Dynamic Live Cell Imaging of Budding Yeast Meiosis.

For over a century, major advances in understanding meiosis have come from the use of microscopy-based methods. Studies using the budding yeast, Saccharomyces cerevisiae, have made important contributions to our understanding of meiosis because of the facility with which budding yeast can be manipulated as a genetic model organism. In contrast, imaging-based approaches with budding yeast have been constrained by the small size of its chromosomes. The advent of advances in fluorescent chromosome tagging techniques has made it possible to use yeast more effectively for imaging-based approaches as well. This protocol describes live cell imaging methods that can be used to monitor chromosome movements throughout meiosis in living yeast cells.

An integrative temperature-controlled microfluidic system for budding yeast heat shock response analysis at the single-cell level.

Cells can respond and adapt to complex forms of environmental change. Budding yeast is widely used as a model system for these stress response studies. In these studies, the precise control of the environment with high temporal resolution is most important. However, there is a lack of single-cell research platforms that enable precise control of the temperature and form of cell growth. This has hindered our understanding of cellular coping strategies in the face of diverse forms of temperature change. Here, we developed a novel temperature-controlled microfluidic platform that integrates a microheater (using liquid metal) and a thermocouple (liquid metal vs. conductive PDMS) on a chip. Three forms of temperature changes (step, gradient, and periodical oscillations) were realized by automated equipment. The platform has the advantages of low cost and a simple fabrication process. Moreover, we investigated the nuclear entry and exit behaviors of the transcription factor Msn2 in yeast in response to heat stress (37 °C) with different heating modes. The feasibility of this temperature-controlled platform for studying the protein dynamic behavior of yeast cells was demonstrated.

The phospholipids cardiolipin and phosphatidylethanolamine differentially regulate MDC biogenesis.

Cells utilize multiple mechanisms to maintain mitochondrial homeostasis. We recently characterized a pathway that remodels mitochondria in response to metabolic alterations and protein overload stress. This remodeling occurs via the formation of large membranous structures from the mitochondrial outer membrane called mitochondrial-derived compartments (MDCs), which are eventually released from mitochondria and degraded. Here, we conducted a microscopy-based screen in budding yeast to identify factors that regulate MDC formation. We found that two phospholipids, cardiolipin (CL) and phosphatidylethanolamine (PE), differentially regulate MDC biogenesis. CL depletion impairs MDC biogenesis, whereas blocking mitochondrial PE production leads to constitutive MDC formation. Additionally, in response to metabolic MDC activators, cellular and mitochondrial PE declines, and overexpressing mitochondrial PE synthesis enzymes suppress MDC biogenesis. Altogether, our data indicate a requirement for CL in MDC biogenesis and suggest that PE depletion may stimulate MDC formation downstream of MDC-inducing metabolic stress.

Robust microtubule dynamics facilitate low-tension kinetochore detachment in metaphase.

During mitosis, sister chromatids are stretched apart at their centromeres via their attachment to oppositely oriented kinetochore microtubules. This stretching generates inwardly directed tension across the separated sister centromeres. The cell leverages this tension signal to detect and then correct potential errors in chromosome segregation, via a mechanical tension signaling pathway that detaches improperly attached kinetochores from their microtubules. However, the sequence of events leading up to these detachment events remains unknown. In this study, we used microfluidics to sustain and observe low-tension budding yeast metaphase spindles over multiple hours, allowing us to elucidate the tension history prior to a detachment event. We found that, under conditions in which kinetochore phosphorylation weakens low-tension kinetochore-microtubule connections, the mechanical forces produced via the dynamic growth and shortening of microtubules is required to efficiently facilitate detachment events. Our findings underscore the critical role of robust kinetochore microtubule dynamics in ensuring the fidelity of chromosome segregation during mitosis.

Performance of xylose-fermenting yeasts in oat and soybean hulls hydrolysate and improvement of ethanol production using immobilized cell systems.

