Budding Yeast Cells Research Articles

Oxidative stress triggers aggregation of GFP-tagged Hsp31p, the budding yeast environmental stress response chaperone, and glyoxalase III

The Saccharomyces cerevisiae Hsp31p protein belongs to the ubiquitous DJ-1/ThiJ/PfpI family. The most prominent member of this family is human DJ-1; defects of this protein are associated with Parkinson’s disease pathogenesis. Numerous recent findings reported by our group and others have revealed the importance of Hsp31p for survival in the post-diauxic phase of cell growth and under diverse environmental stresses. Hsp31p was shown to possess glutathione-independent glyoxalase III activity and to function as a protein chaperone, suggesting that it has multiple cellular roles. Our previous work also revealed that HSP31 gene expression was controlled by multiple stress-related transcription factors, which mediated HSP31 promoter responses to oxidative, osmotic, and thermal stresses, toxic products of glycolysis, and the diauxic shift. Nevertheless, the exact role of Hsp31p within budding yeast cells remains elusive. Here, we aimed to obtain insights into the function of Hsp31p based on its intracellular localization. We have demonstrated that the Hsp31p-GFP fusion protein is localized to the cytosol under most environmental conditions and that it becomes particulate in response to oxidative stress. However, the particles do not colocalize with other granular subcellular structures present in budding yeast cells. The observed particulate localization does not seem to be important for Hsp31p functionality. Instead, it is likely the result of oxidative damage, as the particle abundance increases when Hsp31p is nonfunctional, when the cellular oxidative stress response is affected, or when cellular maintenance systems that optimize the state of the proteome are compromised.

Is Aggregate-Dependent Yeast Aging Fortuitous? A Model of Damage Segregation and Aggregate Dynamics

During cytokinesis in Saccharomyces cerevisiae, damaged proteins are distributed unequally between the daughter and mother cells. The retention of these proteins is correlated with yeast aging. Even though evidence suggests that aggregates are retained due to an underlying molecular mechanism, the debate on whether an active mechanism is necessary for this asymmetry remains unsolved. In particular, passive diffusion and a bud-specific dilution remain as possible explanations. Here, a computational and mathematical model is provided to test whether passive mechanisms alone are sufficient to account for the aggregate distribution patterns and the aggregate kinetics observed in living cells. To this author’s knowledge, this is the most comprehensive model available on this subject and the only one combining key potentially essential passive-only mechanisms proposed in existing bibliography—namely, the geometrical effect of the dividing yeast cell on the diffusion of protein aggregates, and the possibility of aggregate binding and aggregate formation at different rates. Results suggest that although passive processes alone can reproduce certain averaged observables from experimental bibliography, they are insufficient to vindicate aggregate activity observed in living budding yeast cells. These results are complemented by showing that under basic forms of active quality control, discrepancies between the outputs of the model and experimental bibliography are reduced.

Immunoprecipitation of RNA:DNA Hybrids from Budding Yeast.

During transcription, the nascent transcript behind an elongating RNA polymerase (RNAP) can invade the DNA duplex and hybridize with the complementary DNA template strand, generating a three-stranded "R-loop" structure, composed of an RNA:DNA duplex and an unpaired non-template DNA strand. R-loops can be strongly associated with actively transcribed loci by all RNAPs including the mitochondrial RNA polymerase (mtRNAP). In this chapter, we describe two protocols for the detection of RNA:DNA hybrids in living budding yeast cells, one that uses conventional chromatin immunoprecipitation (ChIP-qPCR) and one that uses DNA:RNA immunoprecipitation (DRIP-qPCR). Both protocols make use of the S9.6 antibody, which is believed to recognize the intermediate A/B helical RNA:DNA duplex conformation, with no sequence specificity.

Observation of live chromatin dynamics in cells via 3D localization microscopy using Tetrapod point spread functions.

