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Time-lapse microscopy is a valuable, widely employed technique for investigating cell cycle dynamics. This chapter will guide you in using this tool to determine the duration of anaphase as a measure of mitotic exit kinetics in budding yeast cells. The methodology explained here is based on monitoring the mitotic spindle and provides a comprehensive guide covering all aspects, from sample preparation and microscopy to data analysis., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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.

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., (© 2024 Xiao et al.)

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., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2023 Parmar et al.)

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.

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., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

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.

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., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., Competing Interests: The authors have declared that no competing interests exist.

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% β-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., Competing Interests: Declaration of competing interest All authors declare no conflict of interest. The manuscript was read by all authors and approved for submission., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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.

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 β-galactosidase under FLD promoter, have been constructed., (© 2020 International Federation for Cell Biology.)

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.

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., (Copyright © 2020 Shor et al.)

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.

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 × 10 7 protoplasts/g fresh weight (FW) was obtained after 0.5 h treatment of 1 mg/ml mixed lytic enzymes in KH 2 PO 4 -K 2 HPO 4 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 × 10 3 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.

The Rho GTPase Cdc42 is highly conserved in structure and function. Mechanical or chemical cues in the microenvironment stimulate the localized activation of Cdc42 to rearrange the actin cytoskeleton and establish cell polarity. A role for Cdc42 in cell polarization was first discovered in the budding yeast Saccharomyces cerevisiae , and subsequently shown to also regulate directional motility in animal cells. Accordingly, in cancer Cdc42 promotes migration, invasion, and spread of tumor cells. Therefore, we targeted Cdc42 as a therapeutic strategy to treat metastatic breast cancer and designed the small molecule MBQ-167 as a potent inhibitor against Cdc42 and the homolog Rac. MBQ-167 inhibited cancer cell proliferation and migration in-vitro , and tumor growth and spread in-vivo in a mouse xenograft model of metastatic breast cancer. Since haploid budding yeast express a single Cdc42 gene, and do not express Rac, we used this well characterized model of polarization to define the contribution of Cdc42 inhibition to the effects of MBQ-167 in eukaryotic cells. Growth, budding pattern, and Cdc42 activity was determined in wildtype yeast or cells expressing a conditional knockdown of Cdc42 in response to vehicle or MBQ-167 treatment. As expected, growth and budding polarity were reduced by knocking-down Cdc42, with a parallel effect observed with MBQ-167. Cdc42 activity assays confirmed that MBQ-167 inhibits Cdc42 activation in yeast, and thus, bud polarity. Hence, we have validated MBQ-167 as a Cdc42 inhibitor in another biological context and present a method to screen Cdc42 inhibitors with potential as anti-metastatic cancer drugs.

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)., Competing Interests: The authors declare no competing interests., (© 2020 The Author(s).)

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 barrier to cost-efficient biomanufacturing is the instability of engineered genetic elements, such as plasmids. Instability can also manifest at the whole-genome level, when fungal dikaryons revert to parental species due to nuclear segregation during cell division. Here, we show that by encapsulating Saccharomyces cerevisiae-Pichia stipitis dikaryons in an alginate matrix, we can limit cell division and preserve their expanded metabolic capabilities. As a proxy to cellulosic ethanol production, we tested the capacity of such cells to carry out ethanologenic fermentation of glucose and xylose, examining substrate use, ploidy, and cell viability in relation to planktonic fusants, as well as in relation to planktonic and encapsulated cell cultures consisting of mixtures of these species. Glucose and xylose consumption and ethanol production by encapsulated dikaryons were greater than planktonic controls. Simultaneous co-fermentation did not occur; rather the order and kinetics of glucose and xylose catabolism by encapsulated dikaryons were similar to cultures where the two species were encapsulated together. Over repeated cycles of fed-batch culture, encapsulated S. cerevisiae-P. stipitis fusants exhibited a dramatic increase in genomic stability, relative to planktonic fusants. Encapsulation also increased the stability of antibiotic-resistance plasmids used to mark each species and preserved a fixed ratio of S. cerevisiae to P. stipitis cells in mixed cultures. Our data demonstrate how encapsulating cells in an extracellular matrix restricts cell division and, thereby, preserves the stability and biological activity of entities ranging from genomes to plasmids to mixed populations, each of which can be essential to cost-efficient biomanufacturing., (© 2020 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.)

