Multi-signal regulation of the GSK-3ß homolog Rim11 controls meiosis entry in budding yeast.

Starvation in diploid budding yeast cells triggers a cell-fate program culminating in meiosis and spore formation. Transcriptional activation of early meiotic genes (EMGs) hinges on the master regulator Ime1, its DNA-binding partner Ume6, and GSK-3ß kinase Rim11. Phosphorylation of Ume6 by Rim11 is required for EMG activation. We report here that Rim11 functions as the central signal integrator for controlling Ume6 phosphorylation and EMG transcription. In nutrient-rich conditions, PKA suppresses Rim11 levels, while TORC1 retains Rim11 in the cytoplasm. Inhibition of PKA and TORC1 induces Rim11 expression and nuclear localization. Remarkably, nuclear Rim11 is required, but not sufficient, for Rim11-dependent Ume6 phosphorylation. In addition, Ime1 is an anchor protein enabling Ume6 phosphorylation by Rim11. Subsequently, Ume6-Ime1 coactivator complexes form and induce EMG transcription. Our results demonstrate how various signaling inputs (PKA/TORC1/Ime1) converge through Rim11 to regulate EMG expression and meiosis initiation. We posit that the signaling-regulatory network elucidated here generates robustness in cell-fate control.

Meiotic prophase requires proteolysis of M phase regulators mediated by the meiosis-specific APC/CAma1.

Whereas proliferating cells enter M phase shortly after DNA replication, the first M phase of meiosis is preceded by an extended prophase in which homologous chromosomes undergo recombination. Exit from prophase I is controlled by the recombination checkpoint (RC), which, in yeast, represses the meiosis-specific transcription factor Ndt80 required for the expression of B-type cyclins and other M phase regulators. We show that an extended prophase I additionally requires the suppression of latent, mitotic cell-cycle controls by the anaphase-promoting complex (APC/C) and its meiosis-specific activator Ama1, which trigger the degradation of M phase regulators and Ndd1, a subunit of a mitotic transcription factor. ama1Δ mutants exit from prophase I prematurely and independently of the RC, which results in recombination defects and chromosome missegregation. Thus, control of prophase I by meiotic mechanisms depends on the suppression of the alternative, mitotic mechanisms by a meiosis-specific form of the APC/C.

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.

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.

Towards a Unified Understanding of Lithium Action in Basic Biology and its Significance for Applied Biology.

Lithium has literally been everywhere forever, since it is one of the three elements created in the Big Bang. Lithium concentration in rocks, soil, and fresh water is highly variable from place to place, and has varied widely in specific regions over evolutionary and geologic time. The biological effects of lithium are many and varied. Based on experiments in which animals are deprived of lithium, lithium is an essential nutrient. At the other extreme, at lithium ingestion sufficient to raise blood concentration significantly over 1 mM/, lithium is acutely toxic. There is no consensus regarding optimum levels of lithium intake for populations or individuals-with the single exception that lithium is a generally accepted first-line therapy for bipolar disorder, and specific dosage guidelines for sufferers of that condition are generally agreed on. Epidemiological evidence correlating various markers of social dysfunction and disease vs. lithium level in drinking water suggest benefits of moderately elevated lithium compared to average levels of lithium intake. In contrast to other biologically significant ions, lithium is unusual in not having its concentration in fluids of multicellular animals closely regulated. For hydrogen ions, sodium ions, potassium ions, calcium ions, chloride ions, and magnesium ions, blood and extracellular fluid concentrations are closely and necessarily regulated by systems of highly selective channels, and primary and secondary active transporters. Lithium, while having strong biological activity, is tolerated over body fluid concentrations ranging over many orders of magnitude. The lack of biological regulation of lithium appears due to lack of lithium-specific binding sites and selectivity filters. Rather lithium exerts its myriad physiological and biochemical effects by competing for macromolecular sites that are relatively specific for other cations, most especially for sodium and magnesium. This review will consider what is known about the nature of this competition and suggest using and extending this knowledge towards the goal of a unified understanding of lithium in biology and the application of that understanding in medicine and nutrition.

Deep learning-driven imaging of cell division and cell growth across an entire eukaryotic life cycle.