We report the observation of chromatin dynamics in living budding yeast (Saccharomyces cerevisiae) cells, in three-dimensions (3D). Using dual color localization microscopy and employing a Tetrapod point spread function, we analyze the spatio-temporal dynamics of two fluorescently labeled DNA loci surrounding the GAL locus. From the measured trajectories, we obtain different dynamical characteristics in terms of inter-loci distance and temporal variance; when the GAL locus is activated, the 3D inter-loci distance and temporal variance increase compared to the inactive state. These changes are visible in spite of the large thermally- and biologically-driven heterogeneity in the relative motion of the two loci. Our observations are consistent with current euchromatin vs. heterochromatin models.

Quasi-Newton Stochastic Optimization Algorithm for Parameter Estimation of a Stochastic Model of the Budding Yeast Cell Cycle.

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.

Second-generation method for analysis of chromatin binding with formaldehyde–cross-linking kinetics

Formaldehyde-cross-linking underpins many of the most commonly used experimental approaches in the chromatin field, especially in capturing site-specific protein-DNA interactions. Extending such assays to assess the stability and binding kinetics of protein-DNA interactions is more challenging, requiring absolute measurements with a relatively high degree of physical precision. We previously described an experimental framework called the cross-linking kinetics (CLK) assay, which uses time-dependent formaldehyde-cross-linking data to extract kinetic parameters of chromatin binding. Many aspects of formaldehyde behavior in cells are unknown or undocumented, however, and could potentially affect CLK data analyses. Here, we report biochemical results that better define the properties of formaldehyde-cross-linking in budding yeast cells. These results have the potential to inform interpretations of "standard" chromatin assays, including chromatin immunoprecipitation. Moreover, the chemical complexity we uncovered resulted in the development of an improved method for measuring binding kinetics with the CLK approach. Optimum conditions included an increased formaldehyde concentration and more robust glycine-quench conditions. Notably, we observed that formaldehyde-cross-linking rates can vary dramatically for different protein-DNA interactions in vivo Some interactions were cross-linked much faster than the in vivo macromolecular interactions, making them suitable for kinetic analysis. For other interactions, we found the cross-linking reaction occurred on the same time scale or slower than binding dynamics; for these interactions, it was sometimes possible to compute the in vivo equilibrium-binding constant but not binding on- and off-rates. This improved method yields more accurate in vivo binding kinetics estimates on the minute time scale.

Antibiotic sensitivity pattern of pathogens in children with urinary tract infection in a tertiary care hospital in Kachchh, Gujarat, India

Background: This prospective observational study was conducted in a tertiary care hospital in Kachchh, over a period of 6 months, to know the antibiotic sensitivity pattern of pathogens in children less than 18 years old with Urinary Tract Infection (UTI).Methods: Between December 2016 to June 2017, 186 children met the inclusion criterion. Urine samples were collected and processed for urine routine microscopy, culture and antibiotic sensitivity as per the standard laboratory guidelines. Urine culture was positive in 50 cases, which were further evaluated to find out any renal disease. All patients were given 10-14 days antibiotics course and follow-up urine reports were done. Patients were considered cured when the follow-up urine reports were normal.Results: E. coli was the commonest organism (34%) isolated, found mainly in 1-5 years age group (20%). Enterococci were isolated in 32% cases, most of which were less than 5 years of age (28%). Klebsiella (12%), Methicillin Resistant Staphylococcus Aureus (MRSA) (10%), Coagulase negative staphylococci (6%), Pseudomonas (2%) and Budding yeast cell (4%) were the less frequent organisms isolated. E. coli were found to be less sensitive to different Aminoglycosides (11.7% - 23.5%), Cephalosporins (11.7% - 52.9%), Fluoroquinolones (5.8% - 11.7%), Co-trimoxazole (17.6%) and Piperacillin (17.6%), but were more sensitive (70.6%) to Imipenems. Enterococci were also partially sensitive to Aminoglycosides (6.2-18.7%), Cephalosporins (25-37.5.7%), Fluoroquinolones (6.25-12.5%), Penicillin-G (50%), Piperacillin (31.25%) and Co-trimoxazole (43.75%), but had good sensitivity for Imipenems (68.7%), Linezolid (75%) and Vancomycin (81.2%). Similarly, MRSA was 100% sensitive to Linezolid and Imipenem but partially sensitive (20-60%) to other antibiotics. Klebsiella showed 16.65-33.3% sensitivity to all antibiotics except Imipenem (83.3%) and was 100% resistant to Co-trimoxazole. Coagulase negative Staphylococci (CONS) remained 100% sensitive to all antibiotics and Pseudomonas was resistant to all antibiotics.Conclusions: The study concludes that pathogens for UTI in children have developed resistance, even to the newer generation antibiotics, probably due to the irrational use of antibiotics. In view of emergence of multi drug resistant pathogens, which carry considerable morbidity and mortality, every effort must be taken to use antibiotics judiciously.