Here, we describe a novel peroxin, Pex37, in the yeast Hansenula polymorpha. H. polymorpha Pex37 is a peroxisomal membrane protein, which belongs to a protein family that includes, among others, the Neurospora crassa Woronin body protein Wsc, the human peroxisomal membrane protein PXMP2, the Saccharomyces cerevisiae mitochondrial inner membrane protein Sym1, and its mammalian homologue MPV17. We show that deletion of H. polymorpha PEX37 does not appear to have a significant effect on peroxisome biogenesis or proliferation in cells grown at peroxisome-inducing growth conditions (methanol). However, the absence of Pex37 results in a reduction in peroxisome numbers and a defect in peroxisome segregation in cells grown at peroxisome-repressing conditions (glucose). Conversely, overproduction of Pex37 in glucose-grown cells results in an increase in peroxisome numbers in conjunction with a decrease in their size. The increase in numbers in PEX37-overexpressing cells depends on the dynamin-related protein Dnm1. Together our data suggest that Pex37 is involved in peroxisome fission in glucose-grown cells. Introduction of human PXMP2 in H. polymorpha pex37 cells partially restored the peroxisomal phenotype, indicating that PXMP2 represents a functional homologue of Pex37. H.polymorpha pex37 cells did not show aberrant growth on any of the tested carbon and nitrogen sources that are metabolized by peroxisomal enzymes, suggesting that Pex37 may not fulfill an essential function in transport of these substrates or compounds required for their metabolism across the peroxisomal membrane., (© 2019 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Sensor faults can impede the functionality of monitoring and control systems for bioprocesses. Hence, suitable systems need to be developed to validate the sensor readings prior to their use in monitoring and control systems. This study presents a novel approach for online validation of sensor readings. The basic idea is to compare the original sensor reading with predictions for this sensor reading based on the remaining sensor network's information. Deviations between original and predicted sensor readings are used to indicate sensor faults. Since especially batch processes show varying lengths and different phases (e.g., lag and exponential phase), prediction models that are dependent on process time are necessary. The binary particle swarm optimization algorithm is used to select the best prediction models for each time step. A regularization approach is utilized to avoid overfitting. Models with high complexity and prediction errors are penalized, resulting in optimal predictions for the sensor reading at each time step (5% mean relative prediction error). The sensor reliability is calculated by the Kullback-Leibler divergence between the distribution of model-based predictions and the distribution of a moving window of original sensor readings (moving window size = 10 readings). The developed system allows for the online detection of sensor faults. This is especially important when sensor data are used as input to soft sensors for critical quality attributes or the process control system. The proof-of-concept is exemplarily shown for a turbidity sensor that is used to monitor a Pichia pastoris-batch process.

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.

Production of healthy gametes in meiosis relies on the quality control and proper distribution of both nuclear and cytoplasmic contents. Meiotic differentiation naturally eliminates age-induced cellular damage by an unknown mechanism. Using time-lapse fluorescence microscopy in budding yeast, we found that nuclear senescence factors - including protein aggregates, extrachromosomal ribosomal DNA circles, and abnormal nucleolar material - are sequestered away from chromosomes during meiosis II and subsequently eliminated. A similar sequestration and elimination process occurs for the core subunits of the nuclear pore complex in both young and aged cells. Nuclear envelope remodeling drives the formation of a membranous compartment containing the sequestered material. Importantly, de novo generation of plasma membrane is required for the sequestration event, preventing the inheritance of long-lived nucleoporins and senescence factors into the newly formed gametes. Our study uncovers a new mechanism of nuclear quality control and provides insight into its function in meiotic cellular rejuvenation., Competing Interests: GK, JG, JS, KC, DJ, KM No competing interests declared, EÜ Reviewing editor, eLife, (© 2019, King et al.)