The life cycle of biomedical and agriculturally relevant eukaryotic microorganisms involves complex transitions between proliferative and non-proliferative states such as dormancy, mating, meiosis, and cell division. New drugs, pesticides, and vaccines can be created by targeting specific life cycle stages of parasites and pathogens. However, defining the structure of a microbial life cycle often relies on partial observations that are theoretically assembled in an ideal life cycle path. To create a more quantitative approach to studying complete eukaryotic life cycles, we generated a deep learning-driven imaging framework to track microorganisms across sexually reproducing generations. Our approach combines microfluidic culturing, life cycle stage-specific segmentation of microscopy images using convolutional neural networks, and a novel cell tracking algorithm, FIEST, based on enhancing the overlap of single cell masks in consecutive images through deep learning video frame interpolation. As proof of principle, we used this approach to quantitatively image and compare cell growth and cell cycle regulation across the sexual life cycle of Saccharomyces cerevisiae. We developed a fluorescent reporter system based on a fluorescently labeled Whi5 protein, the yeast analog of mammalian Rb, and a new High-Cdk1 activity sensor, LiCHI, designed to report during DNA replication, mitosis, meiotic homologous recombination, meiosis I, and meiosis II. We found that cell growth preceded the exit from non-proliferative states such as mitotic G1, pre-meiotic G1, and the G0 spore state during germination. A decrease in the total cell concentration of Whi5 characterized the exit from non-proliferative states, which is consistent with a Whi5 dilution model. The nuclear accumulation of Whi5 was developmentally regulated, being at its highest during meiotic exit and spore formation. The temporal coordination of cell division and growth was not significantly different across three sexually reproducing generations. Our framework could be used to quantitatively characterize other single-cell eukaryotic life cycles that remain incompletely described. An off-the-shelf user interface Yeastvision provides free access to our image processing and single-cell tracking algorithms.

Cdc14 plans autophagy for meiotic cell divisions.

The role of meiotic proteasome-mediated degradation has been extensively studied. At the same time, macroautophagy/autophagy only emerged recently as an essential regulator for meiosis progression. Our recent publication showed that autophagy in meiotic cells exhibits a temporal pattern distinct from that in quiescent cells or mitotic cells under prolonged starvation. Importantly, autophagic activity oscillates during meiotic cell divisions, i.e., meiosis I and meiosis II, which can accelerate meiotic progression and increase sporulation efficiency. Our in vitro and in vivo assays revealed that the conserved phosphatase Cdc14 stimulates autophagy initiation during meiotic divisions, specifically in anaphase I and II, when a subpopulation of active Cdc14 relocates to the cytosol and interacts with phagophore assembly sites (PAS) triggering the dephosphorylation of Atg13 to stimulate Atg1 kinase activity and autophagy. Together, our findings reveal a mechanism for the coordination of autophagy activity in the context of meiosis progression.

Cdc14 spatiotemporally dephosphorylates Atg13 to activate autophagy during meiotic divisions.

Autophagy is a conserved eukaryotic lysosomal degradation pathway that responds to environmental and cellular cues. Autophagy is essential for the meiotic exit and sporulation in budding yeast, but the underlying molecular mechanisms remain unknown. Here, we show that autophagy is maintained during meiosis and stimulated in anaphase I and II. Cells with higher levels of autophagy complete meiosis faster, and genetically enhanced autophagy increases meiotic kinetics and sporulation efficiency. Strikingly, our data reveal that the conserved phosphatase Cdc14 regulates meiosis-specific autophagy. Cdc14 is activated in anaphase I and II, accompanying its subcellular relocation from the nucleolus to the cytoplasm, where it dephosphorylates Atg13 to stimulate Atg1 kinase activity and thus autophagy. Together, our findings reveal a meiosis-tailored mechanism that spatiotemporally controls meiotic autophagy activity to ensure meiosis progression, exit, and sporulation.

Live-cell fluorescence spectral imaging as a data science challenge.

Live-cell fluorescence spectral imaging is an evolving modality of microscopy that uses specific properties of fluorophores, such as excitation or emission spectra, to detect multiple molecules and structures in intact cells. The main challenge of analyzing live-cell fluorescence spectral imaging data is the precise quantification of fluorescent molecules despite the weak signals and high noise found when imaging living cells under non-phototoxic conditions. Beyond the optimization of fluorophores and microscopy setups, quantifying multiple fluorophores requires algorithms that separate or unmix the contributions of the numerous fluorescent signals recorded at the single pixel level. This review aims to provide both the experimental scientist and the data analyst with a straightforward description of the evolution of spectral unmixing algorithms for fluorescence live-cell imaging. We show how the initial systems of linear equations used to determine the concentration of fluorophores in a pixel progressively evolved into matrix factorization, clustering, and deep learning approaches. We outline potential future trends on combining fluorescence spectral imaging with label-free detection methods, fluorescence lifetime imaging, and deep learning image analysis.