Molecular Genetic Characterization of Mutagenesis Using a Highly Sensitive Single-Stranded DNA Reporter System in Budding Yeast.

Mutations are permanent alterations to the coding content of DNA. They are starting material for the Darwinian evolution of species by natural selection, which has yielded an amazing diversity of life on Earth. Mutations can also be the fundamental basis of serious human maladies, most notably cancers. In this chapter, I describe a highly sensitive reporter system for the molecular genetic analysis of mutagenesis, featuring controlled generation of long stretches of single-stranded DNA in budding yeast cells. This system is ~100- to ~1000-fold more susceptible to mutation than conventional double-stranded DNA reporters, and is well suited for generating large mutational datasets to investigate the properties of mutagens.

The Adder Phenomenon Emerges from Independent Control of Pre- and Post-Start Phases of the Budding Yeast Cell Cycle

Although it has long been clear that cells actively regulate their size, the molecular mechanisms underlying this regulation have remained poorly understood. In budding yeast, cell size primarily modulates the duration of the cell-division cycle by controlling the G1/S transition known as Start. We have recently shown that the rate of progression through Start increases with cell size, because cell growth dilutes the cell-cycle inhibitor Whi5 in G1. Recent phenomenological studies in yeast and bacteria have shown that these cells add an approximately constant volume during each complete cell cycle, independent of their size at birth. These results seem to be in conflict, as the phenomenological studies suggest that cells measure the amount they grow, rather than their size, and that size control acts over the whole cell cycle, rather than specifically in G1. Here, we propose an integrated model that unifies the adder phenomenology with the molecular mechanism of G1/S cell-size control. We use single-cell microscopy to parameterize a full cell-cycle model based on independent control of pre- and post-Start cell-cycle periods. We find that our model predicts the size-independent amount of cell growth during the full cell cycle. This suggests that the adder phenomenon is an emergent property of the independent regulation of pre- and post-Start cell-cycle periods rather than the consequence of an underlying molecular mechanism measuring a fixed amount of growth.

The abundance of Fob1 modulates the efficiency of rRFBs to stall replication forks.

In eukaryotes, ribosomal genes (rDNA) are organized in tandem repeats localized in one or a few clusters. Each repeat encompasses a transcription unit and a non-transcribed spacer. Replication forks moving in the direction opposite to transcription are blocked at specific sites called replication fork barriers (rRFBs) in the non-transcribed spacer close to the 3′ end of the transcription unit. Here, we investigated and quantified the efficiency of rRFBs in Saccharomyces cerevisiae and to this end transfected budding yeast cells that express dissimilar quantities of Fob1 with circular minichromosomes containing different copies of the minimal 20-bp DNA segment that bind Fob1. To identify fork stalling we used high-resolution 2D agarose gel electrophoresis. The results obtained indicated that neighbor DNA sequences and the relative abundance of Fob1 modulate the efficiency of rRFBs to stall replication forks.