Cell-cycle checkpoints and DNA repair processes protect organisms from potentially lethal mutational damage. Compared to other budding yeasts in the subphylum Saccharomycotina, we noticed that a lineage in the genus Hanseniaspora exhibited very high evolutionary rates, low Guanine-Cytosine (GC) content, small genome sizes, and lower gene numbers. To better understand Hanseniaspora evolution, we analyzed 25 genomes, including 11 newly sequenced, representing 18/21 known species in the genus. Our phylogenomic analyses identify two Hanseniaspora lineages, a faster-evolving lineage (FEL), which began diversifying approximately 87 million years ago (mya), and a slower-evolving lineage (SEL), which began diversifying approximately 54 mya. Remarkably, both lineages lost genes associated with the cell cycle and genome integrity, but these losses were greater in the FEL. E.g., all species lost the cell-cycle regulator WHIskey 5 (WHI5), and the FEL lost components of the spindle checkpoint pathway (e.g., Mitotic Arrest-Deficient 1 [MAD1], Mitotic Arrest-Deficient 2 [MAD2]) and DNA-damage-checkpoint pathway (e.g., Mitosis Entry Checkpoint 3 [MEC3], RADiation sensitive 9 [RAD9]). Similarly, both lineages lost genes involved in DNA repair pathways, including the DNA glycosylase gene 3-MethylAdenine DNA Glycosylase 1 (MAG1), which is part of the base-excision repair pathway, and the DNA photolyase gene PHotoreactivation Repair deficient 1 (PHR1), which is involved in pyrimidine dimer repair. Strikingly, the FEL lost 33 additional genes, including polymerases (i.e., POLymerase 4 [POL4] and POL32) and telomere-associated genes (e.g., Repressor/activator site binding protein-Interacting Factor 1 [RIF1], Replication Factor A 3 [RFA3], Cell Division Cycle 13 [CDC13], Pbp1p Binding Protein [PBP2]). Echoing these losses, molecular evolutionary analyses reveal that, compared to the SEL, the FEL stem lineage underwent a burst of accelerated evolution, which resulted in greater mutational loads, homopolymer instabilities, and higher fractions of mutations associated with the common endogenously damaged base, 8-oxoguanine. We conclude that Hanseniaspora is an ancient lineage that has diversified and thrived, despite lacking many otherwise highly conserved cell-cycle and genome integrity genes and pathways, and may represent a novel, to our knowledge, system for studying cellular life without them., Competing Interests: The authors have declared that no competing interests exist.

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments in yeast are dynamic, and a full understanding of their properties requires time-lapse imaging. Multi-color 4D confocal fluorescence microscopy is a powerful method for tracking the behavior and composition of an intracellular compartment on a time scale of 5-15 min. Rigorous analysis of compartment dynamics requires the capture of thousands of optical sections. To achieve this aim, photobleaching and phototoxicity are minimized by scanning rapidly at very low laser power, and the pixel dimensions and Z-step intervals are set to the largest values that are compatible with sampling the image at full resolution. The resulting 4D data sets are noisy but can be smoothed by deconvolution. Even with high quality data, the analysis phase is challenging because intracellular structures are often numerous, heterogeneous, and mobile. To meet this need, custom ImageJ plugins were written to array 4D data on a computer screen, identify structures of interest, edit the data to isolate individual structures, quantify the fluorescence time courses, and make movies of the projected Z-stacks. 4D movies are particularly useful for distinguishing stable compartments from transient compartments that turn over by maturation. Such movies can also be used to characterize events such as compartment fusion, and to test the effects of specific mutations or other perturbations.

