scholarly journals Single-Cell Time-Lapse Observation Reveals Cell Shrinkage upon Cell Death in Batch Culture of Saccharomyces cerevisiae

mBio ◽  
2021 ◽  
Vol 12 (6) ◽  
Author(s):  
Setsu Kato ◽  
Kenta Suzuki ◽  
Taiki Kenjo ◽  
Junya Kato ◽  
Yoshiteru Aoi ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Death ◽  
Stationary Phase ◽  
Cell Survival ◽  
Single Cell ◽  
Batch Culture ◽  
Time Lapse ◽  
Cell Shrinkage ◽  
Clonal Population ◽  
Content Type

Cells display various behaviors even though they originate from a clonal population. Such diversity is also observed in cell survival in the stationary phase of Saccharomyces cerevisiae .

scholarly journals Connecting the Dots between Mechanosensitive Channel Abundance, Osmotic Shock, and Survival at Single-Cell Resolution

2018 ◽  
Vol 200 (23) ◽  
Author(s):  
Griffin Chure ◽  
Heun Jin Lee ◽  
Akiko Rasmussen ◽  
Rob Phillips
Keyword(s):  
Cell Survival ◽  
Single Cell ◽  
Osmotic Shock ◽  
Single Cells ◽  
Mechanosensitive Channel ◽  
Membrane Tension ◽  
Wild Type ◽  
Content Type ◽  
E Coli ◽  
Structural Methods

ABSTRACTRapid changes in extracellular osmolarity are one of many insults microbial cells face on a daily basis. To protect against such shocks,Escherichia coliand other microbes express several types of transmembrane channels that open and close in response to changes in membrane tension. InE. coli, one of the most abundant channels is the mechanosensitive channel of large conductance (MscL). While this channel has been heavily characterized through structural methods, electrophysiology, and theoretical modeling, our understanding of its physiological role in preventing cell death by alleviating high membrane tension remains tenuous. In this work, we examine the contribution of MscL alone to cell survival after osmotic shock at single-cell resolution using quantitative fluorescence microscopy. We conducted these experiments in anE. colistrain which is lacking all mechanosensitive channel genes save for MscL, whose expression was tuned across 3 orders of magnitude through modifications of the Shine-Dalgarno sequence. While theoretical models suggest that only a few MscL channels would be needed to alleviate even large changes in osmotic pressure, we find that between 500 and 700 channels per cell are needed to convey upwards of 80% survival. This number agrees with the average MscL copy number measured in wild-typeE. colicells through proteomic studies and quantitative Western blotting. Furthermore, we observed zero survival events in cells with fewer than ∼100 channels per cell. This work opens new questions concerning the contribution of other mechanosensitive channels to survival, as well as regulation of their activity.IMPORTANCEMechanosensitive (MS) channels are transmembrane protein complexes which open and close in response to changes in membrane tension as a result of osmotic shock. Despite extensive biophysical characterization, the contribution of these channels to cell survival remains largely unknown. In this work, we used quantitative video microscopy to measure the abundance of a single species of MS channel in single cells, followed by their survival after a large osmotic shock. We observed total death of the population with fewer than ∼100 channels per cell and determined that approximately 500 to 700 channels were needed for 80% survival. The number of channels we found to confer nearly full survival is consistent with the counts of the numbers of channels in wild-type cells in several earlier studies. These results prompt further studies to dissect the contribution of other channel species to survival.

scholarly journals The Stationary-Phase Cells of Saccharomyces cerevisiae Display Dynamic Actin Filaments Required for Processes Extending Chronological Life Span

2015 ◽  
Vol 35 (22) ◽  
pp. 3892-3908 ◽  
Author(s):  
Pavla Vasicova ◽  
Renata Lejskova ◽  
Ivana Malcova ◽  
Jiri Hasek
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Stationary Phase ◽  
Actin Filaments ◽  
Phase Cell ◽  
Content Type ◽  
Chronological Aging ◽  
Yeast Cultures ◽  
Actin Cables ◽  
Mitochondrial Networks

Stationary-growth-phaseSaccharomyces cerevisiaeyeast cultures consist of nondividing cells that undergo chronological aging. For their successful survival, the turnover of proteins and organelles, ensured by autophagy and the activation of mitochondria, is performed. Some of these processes are engaged in by the actin cytoskeleton. InS. cerevisiaestationary-phase cells, F actin has been shown to form static aggregates named actin bodies, subsequently cited to be markers of quiescence. Ourin vivoanalyses revealed that stationary-phase cultures contain cells with dynamic actin filaments, besides the cells with static actin bodies. The cells with dynamic actin displayed active endocytosis and autophagy and well-developed mitochondrial networks. Even more, stationary-phase cell cultures grown under calorie restriction predominantly contained cells with actin cables, confirming that the presence of actin cables is linked to successful adaptation to stationary phase. Cells with actin bodies were inactive in endocytosis and autophagy and displayed aberrations in mitochondrial networks. Notably, cells of the respiratory activity-deficientcox4Δ strain displayed the same mitochondrial aberrations and actin bodies only. Additionally, our results indicate that mitochondrial dysfunction precedes the formation of actin bodies and the appearance of actin bodies corresponds to decreased cell fitness. We conclude that the F-actin status reflects the extent of damage that arises from exponential growth.

scholarly journals The Endoplasmic Reticulum-Mitochondrion Tether ERMES Orchestrates Fungal Immune Evasion, Illuminating Inflammasome Responses to Hyphal Signals

mSphere ◽  
2016 ◽  
Vol 1 (3) ◽  
Author(s):  
Timothy M. Tucey ◽  
Jiyoti Verma-Gaur ◽  
Julie Nguyen ◽  
Victoria L. Hewitt ◽  
Tricia L. Lo ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Wall ◽  
Candida Albicans ