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.

Phospho-regulated Bim1/EB1 interactions trigger Dam1c ring assembly at the budding yeast outer kinetochore.

Kinetochores form the link between chromosomes and microtubules of the mitotic spindle. The heterodecameric Dam1 complex (Dam1c) is a major component of the Saccharomyces cerevisiae outer kinetochore, assembling into 3 MDa-sized microtubule-embracing rings, but how ring assembly is specifically initiated in vivo remains to be understood. Here, we describe a molecular pathway that provides local control of ring assembly during the establishment of sister kinetochore bi-orientation. We show that Dam1c and the general microtubule plus end-associated protein (+TIP) Bim1/EB1 form a stable complex depending on a conserved motif in the Duo1 subunit of Dam1c. EM analyses reveal that Bim1 crosslinks protrusion domains of adjacent Dam1c heterodecamers and promotes the formation of oligomers with defined curvature. Disruption of the Dam1c-Bim1 interaction impairs kinetochore localization of Dam1c in metaphase and delays mitosis. Phosphorylation promotes Dam1c-Bim1 binding by relieving an intramolecular inhibition of the Dam1 C-terminus. In addition, Bim1 recruits Bik1/CLIP-170 to Dam1c and induces formation of full rings even in the absence of microtubules. Our data help to explain how new kinetochore end-on attachments are formed during the process of attachment error correction.

Modeling the START transition in the budding yeast cell cycle.

Budding yeast, Saccharomyces cerevisiae, is widely used as a model organism to study the genetics underlying eukaryotic cellular processes and growth critical to cancer development, such as cell division and cell cycle progression. The budding yeast cell cycle is also one of the best-studied dynamical systems owing to its thoroughly resolved genetics. However, the dynamics underlying the crucial cell cycle decision point called the START transition, at which the cell commits to a new round of DNA replication and cell division, are under-studied. The START machinery involves a central cyclin-dependent kinase; cyclins responsible for starting the transition, bud formation, and initiating DNA synthesis; and their transcriptional regulators. However, evidence has shown that the mechanism is more complicated than a simple irreversible transition switch. Activating a key transcription regulator SBF requires the phosphorylation of its inhibitor, Whi5, or an SBF/MBF monomeric component, Swi6, but not necessarily both. Also, the timing and mechanism of the inhibitor Whi5's nuclear export, while important, are not critical for the timing and execution of START. Therefore, there is a need for a consolidated model for the budding yeast START transition, reconciling regulatory and spatial dynamics. We built a detailed mathematical model (START-BYCC) for the START transition in the budding yeast cell cycle based on established molecular interactions and experimental phenotypes. START-BYCC recapitulates the underlying dynamics and correctly emulates key phenotypic traits of ~150 known START mutants, including regulation of size control, localization of inhibitor/transcription factor complexes, and the nutritional effects on size control. Such a detailed mechanistic understanding of the underlying dynamics gets us closer towards deconvoluting the aberrant cellular development in cancer.

How to Survive without Water: A Short Lesson on the Desiccation Tolerance of Budding Yeast.

Water is essential to all life on earth. It is a major component that makes up living organisms and plays a vital role in multiple biological processes. It provides a medium for chemical and enzymatic reactions in the cell and is a major player in osmoregulation and the maintenance of cell turgidity. Despite this, many organisms, called anhydrobiotes, are capable of surviving under extremely dehydrated conditions. Less is known about how anhydrobiotes adapt and survive under desiccation stress. Studies have shown that morphological and physiological changes occur in anhydrobiotes in response to desiccation stress. Certain disaccharides and proteins, including heat shock proteins, intrinsically disordered proteins, and hydrophilins, play important roles in the desiccation tolerance of anhydrobiotes. In this review, we summarize the recent findings of desiccation tolerance in the budding yeast Saccharomyces cerevisiae. We also propose that the yeast under desiccation could be used as a model to study neurodegenerative disorders.