NOD-Like Receptor P3 Inflammasome Controls Protective Th1/Th17 Immunity against Pulmonary Paracoccidioidomycosis.

The NOD-like receptor P3 (NLRP3) inflammasome is an intracellular multimeric complex that triggers the activation of inflammatory caspases and the maturation of IL-1β and IL-18, important cytokines for the innate immune response against pathogens. The functional NLRP3 inflammasome complex consists of NLRP3, the adaptor protein apoptosis-associated speck-like protein, and caspase-1. Various molecular mechanisms were associated with NLRP3 activation including the presence of extracellular ATP, recognized by the cell surface P2X7 receptor (P2X7R). Several pattern recognition receptors on innate immune cells recognize Paracoccidioides brasiliensis components resulting in diverse responses that influence adaptive immunity and disease outcome. However, the role of NLRP3 inflammasome was scantily investigated in pulmonary paracoccidioidomycosis (PCM), leading us to use an intratracheal (i.t.) model of infection to study the influence of this receptor in anti-fungal immunity and severity of infection. For in vivo studies, C57BL/6 mice deficient for several NLRP3 inflammasome components (Nlrp3−/−, Casp1/11−/−, Asc−/−) as well as deficient for ATP receptor (P2x7r−/−) were infected via i.t. with P. brasiliensis and several parameters of immunity and disease severity analyzed at the acute and chronic periods of infection. Pulmonary PCM was more severe in Nlrp3−/−, Casp1/11−/−, Asc−/−, and P2x7r−/− mice as demonstrated by the increased fungal burdens, mortality rates and tissue pathology developed. The more severe disease developed by NLRP3, ASC, and Caspase-1/11 deficient mice was associated with decreased production of IL-1β and IL-18 and reduced inflammatory reactions mediated by PMN leukocytes and activated CD4+ and CD8+ T cells. The decreased T cell immunity was concomitant with increased expansion of CD4+CD25+Foxp3 regulatory T (Treg) cells. Characterization of intracellular cytokines showed a persistent reduction of CD4+ and CD8+ T cells expressing IFN-γ and IL-17 whereas those producing IL-4 and TGF-β appeared in increased frequencies. Histopathological studies showed that all deficient mouse strains developed more severe lesions containing elevated numbers of budding yeast cells resulting in increased mortality rates. Altogether, these findings led us to conclude that the activation of the NLRP3 inflammasome has a crucial role in the immunoprotection against pulmonary PCM by promoting the expansion of Th1/Th17 immunity and reducing the suppressive control mediated by Treg cells.

Systematic Analysis of the DNA Damage Response Network in Telomere Defective Budding Yeast.

Functional telomeres are critically important to eukaryotic genetic stability. Scores of proteins and pathways are known to affect telomere function. Here, we report a series of related genome-wide genetic interaction screens performed on budding yeast cells with acute or chronic telomere defects. Genetic interactions were examined in cells defective in Cdc13 and Stn1, affecting two components of CST, a single stranded DNA (ssDNA) binding complex that binds telomeric DNA. For comparison, genetic interactions were also examined in cells with defects in Rfa3, affecting the major ssDNA binding protein, RPA, which has overlapping functions with CST at telomeres. In more complex experiments, genetic interactions were measured in cells lacking EXO1 or RAD9, affecting different aspects of the DNA damage response, and containing a cdc13-1 induced telomere defect. Comparing fitness profiles across these data sets helps build a picture of the specific responses to different types of dysfunctional telomeres. The experiments show that each context reveals different genetic interactions, consistent with the idea that each genetic defect causes distinct molecular defects. To help others engage with the large volumes of data, the data are made available via two interactive web-based tools: Profilyzer and DIXY. One particularly striking genetic interaction observed was that the chk1∆ mutation improved fitness of cdc13-1 exo1∆ cells more than other checkpoint mutations (ddc1∆, rad9∆, rad17∆, and rad24∆), whereas, in cdc13-1 cells, the effects of all checkpoint mutations were similar. We show that this can be explained by Chk1 stimulating resection—a new function for Chk1 in the eukaryotic DNA damage response network.