During meiosis, crossing over allows for the exchange of genes between homologous chromosomes, enabling their segregation and leading to genetic variation in the resulting gametes. Spo11, a topoisomerase-like protein expressed in eukaryotes, and diverse accessory factors induce programmed double-strand breaks (DSBs) to initiate meiotic recombination during the early phase of meiosis after DNA replication. DSBs are further repaired via meiosis-specific homologous recombination. Studies on budding yeast have provided insights into meiosis and genetic recombination and have improved our understanding of higher eukaryotic systems. Cohesin, a chromosome-associated multiprotein complex, mediates sister chromatid cohesion (SCC), and is conserved from yeast to humans. Diverse cohesin subunits in budding yeast have been identified in DNA metabolic pathways, such as DNA replication, chromosome segregation, recombination, DNA repair, and gene regulation. During cell cycle, SCC is established by multiple cohesin subunits, which physically bind sister chromatids together and modulate proteins that involve in the capturing and separation of sister chromatids. Cohesin components include at least four core subunits that establish and maintain SCC: two structural maintenance chromosome subunits (Smc1 and Smc3), an α-kleisin subunit (Mcd1/Scc1 during mitosis and Rec8 during meiosis), and Scc3/Irr1 (SA1 and SA2). In addition, the cohesin-associated factors Pds5 and Rad61 regulate structural modifications and cell cyclespecific dynamics of chromatin to ensure accurate chromosome segregation. In this review, we discuss SCC and the recombination pathway, as well as the relationship between the two processes in budding yeast, and we suggest a possible conserved mechanism for meiotic chromosome dynamics from yeast to humans.

We show that when cell-cell feedback is added to a model of the cell cycle for a large population of cells, then instability of the steady state solution occurs in many cases. We show this in the context of a generic agent-based ODE model. If the feedback is positive, then instability of the steady state solution is proved for all parameter values except for a small set on the boundary of parameter space. For negative feedback we prove instability for half the parameter space. We also show by example that instability in the other half may be proved on a case by case basis.

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., Competing Interests: The authors have declared that no competing interests exist.

The nucleolus is a membraneless organelle of the nucleus and the site of rRNA synthesis, maturation, and assembly into preribosomal particles. The nucleolus, organized around arrays of rRNA genes (rDNA), dissolves during prophase of mitosis in metazoans, when rDNA transcription ceases, and reforms in telophase, when rDNA transcription resumes. No such dissolution and reformation cycle exists in budding yeast, and the precise course of nucleolar segregation remains unclear. By quantitative live-cell imaging, we observed that the yeast nucleolus is reorganized in its protein composition during mitosis. Daughter cells received equal shares of preinitiation factors, which bind the RNA polymerase I promoter and the rDNA binding barrier protein Fob1, but only about one-third of RNA polymerase I and the processing factors Nop56 and Nsr1. The distribution bias was diminished in nonpolar chromosome segregation events observable in dyn1 mutants. Unequal distribution, however, was enhanced by defects in RNA polymerase I, suggesting that rDNA transcription supports nucleolar segregation. Indeed, quantification of pre-rRNA levels indicated ongoing rDNA transcription in yeast mitosis. These data, together with photobleaching experiments to measure nucleolar protein dynamics in anaphase, consolidate a model that explains the differential partitioning of nucleolar components in budding yeast mitosis.

Parameter estimation in discrete or continuous deterministic cell cycle models is challenging for several reasons, including the nature of what can be observed, and the accuracy and quantity of those observations. The challenge is even greater for stochastic models, where the number of simulations and amount of empirical data must be even larger to obtain statistically valid parameter estimates. The two main contributions of this work are (1) stochastic model parameter estimation based on directly matching multivariate probability distributions, and (2) a new quasi-Newton algorithm class QNSTOP for stochastic optimization problems. QNSTOP directly uses the random objective function value samples rather than creating ensemble statistics. QNSTOP is used here to directly match empirical and simulated joint probability distributions rather than matching summary statistics. Results are given for a current state-of-the-art stochastic cell cycle model of budding yeast, whose predictions match well some summary statistics and one-dimensional distributions from empirical data, but do not match well the empirical joint distributions. The nature of the mismatch provides insight into the weakness in the stochastic model.

Most cells exhibit a constant ratio between nuclear and cell volume. The mechanism dictating this constant ratio and the nuclear component(s) that scale with cell size are not known. To address this, we examined the consequences to the size and shape of the budding yeast nucleus when cell expansion is inhibited by down-regulating components of the secretory pathway. We find that under conditions where cell size increase is restrained, the nucleus becomes bilobed, with the bulk of the DNA in one lobe and the nucleolus in the other. The formation of bilobed nuclei is dependent on fatty acid and phospholipid synthesis, suggesting that it is associated with nuclear membrane expansion. Bilobed nuclei appeared predominantly after spindle pole body separation, suggesting that nuclear envelope expansion follows cell-cycle cues rather than cell size. Importantly, cells with bilobed nuclei had the same nuclear:cell volume ratio as cells with round nuclei. Therefore, the bilobed nucleus could be a consequence of continued NE expansion as cells traverse the cell cycle without an accompanying increase in nuclear volume due to the inhibition of cell growth. Our data suggest that nuclear volume is not determined by nuclear envelope availability but by one or more nucleoplasmic factors.