Evolutionary biology through the lens of budding yeast comparative genomics.

The budding yeast Saccharomyces cerevisiae is a highly advanced model system for studying genetics, cell biology and systems biology. Over the past decade, the application of high-throughput sequencing technologies to this species has contributed to this yeast also becoming an important model for evolutionary genomics. Indeed, comparative genomic analyses of laboratory, wild and domesticated yeast populations are providing unprecedented detail about many of the processes that govern evolution, including long-term processes, such as reproductive isolation and speciation, and short-term processes, such as adaptation to natural and domestication-related environments.

Cell-size regulation in budding yeast does not depend on linear accumulation of Whi5.

Cells must couple cell-cycle progress to their growth rate to restrict the spread of cell sizes present throughout a population. Linear, rather than exponential, accumulation of Whi5, was proposed to provide this coordination by causing a higher Whi5 concentration in cells born at a smaller size. We tested this model using the inducible GAL1 promoter to make the Whi5 concentration independent of cell size. At an expression level that equalizes the mean cell size with that of wild-type cells, the size distributions of cells with galactose-induced Whi5 expression and wild-type cells are indistinguishable. Fluorescence microscopy confirms that the endogenous and GAL1 promoters produce different relationships between Whi5 concentration and cell volume without diminishing size control in the G1 phase. We also expressed Cln3 from the GAL1 promoter, finding that the spread in cell sizes for an asynchronous population is unaffected by this perturbation. Our findings indicate that size control in budding yeast does not fundamentally originate from the linear accumulation of Whi5, contradicting a previous claim and demonstrating the need for further models of cell-cycle regulation to explain how cell size controls passage through Start.

Modeling the impact of single-cell stochasticity and size control on the population growth rate in asymmetrically dividing cells.

Microbial populations show striking diversity in cell growth morphology and lifecycle; however, our understanding of how these factors influence the growth rate of cell populations remains limited. We use theory and simulations to predict the impact of asymmetric cell division, cell size regulation and single-cell stochasticity on the population growth rate. Our model predicts that coarse-grained noise in the single-cell growth rate λ decreases the population growth rate, as previously seen for symmetrically dividing cells. However, for a given noise in λ we find that dividing asymmetrically can enhance the population growth rate for cells with strong size control (between a "sizer" and an "adder"). To reconcile this finding with the abundance of symmetrically dividing organisms in nature, we propose that additional constraints on cell growth and division must be present which are not included in our model, and we explore the effects of selected extensions thereof. Further, we find that within our model, epigenetically inherited generation times may arise due to size control in asymmetrically dividing cells, providing a possible explanation for recent experimental observations in budding yeast. Taken together, our findings provide insight into the complex effects generated by non-canonical growth morphologies.

Formulation of the biological control yeast Meyerozyma caribbica by electrospraying process: effect on postharvest control of anthracnose in mango (Mangifera indica L.) and papaya (Carica papaya L.).

BACKGROUND: Microorganism for biological control of fruit diseases is an eco-friendly alternative to the use of chemical fungicides. RESULTS: This is the first study evaluating the electrospraying process to encapsulate the biocontrol yeast Meyerozyma caribbica. The effect of encapsulating material [Wey protein concentrate (WPC), Fibersol® and Trehalose], its concentration and storage temperature on the cell viability of M. caribbica, and in vitro and in vivo control of Colletotrichum gloeosporioides was evaluated. The processing with commercial resistant maltodextrin (Fibersol®) 30% (w/v) as encapsulating material showed the highest initial cell viability (95.97 ± 1.01%). The storage at 4 ± 1 °C showed lower losses of viability compared to 25 ± 1 °C. Finally, the encapsulated yeast with Fibersol 30% w/v showed inhibitory activity against anthracnose in the in vitro and in vivo tests, similar to yeast fresh cells. CONCLUSION: Electrospraying was a highly efficient process due to the high cell viability, and consequently, a low quantity of capsules is required for the postharvest treatment of fruits. Additionally, the yeast retained its antagonistic power during storage. © 2021 Society of Chemical Industry.

