When yeast cells change their mind: cell cycle "Start" is reversible under starvation.

Eukaryotic cells decide in late G1 phase of the cell cycle whether to commit to another round of division. This point of cell cycle commitment is termed "Restriction Point" in mammals and "Start" in the budding yeast Saccharomyces cerevisiae. At Start, yeast cells integrate multiple signals such as pheromones and nutrients, and will not pass Start if nutrients are lacking. However, how cells respond to nutrient depletion after the Start decision remains poorly understood. Here, we analyze how post-Start cells respond to nutrient depletion, by monitoring Whi5, the cell cycle inhibitor whose export from the nucleus determines Start. Surprisingly, we find that cells that have passed Start can re-import Whi5 into the nucleus. In these cells, the positive feedback loop activating G1/S transcription is interrupted, and the Whi5 repressor re-binds DNA. Cells which re-import Whi5 become again sensitive to mating pheromone, like pre-Start cells, and CDK activation can occur a second time upon replenishment of nutrients. These results demonstrate that upon starvation, the commitment decision at Start can be reversed. We therefore propose that cell cycle commitment in yeast is a multi-step process, similar to what has been suggested for mammalian cells.

Glucose restriction drives spatial reorganization of mevalonate metabolism.

Eukaryotes compartmentalize metabolic pathways into sub-cellular domains, but the role of inter-organelle contacts in organizing metabolic reactions remains poorly understood. Here, we show that in response to acute glucose restriction (AGR) yeast undergo metabolic remodeling of their mevalonate pathway that is spatially coordinated at nucleus-vacuole junctions (NVJs). The NVJ serves as a metabolic platform by selectively retaining HMG-CoA Reductases (HMGCRs), driving mevalonate pathway flux in an Upc2-dependent manner. Both spatial retention of HMGCRs and increased mevalonate pathway flux during AGR is dependent on NVJ tether Nvj1. Furthermore, we demonstrate that HMGCRs associate into high-molecular-weight assemblies during AGR in an Nvj1-dependent manner. Loss of Nvj1-mediated HMGCR partitioning can be bypassed by artificially multimerizing HMGCRs, indicating NVJ compartmentalization enhances mevalonate pathway flux by promoting the association of HMGCRs in high molecular weight assemblies. Loss of HMGCR compartmentalization perturbs yeast growth following glucose starvation, indicating it promotes adaptive metabolic remodeling. Collectively, we propose a non-canonical mechanism regulating mevalonate metabolism via the spatial compartmentalization of rate-limiting HMGCR enzymes at an inter-organelle contact site.

Transcription levels of a noncoding RNA orchestrate opposing regulatory and cell fate outcomes in yeast.

Transcription through noncoding regions of the genome is pervasive. How these transcription events regulate gene expression remains poorly understood. Here, we report that, in S. cerevisiae, the levels of transcription through a noncoding region, IRT2, located upstream in the promoter of the inducer of meiosis, IME1, regulate opposing chromatin and transcription states. At low levels, the act of IRT2 transcription promotes histone exchange, delivering acetylated histone H3 lysine 56 to chromatin locally. The subsequent open chromatin state directs transcription factor recruitment and induces downstream transcription to repress the IME1 promoter and meiotic entry. Conversely, increasing transcription turns IRT2 into a repressor by promoting transcription-coupled chromatin assembly. The two opposing functions of IRT2 transcription shape a regulatory circuit, which ensures a robust cell-type-specific control of IME1 expression and yeast meiosis. Our data illustrate how intergenic transcription levels are key to controlling local chromatin state, gene expression, and cell fate outcomes.

Nutrient Signaling, Stress Response, and Inter-organelle Communication Are Non-canonical Determinants of Cell Fate.

Isogenic cells manifest distinct cellular fates for a single stress; however, the nongenetic mechanisms driving such fates remain poorly understood. Here, we implement a robust multi-channel live-cell imaging approach to uncover noncanonical factors governing cell fate. We show that in response to acute glucose removal (AGR), budding yeast undergoes distinct fates, becoming either quiescent or senescent. Senescent cells fail to resume mitotic cycles following glucose replenishment but remain responsive to nutrient stimuli. Whereas quiescent cells manifest starvation-induced adaptation, senescent cells display perturbed endomembrane trafficking and defective nucleus-vacuole junction (NVJ) expansion. Surprisingly, senescence occurs even in the absence of lipid droplets. Importantly, we identify the nutrient-sensing kinase Rim15 as a key biomarker predicting cell fates before AGR stress. We propose that isogenic yeast challenged with acute nutrient shortage contains determinants influencing post-stress fate and demonstrate that specific nutrient signaling, stress response, trafficking, and inter-organelle biomarkers are early indicators for long-term fate outcomes.