Prioritization of cell cycle-regulated genes from expression time-profiles is still an open problem. The point at issue is the surprisingly poor overlap among ranked lists obtained from different experimental protocols. Instead of developing a general-purpose computational methodology for detecting periodic signals, we focus on the budding yeast mitotic cell cycle. The reason being that the current availability of a total of 12 datasets, produced by 6 independent groups using 4 different synchronization methods, permits a re-analysis and re-consideration of this problem in a more reliable and extensive data domain. Notably, budding yeast is a model organism for studying cancer and testing new drugs. Here we propose a novel multi-feature score (called PERLA, PERiodicity, Regulation and Lag-Autocorrelation) that integrates different features of cell cycle-regulated gene expression time-profiles. We obtained increased performances on a wide range of benchmarks and, most importantly, a substantially increased overlap of the top ranking genes among different datasets, thus proving the effectiveness of the proposed prioritization algorithm. Examples on how to use PERLA to gain new insight into the biology of the cell cycle, are provided in a final dedicated section., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The size of a cell sets the scale for all biochemical processes within it, thereby affecting cellular fitness and survival. Hence, cell size needs to be kept within certain limits and relatively constant over multiple generations. However, how cells measure their size and use this information to regulate growth and division remains controversial. Here, we present two mechanistic mathematical models of the budding yeast (S. cerevisiae) cell cycle to investigate competing hypotheses on size control: inhibitor dilution and titration of nuclear sites. Our results suggest that an inhibitor-dilution mechanism, in which cell growth dilutes the transcriptional inhibitor Whi5 against the constant activator Cln3, can facilitate size homeostasis. This is achieved by utilising a positive feedback loop to establish a fixed size threshold for the Start transition, which efficiently couples cell growth to cell cycle progression. Yet, we show that inhibitor dilution cannot reproduce the size of mutants that alter the cell's overall ploidy and WHI5 gene copy number. By contrast, size control through titration of Cln3 against a constant number of genomic binding sites for the transcription factor SBF recapitulates both size homeostasis and the size of these mutant strains. Moreover, this model produces an imperfect 'sizer' behaviour in G1 and a 'timer' in S/G2/M, which combine to yield an 'adder' over the whole cell cycle; an observation recently made in experiments. Hence, our model connects these phenomenological data with the molecular details of the cell cycle, providing a systems-level perspective of budding yeast size control., Competing Interests: The authors have declared that no competing interests exist.

In yeast, the accumulation of unfolded proteins in the ER triggers the unfolded protein response (UPR) pathway, which is mediated by Hac1 transcription factor. Here, we characterized the function of a gene encoding Hac1 in the thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656 (OtHAC1). OtHAC1 mRNA contains a non-canonical intron of 176 nt, which was demonstrated to be spliced by RT-PCR. To characterize the function of this gene, we compared the proteome of a Othac1 mutant with wild-type. A total of 463 proteins with differential abundance were detected. The functions of these proteins were annotated in oxidative stress, metabolic pathways, transcription, translation, and of particular interest in secretory pathway. While many intracellular proteins differentially expressed in the mutant were similar to proteins with altered expression in UPR-stressed Saccharomyces cerevisiae, two novel OtHAC1-dependent proteins (Iml1 and Npr2) were identified that are potentially involved in the regulation of autophagy. The data show that OtHAC1 is an important regulator of several different processes in O. thermomethanolica TBRC656.