Evolutionary engineering to improve Wickerhamomyces subpelliculosus and Kazachstania gamospora for baking.

The conventional baker's yeast, Saccharomyces cerevisiae, is the indispensable baking yeast of all times. Its monopoly coupled to its major drawbacks, such as streamlined carbon substrate utilisation base and a poor ability to withstand a number of baking associated stresses, prompt the need to search for alternative yeasts to leaven bread in the era of increasingly complex consumer lifestyles. Our previous work identified the inefficient baking attributes of Wickerhamomyces subpelliculosus and Kazachstania gamospora as well as preliminarily observations of improving the fermentative capacity of these potential alternative baker's yeasts using evolutionary engineering. Here we report on the characterisation and improvement in baking traits in five out of six independently evolved lines incubated for longer time and passaged for at least 60 passages relative to their parental strains as well as the conventional baker's yeast. In addition, the evolved clones produced bread with a higher loaf volume when compared to bread baked with either the ancestral strain or the control conventional baker's yeast. Remarkably, our approach improved the yeasts' ability to withstand baking associated stresses, a key baking trait exhibited poorly in both the conventional baker's yeast and their ancestral strains. W. subpelliculosus evolved the best characteristics attractive for alternative baker's yeasts as compared to the evolved K. gamospora strains. These results demonstrate the robustness of evolutionary engineering in development of alternative baker's yeasts.

Genome editing in the yeast Nakaseomyces delphensis and description of its complete sexual cycle.

The environmental yeast Nakaseomyces delphensis is, phylogenetically, the closest known species to Candida glabrata, a major fungal pathogen of humans. C. glabrata is haploid and described as asexual, while N. delphensis is also haploid, but has been described as competent for mating and meiosis. Both genomes contain homologues of all the genes necessary for sexual reproduction and also the genes for Ho-dependent mating-type switching, like Saccharomyces cerevisiae. We first report the construction of genetically engineered strains of N. delphensis, including by CRISPR-Cas 9 gene editing. We also report the description of the sexual cycle of N. delphensis. We show that it undergoes Ho-dependent mating-type switching in culture and that deletion of the HO gene prevents such switching and allows maintenance of stable, separate, MATa and MATalpha haploid strains. Rare, genetically selected diploids can be obtained through mating of haploid strains, mutated or not for the HO gene. In contrast to HO/HO diploids, which behave as expected, Δho/Δho diploids exhibit unusual profiles in flow cytometry. Both types of diploids can produce recombined haploid cells, which grow like the original haploid-type strain. Our experiments thus allow the genetic manipulation of N. delphensis and the reconstruction, in the laboratory, of its entire life cycle.

Identification of a novel gene controlling homothallism in the yeast Kazachstania naganishii isolated in Japan.

A novel gene controlling homothallic life cycle was identified in the yeast Kazachstania naganishii isolated in Japan. This gene was isolated by means of complementing a mutation, mti1, which had led to heterothallism from original homothallism in the yeast. The configuration of original mutation in MTI1 gene revealed that a truncated product is formed due to occurrence of a stop codon by a nucleotide insertion. When the gene was disrupted with a marker, the disruptant spore clone was haploid and stably heterothallic. Disfunction of the gene caused inability to self-diploidize due to defect of mating-type interconversion. The gene MTI1 (for Mating Type Interconversion) is a weak homolog of the Saccharomyces cerevisiae VID22/ENV11, which has been reported to function in vacuolar protein processing. K. naganishii has a gene representing significant homology with the HO gene of S. cerevisiae on chromosome V, which has not been clarified to be involved in regulation of life cycle in K. naganishii. The MTI1 gene defined in this study is located on K. naganishii chromosome IV and does not represent significant homology to the above ScHO-like gene and any other genes concerning life cycles of yeasts. From the viewpoint of gene evolution, it is extremely interesting that the MTI1 gene is a new type of gene controlling homothallism in addition to an HO-type gene, leading to discovery of an unknown mechanism regulating life cycles in yeasts.