Antihyperlipidemic activity of Cassia auriculata flower extract in oleic acid induced hyperlipidemia in Saccharomyces cerevisiae.

The Cassia auriculataherb has been traditionally used in India for medicinal purposes to treat hyperglycemia, diabetes, rheumatism, asthma, and skin diseases. In the present study, ethanolic extract ofCassia auriculataflower (Et-CAF) depicted anti-hyperlipidemic effect in the budding yeast cells. The hyperlipidemic conditions were induced in the yeast cells with oleic acid which showed an increase in triacylglycerol (TAG) and sterol esters (SE), and was supported by the mRNA expression of LRO1 and DGA1 (involved in TAG formation); as well as ARE1 and ARE2 (involved in SE formation). The anti-hyperlipidemic effect by the Et-CAF was compared with the commercial drug Atorvastatin. The lipid droplets were increased in the hyperlipidemic yeast cell that was observed under the confocal microscope with BODIPY staining; Atorvastatin and Et-CAF reduced the lipid droplets. This study revealed that the anti-hyperlipidemic effect in Et-CAF has gained importance and might be used to fill the gap created by the allopathic drugs.

An intact Mcm10 coiled-coil interaction surface is important for origin melting, helicase assembly and the recruitment of Pol-α to Mcm2-7.

Mcm10 is an essential eukaryotic factor required for DNA replication. The replication fork helicase is composed of Cdc45, Mcm2–7 and GINS (CMG). DDK is an S-phase-specific kinase required for replication initiation, and the DNA primase-polymerase in eukaryotes is pol α. Mcm10 forms oligomers in vitro, mediated by the coiled-coil domain at the N-terminal region of the protein. We characterized an Mcm10 mutant at the N-terminal Domain (NTD), Mcm10-4A, defective for self-interaction. We found that the Mcm10-4A mutant was defective for stimulating DDK phosphorylation of Mcm2, binding to eighty-nucleotide ssDNA, and recruiting pol α to Mcm2–7 in vitro. Expression of wild-type levels of mcm10-4A resulted in severe growth and DNA replication defects in budding yeast cells, with diminished DDK phosphorylation of Mcm2. We then expressed the mcm10-4A in mcm5-bob1 mutant cells to bypass the defects mediated by diminished stimulation of DDK phosphorylation of Mcm2. Expression of wild-type levels of mcm10-4A in mcm5-bob1 mutant cells resulted in severe growth and DNA replication defects, along with diminished RPA signal at replication origins. We also detected diminished GINS and pol-α recruitment to the Mcm2–7 complex. We conclude that an intact Mcm10 coiled-coil interaction surface is important for origin melting, helicase assembly, and the recruitment of pol α to Mcm2–7.

Dpb11 may function with RPA and DNA to initiate DNA replication.

Dpb11 is required for the initiation of DNA replication in budding yeast. We found that Dpb11 binds tightly to single-stranded DNA (ssDNA) or branched DNA structures, while its human homolog, TopBP1, binds tightly to branched-DNA structures. We also found that Dpb11 binds stably to CDK-phosphorylated RPA, the eukaryotic ssDNA binding protein, in the presence of branched DNA. A Dpb11 mutant specifically defective for DNA binding did not exhibit tight binding to RPA in the presence of DNA, suggesting that Dpb11-interaction with DNA may promote the recruitment of RPA to melted DNA. We then characterized a mutant of Dpb11 that is specifically defective in DNA binding in budding yeast cells. Expression of dpb11-m1,2,3,5,ΔC results in a substantial decrease in RPA recruitment to origins, suggesting that Dpb11 interaction with DNA may be required for RPA recruitment to origins. Expression of dpb11-m1,2,3,5,ΔC also results in diminished GINS interaction with Mcm2-7 during S phase, while Cdc45 interaction with Mcm2-7 is like wild-type. The reduced GINS interaction with Mcm2-7 may be an indirect consequence of diminished origin melting. We propose that the tight interaction between Dpb11, CDK-phosphorylated RPA, and branched-DNA may be required for the essential function of stabilizing melted origin DNA in vivo. We also propose an alternative model, wherein Dpb11-DNA interaction is required for some other function in DNA replication initiation, such as helicase activation.