Budding yeast cells produce a finite number of daughter cells before they die. Why old yeast cells stop dividing and die is unclear. We found that age-induced accumulation of the G1/S-phase inhibitor Whi5 and defects in G1/S cyclin transcription cause cell cycle delays and genomic instability that result in cell death. We further identified extrachromosomal rDNA (ribosomal DNA) circles (ERCs) to cause the G1/S cyclin expression defect in old cells. Spontaneous segregation of Whi5 and ERCs into daughter cells rejuvenates old mothers, but daughters that inherit these aging factors die rapidly. Our results identify deregulation of the G1/S-phase transition as the proximal cause of age-induced proliferation decline and cell death in budding yeast., (© 2018 Neurohr et al.; Published by Cold Spring Harbor Laboratory Press.)

Asymmetric cell division generates cell diversity and contributes to cellular aging and rejuvenation. Here, we review the molecular mechanisms enabling budding yeast to recognize spindle pole bodies (SPB, centrosome equivalent) based on their age, and guide their non-random mitotic segregation: SPB inheritance requires the distinction of old from new SPBs and is regulated by the SPB-inheritance network (SPIN) and the mitotic exit network (MEN). The SPIN marks the pre-existing SPB as old and the MEN recognizes these marks translating them into spindle orientation. We next revisit other molecules and structures that partition depending on their age rather than their abundance at mitosis as, for example, DNA, centrosomes, mitochondria, and histones in yeast and other systems. The recurrence of this differential behavior suggests a functional significance for numerous cell types, which we then discuss. We conclude that non-random segregation may facilitate asymmetric cell fate determination and thereby indirectly aging and rejuvenation. Also see the video abstract here: https://youtu.be/1sQ4rAomnWY., (© 2018 ETH Zurich. BioEssays Published by Wiley Periodicals, Inc.)

The budding yeast centrosome, often called the spindle pole body (SPB), nucleates microtubules for chromosome segregation during cell division. An appendage, called the half bridge, attaches to one side of the SPB and regulates SPB duplication and separation. Like DNA, the SPB is duplicated only once per cell cycle. During meiosis, however, after one round of DNA replication, two rounds of SPB duplication and separation are coupled with homologue segregation in meiosis I and sister-chromatid segregation in meiosis II. How SPB duplication and separation are regulated during meiosis remains to be elucidated, and whether regulation in meiosis differs from that in mitosis is unclear. Here we show that overproduction of the half-bridge component Kar1 leads to premature SPB separation during meiosis. Furthermore, excessive Kar1 induces SPB overduplication to form supernumerary SPBs, leading to chromosome missegregation and erroneous ascospore formation. Kar1--mediated SPB duplication bypasses the requirement of dephosphorylation of Sfi1, another half-bridge component previously identified as a licensing factor. Our results therefore reveal an unexpected role of Kar1 in licensing meiotic SPB duplication and suggest a unique mechanism of SPB regulation during budding yeast meiosis.

The causal relationship between genomic instability and replicative aging is unclear. We reveal here that genomic instability at the budding yeast ribosomal DNA (rDNA) locus increases during aging, potentially due to the reduced cohesion that we uncovered during aging caused by the reduced abundance of multiple cohesin subunits, promoting increased global chromosomal instability. In agreement, cohesion is lost during aging at other chromosomal locations in addition to the rDNA, including centromeres. The genomic instability in old cells is exacerbated by a defect in DNA double-strand break (DSB) repair that we uncovered in old yeast. This was due to limiting levels of key homologous recombination proteins because overexpression of Rad51 or Mre11 reduced the accumulation of DSBs and largely restored DSB repair in old cells. We propose that increased rDNA instability and the reduced DSB repair capacity of old cells contribute to the progressive accumulation of global chromosomal DNA breaks, where exceeding a threshold of genomic DNA damage ends the replicative life span.