The budding yeast life cycle: More complex than anticipated?

The budding yeast, Saccharomyces cerevisiae, has served as a model for nearly a century to understand the principles of the eukaryotic life cycle. The canonical life cycle of S. cerevisiae comprises a regular alternation between haploid and diploid phases. Haploid gametes generated by sporulation are expected to quickly restore the diploid phase mainly through inbreeding via intratetrad mating or haploselfing, thereby promoting genome homozygotization. However, recent large population genomics data unveiled that heterozygosity and polyploidy are unexpectedly common. This raises the interesting paradox of a haplo-diplobiontic species being well-adapted to inbreeding and able to maintain high levels of heterozygosity and polyploidy, thereby suggesting an unanticipated complexity of the yeast life cycle. Here, we propose that unprogrammed mating type switching, heterothallism, reduced spore formation and viability, cell-cell fusion and dioecy could play key and uncharted contributions to generate and maintain heterozygosity through polyploidization.

Cellular quiescence in budding yeast.

Cellular quiescence, the temporary and reversible exit from proliferative growth, is the predominant state of all cells. However, our understanding of the biological processes and molecular mechanisms that underlie cell quiescence remains incomplete. As with the mitotic cell cycle, budding and fission yeast are preeminent model systems for studying cellular quiescence owing to their rich experimental toolboxes and the evolutionary conservation across eukaryotes of pathways and processes that control quiescence. Here, we review current knowledge of cell quiescence in budding yeast and how it pertains to cellular quiescence in other organisms, including multicellular animals. Quiescence entails large-scale remodeling of virtually every cellular process, organelle, gene expression, and metabolic state that is executed dynamically as cells undergo the initiation, maintenance, and exit from quiescence. We review these major transitions, our current understanding of their molecular bases, and highlight unresolved questions. We summarize the primary methods employed for quiescence studies in yeast and discuss their relative merits. Understanding cell quiescence has important consequences for human disease as quiescent single-celled microbes are notoriously difficult to kill and quiescent human cells play important roles in diseases such as cancer. We argue that research on cellular quiescence will be accelerated through the adoption of common criteria, and methods, for defining cell quiescence. An integrated approach to studying cell quiescence, and a focus on the behavior of individual cells, will yield new insights into the pathways and processes that underlie cell quiescence leading to a more complete understanding of the life cycle of cells. TAKE AWAY: Quiescent cells are viable cells that have reversibly exited the cell cycle Quiescence is induced in response to a variety of nutrient starvation signals Quiescence is executed dynamically through three phases: initiation, maintenance, and exit Quiescence entails large-scale remodeling of gene expression, organelles, and metabolism Single-cell approaches are required to address heterogeneity among quiescent cells.

The budding yeast transition to quiescence.

A subset of Saccharomyces cerevisiae cells in a stationary phase culture achieve a unique quiescent state characterized by increased cell density, stress tolerance, and longevity. Trehalose accumulation is necessary but not sufficient for conferring this state, and it is not recapitulated by abrupt starvation. The fraction of cells that achieve this state varies widely in haploids and diploids and can approach 100%, indicating that both mother and daughter cells can enter quiescence. The transition begins when about half the glucose has been taken up from the medium. The high affinity glucose transporters are turned on, glycogen storage begins, the Rim15 kinase enters the nucleus and the accumulation of cells in G1 is initiated. After the diauxic shift (DS), when glucose is exhausted from the medium, growth promoting genes are repressed by the recruitment of the histone deacetylase Rpd3 by quiescence-specific repressors. The final division that takes place post-DS is highly asymmetrical and G1 arrest is complete after 48 h. The timing of these events can vary considerably, but they are tightly correlated with total biomass of the culture, suggesting that the transition to quiescence is tightly linked to changes in external glucose levels. After 7 days in culture, there are massive morphological changes at the protein and organelle level. There are global changes in histone modification. An extensive array of condensin-dependent, long-range chromatin interactions lead to genome-wide chromatin compaction that is conserved in yeast and human cells. These interactions are required for the global transcriptional repression that occurs in quiescent yeast.