Functional interrelationships between carbohydrate and lipid storage, and mitochondrial activity during sporulation in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae under conditions of nutrient stress, meiosis precedes the formation of spores. Although the molecular mechanisms that regulate meiosis, such as meiotic recombination and nuclear divisions, have been extensively studied, the metabolic factors that determine the efficiency of sporulation are less understood. Here, we have directly assessed the relationship between metabolic stores and sporulation in S. cerevisiae by genetically disrupting the synthetic pathways for the carbohydrate stores, glycogen (gsy1/2Δ cells), trehalose (tps1Δ cells), or both (gsy1/2Δ and tps1Δ cells). We show that storage carbohydrate-deficient strains are highly inefficient in sporulation. Although glycogen and trehalose stores can partially compensate for each other, they have differential effects on sporulation rate and spore number. Interestingly, deletion of the G1 cyclin, CLN3, which resulted in an increase in cell size, mitochondria and lipid stores, partially rescued meiosis progression and spore ascus formation but not spore number in storage carbohydrate-deficient strains. Sporulation efficiency in the carbohydrate-deficient strain exhibited a greater dependency on mitochondrial activity and lipid stores than wild-type yeast. Taken together, our results provide new insights into the complex crosstalk between metabolic factors that support gametogenesis.

Yeast Ataxin-2 Forms an Intracellular Condensate Required for the Inhibition of TORC1 Signaling during Respiratory Growth.

Yeast ataxin-2, also known as Pbp1 (polyA binding protein-binding protein 1), is an intrinsically disordered protein implicated in stress granule formation, RNA biology, and neurodegenerative disease. To understand the endogenous function of this protein, we identify Pbp1 as a dedicated regulator of TORC1 signaling and autophagy under conditions that require mitochondrial respiration. Pbp1 binds to TORC1 specifically during respiratory growth, but utilizes an additional methionine-rich, low complexity (LC) region to inhibit TORC1. This LC region causes phase separation, forms reversible fibrils, and enables self-association into assemblies required for TORC1 inhibition. Mutants that weaken phase separation in vitro exhibit reduced capacity to inhibit TORC1 and induce autophagy. Loss of Pbp1 leads to mitochondrial dysfunction and reduced fitness during nutritional stress. Thus, Pbp1 forms a condensate in response to respiratory status to regulate TORC1 signaling.

A fully-automated, robust, and versatile algorithm for long-term budding yeast segmentation and tracking.

Live cell time-lapse microscopy, a widely-used technique to study gene expression and protein dynamics in single cells, relies on segmentation and tracking of individual cells for data generation. The potential of the data that can be extracted from this technique is limited by the inability to accurately segment a large number of cells from such microscopy images and track them over long periods of time. Existing segmentation and tracking algorithms either require additional dyes or markers specific to segmentation or they are highly specific to one imaging condition and cell morphology and/or necessitate manual correction. Here we introduce a fully automated, fast and robust segmentation and tracking algorithm for budding yeast that overcomes these limitations. Full automatization is achieved through a novel automated seeding method, which first generates coarse seeds, then automatically fine-tunes cell boundaries using these seeds and automatically corrects segmentation mistakes. Our algorithm can accurately segment and track individual yeast cells without any specific dye or biomarker. Moreover, we show how existing channels devoted to a biological process of interest can be used to improve the segmentation. The algorithm is versatile in that it accurately segments not only cycling cells with smooth elliptical shapes, but also cells with arbitrary morphologies (e.g. sporulating and pheromone treated cells). In addition, the algorithm is independent of the specific imaging method (bright-field/phase) and objective used (40X/63X/100X). We validate our algorithm's performance on 9 cases each entailing a different imaging condition, objective magnification and/or cell morphology. Taken together, our algorithm presents a powerful segmentation and tracking tool that can be adapted to numerous budding yeast single-cell studies.

Integration of Multiple Metabolic Signals Determines Cell Fate Prior to Commitment.

Cell-fate decisions are central to the survival and development of both uni- and multicellular organisms. It remains unclear when and to what degree cells can decide on future fates prior to commitment. This uncertainty stems from experimental and theoretical limitations in measuring and integrating multiple signals at the single-cell level during a decision process. Here, we combine six-color live-cell imaging with the Bayesian method of statistical evidence to study the meiosis/quiescence decision in budding yeast. Integration of multiple upstream metabolic signals predicts individual cell fates with high probability well before commitment. Cells "decide" their fates before birth, well before the activation of pathways characteristic of downstream cell fates. This decision, which remains stable through several cell cycles, occurs when multiple metabolic parameters simultaneously cross cell-fate-specific thresholds. Taken together, our results show that cells can decide their future fates long before commitment mechanisms are activated.

A regulatory circuit of two lncRNAs and a master regulator directs cell fate in yeast.

Transcription of long noncoding RNAs (lncRNAs) regulates local gene expression in eukaryotes. Many examples of how a single lncRNA controls the expression of an adjacent or nearby protein-coding gene have been described. Here we examine the regulation of a locus consisting of two contiguous lncRNAs and the master regulator for entry into yeast meiosis, IME1. We find that the cluster of two lncRNAs together with several transcription factors form a regulatory circuit by which IME1 controls its own promoter and thereby promotes its own expression. Inhibition or stimulation of this unusual feedback circuit affects timing and rate of IME1 accumulation, and hence the ability for cells to enter meiosis. Our data demonstrate that orchestrated transcription through two contiguous lncRNAs promotes local gene expression and determines a critical cell fate decision.