Motivation: Although high-content image cytometry is becoming increasingly routine, processing the large amount of data acquired during time-lapse experiments remains a challenge. The majority of approaches for automated single-cell segmentation focus on flat, uniform fields of view covered with a single layer of cells. In the increasingly popular microfluidic devices that trap individual cells for long term imaging, these conditions are not met. Consequently, most techniques for segmentation perform poorly. Although potentially constraining the generalizability of software, incorporating information about the microfluidic features, flow of media and the morphology of the cells can substantially improve performance., Results: Here we present DISCO (Data Informed Segmentation of Cell Objects), a framework for using the physical constraints imposed by microfluidic traps, the shape based morphological constraints of budding yeast and temporal information about cell growth and motion to allow tracking and segmentation of cells in microfluidic devices. Using manually curated datasets, we demonstrate substantial improvements in both tracking and segmentation when compared with existing software., Availability and Implementation: The MATLAB code for the algorithm and for measuring performance is available at https://github.com/pswain/segmentation-software and the test images and the curated ground-truth results used for comparing the algorithms are available at http://datashare.is.ed.ac.uk/handle/10283/2002., Contact: mcrane2@uw.edu., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com)

Although a lot of effort has been put into the search for factors responsible for aging in yeast mother cells, our knowledge of cellular changes in daughter cells originating from old mothers is still very limited. It has been shown that an old mother is not able to compensate for all negative changes within its cell and therefore transfers them to the bud. In this paper, we show for the first time that daughter cells of an old mother have a reset lifespan expressed in units of time despite drastic reduction of their budding lifespan, which suggests that a single yeast cell has a fixed programmed longevity regardless of the time point at which it was originated. Moreover, in our study we found that longevity parameters are not correlated with the rDNA level, DNA damage, chromosome structure or aging parameters (budding lifespan and total lifespan).

Ribonucleotide reductase (RNR) provides the precursors for the generation of dNTPs, which are required for DNA synthesis and repair. Here, we investigated the function of the major RNR subunits Rnr1 and Rnr3 in telomere elongation in budding yeast. We show that Rnr1 is essential for the sustained elongation of short telomeres by telomerase. In the absence of Rnr1, cells harbor very short, but functional, telomeres, which cannot become elongated by increased telomerase activity or by tethering of telomerase to telomeres. Furthermore, we demonstrate that Rnr1 function is critical to prevent an early onset of replicative senescence and premature survivor formation in telomerase-negative cells but dispensable for telomere elongation by Homology-Directed-Repair. Our results suggest that telomerase has a "basal activity" mode that is sufficient to compensate for the "end-replication-problem" and does not require the presence of Rnr1 and a different "sustained activity" mode necessary for the elongation of short telomeres, which requires an upregulation of dNTP levels and dGTP ratios specifically through Rnr1 function. By analyzing telomere length and dNTP levels in different mutants showing changes in RNR complex composition and activity we provide evidence that the Mec1ATR checkpoint protein promotes telomere elongation by increasing both dNTP levels and dGTP ratios through Rnr1 upregulation in a mechanism that cannot be replaced by its homolog Rnr3.

Genome replication, a key process for a cell, relies on stochastic initiation by replication origins, causing a variability of replication timing from cell to cell. While stochastic models of eukaryotic replication are widely available, the link between the key parameters and overall replication timing has not been addressed systematically. We use a combined analytical and computational approach to calculate how positions and strength of many origins lead to a given cell-to-cell variability of total duration of the replication of a large region, a chromosome or the entire genome. Specifically, the total replication timing can be framed as an extreme-value problem, since it is due to the last region that replicates in each cell. Our calculations identify two regimes based on the spread between characteristic completion times of all inter-origin regions of a genome. For widely different completion times, timing is set by the single specific region that is typically the last to replicate in all cells. Conversely, when the completion time of all regions are comparable, an extreme-value estimate shows that the cell-to-cell variability of genome replication timing has universal properties. Comparison with available data shows that the replication program of three yeast species falls in this extreme-value regime., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Cells under stress transition from a growth to a quiescent state. The conventional thinking is that this is achieved through transcriptional programs, translational regulation, protein degradation, and post-translational modifications. However, there is an increasing realization that stress adaptation also goes along with dramatic changes in the architecture and organization of cells. In particular, it seems to involve the formation of membrane-less compartments and macromolecular assemblies. We propose that cells make widespread use of this ability to change macromolecular organization to adapt to stress conditions and protect themselves. Here, we address what triggers the formation of these assemblies under stress conditions. We present examples illustrating that in some cases, sophisticated signaling pathways transmit environmental fluctuations from the outside to the inside and in others, that external fluctuations directly affect the internal conditions in cells. We further argue that changes in the organization of the cytoplasm and the formation of membrane-less compartments have many advantages over other ways of altering protein function, such as protein degradation, translation or transcription. Furthermore, membrane-less compartments may act as protective devices for key cellular components., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