We investigated the fermentation of a mixture of oat and soybean hulls (1:1) subjected to acid (AH) or enzymatic (EH) hydrolyses, with both showing high osmotic pressures (> 1200 Osm kg-1) for the production of ethanol. Yeasts of genera Spathaspora, Scheffersomyces, Sugiymaella, and Candida, most of them biodiverse Brazilian isolates and previously untested in bioprocesses, were cultivated in these hydrolysates. Spathaspora passalidarum UFMG-CM-469 showed the best ethanol production kinetics in suspended cells cultures in acid hydrolysate, under microaerobic and anaerobic conditions. This strain was immobilized in LentiKats® (polyvinyl alcohol) and cultured in AH and EH. Supplementation of hydrolysates with crude yeast extract and peptone was also performed. The highest ethanol production was obtained using hydrolysates supplemented with crude yeast extract (AH-CYE and EH-CYE) showing yields of 0.40 and 0.44 g g-1, and productivities of 0.39 and 0.29 g (L h)-1, respectively. The reuse of the immobilized cells was tested in sequential fermentations of AH-CYE, EH-CYE, and a mixture of acid and enzymatic hydrolysates (AEH-CYE) operated under batch fluidized bed, with ethanol yields ranging from 0.31 to 0.40 g g-1 and productivities from 0.14 to 0.23 g (L h)-1. These results warrant further research using Spathaspora yeasts for second-generation ethanol production.

The Effects of Photosensitizing Dyes Fagopyrin and Hypericin on Planktonic Growth and Multicellular Life in Budding Yeast.

Naphthodianthrones such as fagopyrin and hypericin found mainly in buckwheat (Fagopyrum spp.) and St. John's wort (SJW) (Hypericum perforatum L.) are natural photosensitizers inside the cell. The effect of photosensitizers was studied under dark conditions on growth, morphogenesis and induction of death in Saccharomyces cerevisiae. Fagopyrin and hypericin induced a biphasic and triphasic dose response in cellular growth, respectively, over a 10-fold concentration change. In fagopyrin-treated cells, disruptions in the normal cell cycle progression were evident by microscopy. DAPI staining revealed several cells that underwent premature mitosis without budding, a striking morphological abnormality. Flow Cytometric (FC) analysis using a concentration of 100 µM showed reduced cell viability by 41% in fagopyrin-treated cells and by 15% in hypericin-treated cells. FC revealed the development of a secondary population of G1 cells in photosensitizer-treated cultures characterized by small size and dense structures. Further, we show that fagopyrin and the closely related hypericin altered the shape and the associated fluorescence of biofilm-like structures. Colonies grown on solid medium containing photosensitizer had restricted growth, while cell-to-cell adherence within the colony was also affected. In conclusion, the photosensitizers under dark conditions affected culture growth, caused toxicity, and disrupted multicellular growth, albeit with different efficiencies.

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.

Changes in growth kinetic parameters, morphology and mitotic activity of yeasts Candida guilliermondii exposed to the low-intensity waves of 51.8-GHz frequency.

Under the influence of electromagnetic waves of millimeter range with the frequency of 51.8 GHz, changes in the morphology, growth parameters and mitotic activity of yeasts C. guilliermondii NP-4 are revealed. Filamentous and giant cells appeared in a population of exposed yeasts. The sigmoid shape of the growth curve remained but the lag phase duration was increased by 2 h in comparison with non-exposed yeasts; accordingly, the log and stationary phases followed 2 h later. The specific growth rate in the log growth phase and colony-forming ability of exposed yeasts was decreased. It is suggested that yeasts have some response mechanisms to 51.8-GHz frequency electromagnetic waves. The results can be used to understand the response mechanisms of microorganisms to non-ionizing radiation, as well as to develop approaches to protect living organisms from it. The effect of electromagnetic waves of 51.8-GHz frequency to suppress yeasts can be applied in biotechnology and medicine.

Nitrogen supplementation ameliorates product quality and quantity during high cell density bioreactor studies of Pichia pastoris: A case study with proteolysis prone streptokinase.

Streptokinase is a well-established cost-effective therapeutic molecule for thrombo-embolic complications. In the current study, a tag-free variant of streptokinase with a native N-terminus (N-rSK) was developed using the Pichia expression system. A three-copy clone was screened that secreted 1062 mg/L of N-rSK in the complex medium at shake flask level. The biologically active (67,552.61 IU/mg) N-rSK recovered by anion exchange chromatography was predicted to contain 15.43% α-helices, 26.43% ß-sheets. The fermentation run in a complex medium yielded a poor quality product due to excessive N-rSK degradation. Therefore, modified basal salt medium was also employed during fermentation operations to reduce the proteolytic processing of the recombinant product. The concomitant feeding of 1 g/L/h soya flour hydrolysate with methanol during the protein synthesis phase reduced the proteolysis and yielded 2.29 g/L of N-rSK. The fermentation medium was also supplemented with urea during growth and induction phases. The combined feeding approach of nitrogen-rich soya flour hydrolysate and urea during bioreactor operations showed significant improvement in protein stability and resulted in a 4-fold increase in N-rSK concentration to a level of 4.03 g/L over shake flask. Under optimized conditions, the volumetric productivity and specific product yield were 52.33 mg/L/h and 33.24 mg/g DCW, respectively.