Biodiversity of yeasts isolated during spontaneous fermentation of cool climate grape musts.

Biodiversity of native yeasts, especially in winemaking, has hidden potential. In order to use the value of non-Saccharomyces strains in wine production and to minimise the possibility of its deterioration, it is necessary to thoroughly study the yeast cultures present on grape fruits and in grape must, as well as their metabolic properties. The aim of the study was to characterise the yeast microbiota found during spontaneous fermentation of grape musts obtained from grape varieties 'Rondo', 'Regent' and 'Johanniter'. Grapes from two vineyards (Srebrna Góra and Zadora) located in southern Poland were used for the research. Succession of subsequent groups of yeasts was observed during the process. Metschnikowia pulcherrima yeasts were identified both at the beginning and the end of the process. Hanseniaspora uvarum, Wickerhamomyces onychis and Torulaspora delbrueckii strains were also identified during the fermentation. Torulaspora delbrueckii and Wickerhamomyces onychis strains were identified only in grape musts obtained from grapes of the Zadora vineyard. These strains may be characteristic of this vineyard and shape the identity of wines formed in it. Our research has provided specific knowledge on the biodiversity of yeast cultures on grapes and during their spontaneous fermentation. The research results presented indicate the possibility of using native strains for fermentation of grape musts, allowing to obtain a product with favourable chemical composition and sensory profile.

Augmented peroxisomal ROS buffering capacity renders oxidative and thermal stress cross-tolerance in yeast.

BACKGROUND: Thermotolerant yeast has outstanding potential in industrial applications. Komagataella phaffii (Pichia pastoris) is a common cell factory for industrial production of heterologous proteins. RESULTS: Herein, we obtained a thermotolerant K. phaffii mutant G14 by mutagenesis and adaptive evolution. G14 exhibited oxidative and thermal stress cross-tolerance and high heterologous protein production efficiency. The reactive oxygen species (ROS) level and lipid peroxidation in G14 were reduced compared to the parent. Oxidative stress response (OSR) and heat shock response (HSR) are two major responses to thermal stress, but the activation of them was different in G14 and its parent. Compared with the parent, G14 acquired the better performance owing to its stronger OSR. Peroxisomes, as the main cellular site for cellular ROS generation and detoxification, had larger volume in G14 than the parent. And, the peroxisomal catalase activity and expression level in G14 was also higher than that of the parent. Excitingly, the gene knockdown of CAT encoding peroxisomal catalase by dCas9 severely reduced the oxidative and thermal stress cross-tolerance of G14. These results suggested that the augmented OSR was responsible for the oxidative and thermal stress cross-tolerance of G14. Nevertheless, OSR was not strong enough to protect the parent from thermal stress, even when HSR was initiated. Therefore, the parent cannot recover, thereby inducing the autophagy pathway and resulting in severe cell death. CONCLUSIONS: Our findings indicate the importance of peroxisome and the significance of redox balance in thermotolerance of yeasts.

Species coexistence through simultaneous fluctuation-dependent mechanisms.