The budding yeast Polo-like kinase Cdc5 is a key regulator of many mitotic events. Cdc5 coordinates its functions spatially and temporally by changing its localization during the cell cycle: Cdc5 is imported into the nucleus in G2 phase and released to the cytoplasm in anaphase, where it accumulates at the bud neck. Cdc5 also localizes to the spindle pole bodies (SPBs) from S phase until the end of mitosis. Whether Cdc5 changes its SPB population during the cell cycle is not known. We find that Cdc5 localizes to distinct SPB subpopulations, depending on the mitotic stage. Cdc5 localizes to the nuclear side of the SPBs during metaphase and early anaphase and to the cytoplasmic surface of the SPBs during late anaphase. Cdc14 is necessary to relocalize Cdc5 from the nuclear SPB plaque. Accumulation of Cdc5 at the daughter SPB in late anaphase is controlled by Bfa1. We also show that Cdc5 and Bfa1 are found in spatially distinct locations at the SPBs during G2/M arrest after DNA damage. Collectively our data reveal that Cdc5 is a dynamic component of the SPBs during mitosis and provide new insight into its regulation during both late mitotic events and DNA damage-induced G2/M arrest., (© 2017 Botchkarev et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

The highly conserved family of Phosphoprotein phosphatases (PPP) regulates several major physiological processes in yeast. However, very little is known about the PPP orthologs from the yeast species inhabiting extreme environmental niches. In the present study we have identified DhSIT4, a member of PPP6 class of serine threonine phosphatases from the halotolerant yeast Debaryomyces hansenii. Deletion of DhSIT4 in D. hansenii was not lethal but the mutant exhibited reduced growth due to its effect on the cell cycle. The knock out mutant Dhsit4Δ showed sensitivity towards Li + , Na + and cell wall damaging agents. The expression of DhSit4p rescued salt, caffeine and calcofluor white sensitivity of Dhmpk1Δ strain and thereby indicating a genetic interaction of this phosphatase with the cell wall integrity pathway in this species. Our study also demonstrated the antagonistic roles of DhSit4p and DhPpz1p in maintaining the cell cycle and ion homeostasis in D. hansenii., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Parameter estimation in systems biology is typically done by enforcing experimental observations through an objective function as the parameter space of a model is explored by numerical simulations. Past studies have shown that one usually finds a set of "feasible" parameter vectors that fit the available experimental data equally well, and that these alternative vectors can make different predictions under novel experimental conditions. In this study, we characterize the feasible region of a complex model of the budding yeast cell cycle under a large set of discrete experimental constraints in order to test whether the statistical features of relative protein abundance predictions are influenced by the topology of the cell cycle regulatory network., Results: Using differential evolution, we generate an ensemble of feasible parameter vectors that reproduce the phenotypes (viable or inviable) of wild-type yeast cells and 110 mutant strains. We use this ensemble to predict the phenotypes of 129 mutant strains for which experimental data is not available. We identify 86 novel mutants that are predicted to be viable and then rank the cell cycle proteins in terms of their contributions to cumulative variability of relative protein abundance predictions. Proteins involved in "regulation of cell size" and "regulation of G1/S transition" contribute most to predictive variability, whereas proteins involved in "positive regulation of transcription involved in exit from mitosis," "mitotic spindle assembly checkpoint" and "negative regulation of cyclin-dependent protein kinase by cyclin degradation" contribute the least. These results suggest that the statistics of these predictions may be generating patterns specific to individual network modules (START, S/G2/M, and EXIT). To test this hypothesis, we develop random forest models for predicting the network modules of cell cycle regulators using relative abundance statistics as model inputs. Predictive performance is assessed by the areas under receiver operating characteristics curves (AUC). Our models generate an AUC range of 0.83-0.87 as opposed to randomized models with AUC values around 0.50., Conclusions: By using differential evolution and random forest modeling, we show that the model prediction statistics generate distinct network module-specific patterns within the cell cycle network.