Investigation of daughter cell dissection coincidence of single budding yeast cells immobilized in microfluidic traps.

Microfluidic methodologies allow for automatic and high-throughput replicative lifespan (RLS) determination of single budding yeast cells. However, the resulted RLS is highly impacted by the robustness of experimental conditions, especially the microfluidic yeast-trapping structures, which are designed for cell retention, growth, budding, and daughter cell dissection. In this work, four microfluidic yeast-trapping structures, which were commonly used to immobilize mother cells and remove daughter cells for entire lifespan of budding yeast, were systematically investigated by means of finite element modeling (FEM). The results from this analysis led us to propose an optimized design, the yeast rotation (YRot) trap, which is a "leaky bowl"-shaped structure composed of two mirrored microcolumns facing each other. The YRot trap enables stable retention of mother cells in its "bowl" and hydrodynamic rotation of buds into its "leaky orifice" such that matured progenies can be dissected in a coincident direction. We validated the functions of the YRot trap in terms of cell rotation and daughter dissection by both FEM simulations and experiments. With the integration of denser YRot traps in microchannels, the microfluidic platform with stable single-yeast immobilization, long-term cell culturing, and coincident daughter dissection could potentially improve the robustness of experimental conditions for precise RLS determination in yeast aging studies.

A Novel Hyperactive Nud1 Mitotic Exit Network Scaffold Causes Spindle Position Checkpoint Bypass in Budding Yeast.

Mitotic exit is a critical cell cycle transition that requires the careful coordination of nuclear positioning and cyclin B destruction in budding yeast for the maintenance of genome integrity. The mitotic exit network (MEN) is a Ras-like signal transduction pathway that promotes this process during anaphase. A crucial step in MEN activation occurs when the Dbf2-Mob1 protein kinase complex associates with the Nud1 scaffold protein at the yeast spindle pole bodies (SPBs; centrosome equivalents) and thereby becomes activated. This requires prior priming phosphorylation of Nud1 by Cdc15 at SPBs. Cdc15 activation, in turn, requires both the Tem1 GTPase and the Polo kinase Cdc5, but how Cdc15 associates with SPBs is not well understood. We have identified a hyperactive allele of NUD1, nud1-A308T, that recruits Cdc15 to SPBs in all stages of the cell cycle in a CDC5-independent manner. This allele leads to early recruitment of Dbf2-Mob1 during metaphase and requires known Cdc15 phospho-sites on Nud1. The presence of nud1-A308T leads to loss of coupling between nuclear position and mitotic exit in cells with mispositioned spindles. Our findings highlight the importance of scaffold regulation in signaling pathways to prevent improper activation.

Fructose-1,6-bisphosphatase degradation in the methylotrophic yeast Komagataella phaffii occurs in autophagy pathway.

Many enzymes of methanol metabolism of methylotrophic yeasts are located in peroxisomes whereas some of them have the cytosolic localization. After shift of methanol-grown cells of methylotrophic yeasts to glucose medium, a decrease in the activity of cytosolic (formaldehyde dehydrogenase, formate dehydrogenase, and fructose-1,6-bisphosphatase [FBP]) along with peroxisomal enzymes of methanol metabolism is observed. Mechanisms of inactivation of cytosolic enzymes remain unknown. To study the mechanism of FBP inactivation, the changes in its specific activity of the wild type strain GS200, the strain with the deletion of the GSS1 hexose sensor gene and strain defected in autophagy pathway SMD1163 of Komagataella phaffii with or without the addition of the MG132 (proteasome degradation inhibitor) were investigated after shift of methanol-grown cells in glucose medium. Western blot analysis showed that inactivation of FBP in GS200 occurred due to protein degradation whereas inactivation in the strains SMD1163 and gss1Δ was negligible in such conditions. The effect of the proteasome inhibitor MG132 on FBP inactivation was insignificant. To confirm FBP degradation pathway, the recombinant strains with GFP-labeled Fbp1 of K. phaffii and red fluorescent protein-labeled peroxisomes were constructed on the background of GS200 and SMD1163. The fluorescent microscopy analysis of the constructed strains was performed using the vacuolar membrane dye FM4-64. Microscopic data confirmed that Fbp1 degrades by autophagy pathway in K. phaffii. K. phaffii transformants, which express heterologous ß-galactosidase under FLD promoter, have been constructed.