The Replication Kinase Cdc7 Marks Histones to Regulate Biosynthesis Genes

Post‐translational histone modifications (PTMs) play important roles in regulating various DNA‐templated cellular functions including transcription, DNA damage repair, DNA replication and telomeric silencing. Histone H3 Threonine 45 phosphorylation (H3T45ph) is a modification that is enriched during S‐phase of the cell cycle in Saccharomyces cerevisiae and is carried out by the kinase Cdc7‐Dbf4 (DDK) in vivo and in vitro. To understand the function of this mark we identified the genomic H3T45ph locations by performing chromatin immunoprecipitation followed by high throughput sequencing (ChIP‐Seq) of wild‐type budding yeast cell chromatin. Remarkably, this PTM is observed not only at origins of DNA replication, but is also uniquely distributed on the promoters of highly transcribed RNA polymerase I, II and III‐regulated genes dedicated to protein synthesis and glycolysis. Here we show that H3T45 phosphorylation, mediated by Cdc7‐Dbf4, is required for polymerase recruitment and full expression of ribosome protein (RP) and tRNA genes as well as for the regulation of cohesin loading. Collectively, we identify an unexpected gene regulatory function for the DNA‐replication kinase Cdc7‐Dbf4 and identify a novel histone modification regulating the protein biosynthesis gene machinery.Support or Funding InformationThis work is supported by NIH award R01 GM111911.

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells.

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Crosstalk of cell polarity signaling pathways.

Cell polarity, the asymmetric organization of cellular components along one or multiple axes, is present in most cells. From budding yeast cell polarization induced by pheromone signaling, oocyte polarization at fertilization to polarized epithelia and neuronal cells in multicellular organisms, similar mechanisms are used to determine cell polarity. Crucial role in this process is played by signaling lipid molecules, small Rho family GTPases and Par proteins. All these signaling circuits finally govern the cytoskeleton, which is responsible for oriented cell migration, cell shape changes, and polarized membrane and organelle trafficking. Thus, typically in the process of cell polarization, most cellular constituents become polarized, including plasma membrane lipid composition, ion concentrations, membrane receptors, and proteins in general, mRNA, vesicle trafficking, or intracellular organelles. This review gives a brief overview how these systems talk to each other both during initial symmetry breaking and within the signaling feedback loop mechanisms used to preserve the polarized state.

A Clb/Cdk1-mediated regulation of Fkh2 synchronizes CLB expression in the budding yeast cell cycle

Precise timing of cell division is achieved by coupling waves of cyclin-dependent kinase (Cdk) activity with a transcriptional oscillator throughout cell cycle progression. Although details of transcription of cyclin genes are known, it is unclear which is the transcriptional cascade that modulates their expression in a timely fashion. Here, we demonstrate that a Clb/Cdk1-mediated regulation of the Fkh2 transcription factor synchronizes the temporal mitotic CLB expression in budding yeast. A simplified kinetic model of the cyclin/Cdk network predicts a linear cascade where a Clb/Cdk1-mediated regulation of an activator molecule drives CLB3 and CLB2 expression. Experimental validation highlights Fkh2 as modulator of CLB3 transcript levels, besides its role in regulating CLB2 expression. A Boolean model based on the minimal number of interactions needed to capture the information flow of the Clb/Cdk1 network supports the role of an activator molecule in the sequential activation, and oscillatory behavior, of mitotic Clb cyclins. This work illustrates how transcription and phosphorylation networks can be coupled by a Clb/Cdk1-mediated regulation that synchronizes them.