a dormant state in which their metabolism slows down to conserve vital energy. When the environmental conditions improve, the organisms can return to their normal state and continue to grow. The interior of cells is known as the cytoplasm. It is very crowded and contains many molecules and compartments called organelles that carry out a variety of vital processes. The cytoplasm has long been considered to be fluid-like in nature, but recent evidence suggests that in bacterial cells it can solidify to resemble a soft glass-type material under certain conditions. When cells become dormant they stop dividing and reorganise their cytoplasm in several ways; for example, the water content drops and many essential proteins form storage compartments. However, it was not clear how cells regulate the structure of the cytoplasm to enter into or exit from dormancy. Now, Munder et al. analyse the changes that occur in the cytoplasm when baker’s yeast cells enter a dormant state. The experiments show that when yeast cells are deprived of energy – as happens during dormancy – the cytoplasm becomes more acidic than normal. This limits the ability of molecules and organelles to move around the cytoplasm. Similar results were also seen in other types of fungi and an amoeba. Munder et al. found that this increase in acidity during dormancy causes many proteins to interact with each other and form large clumps or filament structures that result in the cytoplasm becoming stiffer. A separate study by Joyner et al. found that when yeast cells are starved of sugar, two large molecules are less able to move around the cell interior. Together, the findings of the studies suggest that the interior of cells can undergo a transition from a fluid-like to a more solid-like state to protect the cells from damage when energy is in short supply. The next challenge is to understand the molecular mechanisms that cause the physical properties of the cytoplasm to change under different conditions. DOI: http://dx.doi.org/10.7554/eLife.09347.002

The deposition of misfolded proteins in cytoplasmic protein bodies requires the concerted action of stress-inducible protein-sorting factors and molecular chaperones. Protein sequestration during acute stress is a cellular strategy that adjusts the flux of misfolded proteins to the capacities of the protein quality control system., 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.

In search of alternative expression platforms heterologous protein production in microalgae has gained increasing importance in the last years. Particularly, the chloroplast of the green alga Chlamydomonas reinhardtii has been adopted to successfully express foreign proteins like vaccines and antibodies. However, when compared with other expression systems, the development of the algal chloroplast to a powerful production platform for recombinant proteins is still in its early stages. In an effort to further improve methods for a reliable and rapid generation of transplastomic Chlamydomonas strains we constructed the key plasmid pMM2 containing the psbA gene and a multiple cloning site for foreign gene insertion. The psbA gene allows a marker-free selection procedure using as a recipient the Fud7 strain of Chlamydomonas, which grows on media containing acetate as a carbon source, but is unable to grow photoautotrophically due to the lack of an intact psbA gene. Biolistic transformation of Fud7 with vectors containing this gene restores photoautotrophic growth and thus permits selection in the light on media without carbon sources and antibiotics. The multiple cloning site with a BsaI recognition sequence allows type IIs restriction enzyme-based modular cloning which rapidly generates new gene constructs without sequences, which could influence the expression and characteristics of the foreign protein. In order to demonstrate the feasibility of this approach, a codon optimized version of the gene for the bacterial protein MPT64 has been integrated into the plastome. Several strains with different promoter/UTR combinations show a stable expression of the HA tagged MPT64 protein in Chlamydomonas chloroplasts.

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. DOI: http://dx.doi.org/10.7554/eLife.02409.001, eLife digest Life is based on a series of chemical reactions that control how cells live, grow, and divide. Various metabolic enzymes allow cells to control the rate at which these reactions occur. Recently, researchers have noticed that metabolic enzymes can form filaments in cells, usually when the cells are deprived of energy or nutrients. Petrovska, Nüske et al. now reveal more about how and why an enzyme called glutamine synthetase (Gln1) forms filaments in yeast cells. Gln1 has a cylindrical shape. This shape means that stacking the enzymes end-to-end under the right conditions is enough to make them bond into a long filament. In addition, a zip-like mechanism enables neighboring filaments to fuse to create thicker fibres. These filaments are more likely to form if there are high concentrations of large background molecules around—a condition known as macromolecular crowding. Petrovska, Nüske et al. also found evidence that the filaments are part of a strategy to help cells survive starvation. Enzymes were more likely to construct filaments when the cell division cycle had stopped, which commonly occurs due to a lack of nutrients. In addition, Gln1 filaments only form if the cytoplasm of the cell becomes acidic—which is a response to the cell starving. This has been seen for other metabolic enzymes as well, suggesting that acidification is a signal to reprogram the metabolism of a cell. The Gln1 enzymes in a filament are inactivated, but become active again after the filament breaks up. In addition, preventing Gln1 filament formation makes it harder for cells to recover from a period of starvation. Petrovska, Nüske et al. therefore suggest that the filaments act as a storage depot for the enzymes during starvation. Further experiments are now needed to uncover exactly how these manage to help the starving cell to survive and recover. DOI: http://dx.doi.org/10.7554/eLife.02409.002