R-loops at centromeric chromatin contribute to defects in kinetochore integrity and chromosomal instability in budding yeast.

R-loops, the byproduct of DNA-RNA hybridization and the displaced single-stranded DNA (ssDNA), have been identified in bacteria, yeasts, and other eukaryotic organisms. The persistent presence of R-loops contributes to defects in DNA replication and repair, gene expression, and genomic integrity. R-loops have not been detected at centromeric (CEN) chromatin in wild-type budding yeast. Here we used an hpr1∆ strain that accumulates R-loops to investigate the consequences of R-loops at CEN chromatin and chromosome segregation. We show that Hpr1 interacts with the CEN-histone H3 variant, Cse4, and prevents the accumulation of R-loops at CEN chromatin for chromosomal stability. DNA-RNA immunoprecipitation (DRIP) analysis showed an accumulation of R-loops at CEN chromatin that was reduced by overexpression of RNH1 in hpr1∆ strains. Increased levels of ssDNA, reduced levels of Cse4 and its assembly factor Scm3, and mislocalization of histone H3 at CEN chromatin were observed in hpr1∆ strains. We determined that accumulation of R-loops at CEN chromatin contributes to defects in kinetochore biorientation and chromosomal instability (CIN) and these phenotypes are suppressed by RNH1 overexpression in hpr1∆ strains. In summary, our studies provide mechanistic insights into how accumulation of R-loops at CEN contributes to defects in kinetochore integrity and CIN.

A Noncanonical DNA Damage Checkpoint Response in a Major Fungal Pathogen.

DNA damage checkpoints are key guardians of genome integrity. Eukaryotic cells respond to DNA damage by triggering extensive phosphorylation of Rad53/CHK2 effector kinase, whereupon activated Rad53/CHK2 mediates further aspects of checkpoint activation, including cell cycle arrest and transcriptional changes. Budding yeast Candida glabrata, closely related to model eukaryote Saccharomyces cerevisiae, is an opportunistic pathogen characterized by high genetic diversity and rapid emergence of drug-resistant mutants. However, the mechanisms underlying this genetic variability are unclear. We used Western blotting and mass spectrometry to show that, unlike S. cerevisiae, C. glabrata cells exposed to DNA damage did not induce C. glabrata Rad53 (CgRad53) phosphorylation. Furthermore, flow cytometry analysis showed that, unlike S. cerevisiae, C. glabrata cells did not accumulate in S phase upon DNA damage. Consistent with these observations, time-lapse microscopy showed C. glabrata cells continuing to divide in the presence of DNA damage, resulting in mitotic errors and cell death. Finally, transcriptome sequencing (RNAseq) analysis revealed transcriptional rewiring of the DNA damage response in C. glabrata and identified several key protectors of genome stability upregulated by DNA damage in S. cerevisiae but downregulated in C. glabrata, including proliferating cell nuclear antigen (PCNA). Together, our results reveal a noncanonical fungal DNA damage response in C. glabrata, which may contribute to rapidly generating genetic change and drug resistance.IMPORTANCE In order to preserve genome integrity, all cells must mount appropriate responses to DNA damage, including slowing down or arresting the cell cycle to give the cells time to repair the damage and changing gene expression, for example to induce genes involved in DNA repair. The Rad53 protein kinase is a conserved central mediator of these responses in eukaryotic cells, and its extensive phosphorylation upon DNA damage is necessary for its activation and subsequent activity. Interestingly, here we show that in the opportunistic fungal pathogen Candida glabrata, Rad53 phosphorylation is not induced by DNA damage, nor do these cells arrest in S phase under these conditions, in contrast to the closely related yeast Saccharomyces cerevisiae Instead, C. glabrata cells continue to divide in the presence of DNA damage, resulting in significant cell lethality. Finally, we show that a number of genes involved in DNA repair are strongly induced by DNA damage in S. cerevisiae but repressed in C. glabrata Together, these findings shed new light on mechanisms regulating genome stability in fungal pathogens.

Nitrogen Starvation and Stationary Phase Lipophagy Have Distinct Molecular Mechanisms.