Understanding the origins and maintenance of biodiversity remains one of biology's grand challenges. From theory and observational evidence, we know that variability in environmental conditions through time is likely critical to the coexistence of competing species. Nevertheless, experimental tests of fluctuation-driven coexistence are rare and have typically focused on just one of two potential mechanisms, the temporal storage effect, to the neglect of the theoretically equally plausible mechanism known as relative nonlinearity of competition. We combined experiments and simulations in a system of nectar yeasts to quantify the relative contribution of the two mechanisms to coexistence. Resource competition models parameterized from single-species assays predicted the outcomes of mixed-culture competition experiments with 83% accuracy. Model simulations revealed that both mechanisms have measurable effects on coexistence and that relative nonlinearity can be equal or greater in magnitude to the temporal storage effect. In addition, we show that their effect on coexistence can be both antagonistic and complementary. These results falsify the common assumption that relative nonlinearity is of negligible importance, and in doing so reveal the importance of testing coexistence mechanisms in combination.

Association mapping, transcriptomics, and transient expression identify candidate genes mediating plant-pathogen interactions in a tree.

Invasive microbes causing diseases such as sudden oak death negatively affect ecosystems and economies around the world. The deployment of resistant genotypes for combating introduced diseases typically relies on breeding programs that can take decades to complete. To demonstrate how this process can be accelerated, we employed a genome-wide association mapping of ca 1,000 resequenced Populus trichocarpa trees individually challenged with Sphaerulina musiva, an invasive fungal pathogen. Among significant associations, three loci associated with resistance were identified and predicted to encode one putative membrane-bound L-type receptor-like kinase and two receptor-like proteins. A susceptibility-associated locus was predicted to encode a putative G-type D-mannose-binding receptor-like kinase. Multiple lines of evidence, including allele analysis, transcriptomics, binding assays, and overexpression, support the hypothesized function of these candidate genes in the P. trichocarpa response to S. musiva.

Influence of Non-Saccharomyces on Wine Chemistry: A Focus on Aroma-Related Compounds.

Wine fermentation processes are driven by complex microbial systems, which comprise eukaryotic and prokaryotic microorganisms that participate in several biochemical interactions with the must and wine chemicals and modulate the organoleptic properties of wine. Among these, yeasts play a fundamental role, since they carry out the alcoholic fermentation (AF), converting sugars to ethanol and CO2 together with a wide range of volatile organic compounds. The contribution of Saccharomyces cerevisiae, the reference organism associated with AF, has been extensively studied. However, in the last decade, selected non-Saccharomyces strains received considerable commercial and oenological interest due to their specific pro-technological aptitudes and the positive influence on sensory quality. This review aims to highlight the inter-specific variability within the heterogeneous class of non-Saccharomyces in terms of synthesis and release of volatile organic compounds during controlled AF in wine. In particular, we reported findings on the presence of model non-Saccharomyces organisms, including Torulaspora delbrueckii, Hanseniaspora spp,Lachancea thermotolerans, Metschnikowia pulcherrima, Pichia spp. and Candida zemplinina, in combination with S. cerevisiae. The evidence is discussed from both basic and applicative scientific perspective. In particular, the oenological significance in different kind of wines has been underlined.

Physiological performance of Kazachstania unispora in sourdough environments.

In this work we explored the potential of several strains of Kazachstania unispora to be used as non-conventional yeasts in sourdough fermentation. Properties such as carbohydrate source utilization, tolerance to different environmental factors and the performance in fermentation were evaluated. The K. unispora strains are characterized by rather restricted substrate utilization: only glucose and fructose supported the growth of the strains. However, the growth in presence of fructose was higher compared to a Saccharomyces cerevisiae commercial strain. Moreover, the inability to ferment maltose can be considered a positive characteristic in sourdoughs, where the yeasts can form a nutritional mutualism with maltose-positive Lactic Acid Bacteria. Tolerance assays showed that K. unispora strains are adapted to a sourdough environment: they were able to grow in conditions of high osmolarity, high acidity and in presence of organic acids, ethanol and salt. Finally, the performance in fermentation was comparable with the S. cerevisiae commercial strain. Moreover, the growth was more efficient, which is an advantage in obtaining the biomass in an industrial scale. Our data show that K. unispora strains have positive properties that should be explored further in bakery sector.