Cell cycle-independent integration of stress signals by Xbp1 promotes Non-G1/G0 quiescence entry.

Cellular quiescence is a nonproliferative state required for cell survival under stress and during development. In most quiescent cells, proliferation is stopped in a reversible state of low Cdk1 kinase activity; in many organisms, however, quiescent states with high-Cdk1 activity can also be established through still uncharacterized stress or developmental mechanisms. Here, we used a microfluidics approach coupled to phenotypic classification by machine learning to identify stress pathways associated with starvation-triggered high-Cdk1 quiescent states in Saccharomyces cerevisiae. We found that low- and high-Cdk1 quiescent states shared a core of stress-associated processes, such as autophagy, protein aggregation, and mitochondrial up-regulation, but differed in the nuclear accumulation of the stress transcription factors Xbp1, Gln3, and Sfp1. The decision between low- or high-Cdk1 quiescence was controlled by cell cycle-independent accumulation of Xbp1, which acted as a time-delayed integrator of the duration of stress stimuli. Our results show how cell cycle-independent stress-activated factors promote cellular quiescence outside G1/G0.

Meiosis Research in Orphan and Non-orphan Tropical Crops.

Plant breeding is directly linked to the development of crops that can effectively adapt to challenging conditions such as soil nutrient depletion, water pollution, drought, and anthropogenic climate change. These conditions are extremely relevant in developing countries already burdened with population growth and unchecked urban expansion, especially in the tropical global southern hemisphere. Engineering new crops thus has potential to enhance food security, prevent hunger, and spur sustainable agricultural growth. A major tool for the improvement of plant varieties in this context could be the manipulation of homologous recombination and genome haploidization during meiosis. The isolation or the design of mutations in key meiotic genes may facilitate DNA recombination and transmission of important genes quickly and efficiently. Genome haploidization through centromeric histone mutants could be an option to create new crosses rapidly. This review covers technical approaches to engineer key meiotic genes in tropical crops as a blueprint for future work and examples of tropical crops in which such strategies could be applied are given.

Biogenic silica-based microparticles obtained as a sub-product of the nanocellulose extraction process from pineapple peels.

Silica in plant tissues has been suggested as a component for enhancing mechanical properties, and as a physical barrier. Pineapples present in their shell and bracts rosette-like microparticles that could be associated to biogenic silica. In this study, we show for the first time that silica-based microparticles are co-purified during the extraction process of nanocellulose from pineapple (Ananas comosus). This shows that vegetable biomass could be an underappreciated source, not only for nanocellulose, but also for a highly valuable sub-product, like 10 µm biogenic rosette-like silica-based microparticles. The recovery yield obtained was 7.2 wt.%; based on the dried initial solid. Due to their size and morphology, the microparticles have potential applications as reinforcement in adhesives, polymer composites, in the biomedical field, and even as a source of silica for fertilizers.

Casein Kinase 1 Coordinates Cohesin Cleavage, Gametogenesis, and Exit from M Phase in Meiosis II.

Meiosis consists of DNA replication followed by two consecutive nuclear divisions and gametogenesis or spore formation. While meiosis I has been studied extensively, less is known about the regulation of meiosis II. Here we show that Hrr25, the conserved casein kinase 1δ of budding yeast, links three mutually independent key processes of meiosis II. First, Hrr25 induces nuclear division by priming centromeric cohesin for cleavage by separase. Hrr25 simultaneously phosphorylates Rec8, the cleavable subunit of cohesin, and removes from centromeres the cohesin protector composed of shugoshin and the phosphatase PP2A. Second, Hrr25 initiates the sporulation program by inducing the synthesis of membranes that engulf the emerging nuclei at anaphase II. Third, Hrr25 mediates exit from meiosis II by activating pathways that trigger the destruction of M-phase-promoting kinases. Thus, Hrr25 synchronizes formation of the single-copy genome with gamete differentiation and termination of meiosis.

Interchromatidal central ridge and transversal symmetry in early metaphasic human chromosome one.

The topographic structure of Giemsa-banded (G-banded) early metaphase human chromosomes adsorbed on glass was analyzed by atomic force microscope using amplitude modulation mode (AM-AFM). Longitudinal height measurements for early metaphasic human chromosomes showed a central ridge that was further characterized by transversal height measurements. The heterochromatic regions displayed a high level of transversal symmetry, while the euchromatic ones presented several peaks across the transversal height measurements. We suggest that this central ridge and symmetry patterns point out a transitional arrangement of the early metaphase chromosome and support evidence for interchromatidal interactions prior to disjunction.