In yeast, the selective autophagy of intracellular lipid droplets (LDs) or lipophagy can be induced by either nitrogen (N) starvation or carbon limitation (e.g., in the stationary (S) phase). We developed the yeast, Komagataella phaffii (formerly Pichia pastoris), as a new lipophagy model and compared the N-starvation and S-phase lipophagy in over 30 autophagy-related mutants using the Erg6-GFP processing assay. Surprisingly, two lipophagy pathways had hardly overlapping stringent molecular requirements. While the N-starvation lipophagy strictly depended on the core autophagic machinery (Atg1-Atg9, Atg18, and Vps15), vacuole fusion machinery (Vam7 and Ypt7), and vacuolar proteolysis (proteinases A and B), only Atg6 and proteinases A and B were essential for the S-phase lipophagy. The rest of the proteins were only partially required in the S-phase. Moreover, we isolated the prl1 (for the positive regulator of lipophagy 1) mutant affected in the S-phase lipophagy, but not N-starvation lipophagy. The prl1 defect was at a stage of delivery of the LDs from the cytoplasm to the vacuole, further supporting the mechanistically different nature of the two lipophagy pathways. Taken together, our results suggest that N-starvation and S-phase lipophagy have distinct molecular mechanisms.

Protocol for Titrating Gene Expression Levels in Budding Yeast.

The biological phenotype is affected by the level of gene expression. Here, we provide a step-by-step protocol for precisely titrating and quantitatively observing the target gene expression level in budding yeast by manipulating its copy number in the genome. Using this method, we construct various strains with different gene copy numbers of the cell cycle inhibitor Whi5. This protocol enables stable and inherent control of gene expression at the expected level with fluorescent intensity as the quantitative readout. For complete details on the use and execution of this protocol, please refer to Qu et al. (2019).

Enhancement of protoplast preparation and regeneration of Hirsutella sinensis based on process optimization.

OBJECTIVE: To explore the optimal methods for the protoplast preparation and regeneration of Hirsutella sinensis by optimizing the limiting factors. RESULTS: During the treatment of enzymatic protoplast preparation, mycelium cultured for 7 days was the optimal start material. The maximum protoplast preparation rate of 4.3 × 107 protoplasts/g fresh weight (FW) was obtained after 0.5 h treatment of 1 mg/ml mixed lytic enzymes in KH2PO4-K2HPO4 buffer (pH 5.5) with 0.6 M KCl at 18 °C. As for the protoplast regeneration, the maximum protoplast regeneration rate reached 12.32% through 5 × 103 protoplasts mL-1 cultivated for 20 days in the regeneration medium with 0.6 M mannitol and 1.5% agar. CONCLUSIONS: The preparation and regeneration of H. sinensis protoplasts was firstly established based on process optimization and it provided a foundation for the study of H. sinensis mutagenesis.

Cell size sets the diameter of the budding yeast contractile ring.

The formation and maintenance of subcellular structures and organelles with a well-defined size is a key requirement for cell function, yet our understanding of the underlying size control mechanisms is limited. While budding yeast cell polarization and subsequent assembly of a septin ring at the site of bud formation has been successfully used as a model for biological self-assembly processes, the mechanisms that set the size of the septin ring at the bud neck are unknown. Here, we use live-cell imaging and genetic manipulation of cell volume to show that the septin ring diameter increases with cell volume. This cell-volume-dependence largely accounts for modulations of ring size due to changes in ploidy and genetic manipulation of cell polarization. Our findings suggest that the ring diameter is set through the dynamic interplay of septin recruitment and Cdc42 polarization, establishing it as a model for size homeostasis of self-assembling organelles.

A white-to-opaque-like phenotypic switch in the yeast Torulaspora microellipsoides.

Torulaspora microellipsoides is an under-characterized budding yeast of the Saccharomycetaceae family that is primarily associated with viticulture. Here we report for the first time to our knowledge that T. microellipsoides undergoes a low-frequency morphological switch from small budding haploid (white) yeast to larger, higher ploidy (opaque) yeast. Comparison of transcriptomes by mRNA-seq revealed 511 differentially regulated genes, with white cells having greater expression of genes involved in stress resistance and complex carbohydrate utilization, and opaque cells up-regulating genes involved in ribosome biogenesis. Growth assays showed that white cells are physiologically more resistant to stationary-phase conditions and oxidative stress, whereas opaque cells exhibited greater cold tolerance. We propose that phenotypic switching in T. microellipsoides is an ecological adaptation, as has been suggested for similar morphological switching in distantly related species like Candida albicans, and we propose that this switching is a more broadly utilized biological strategy among yeasts than previously thought.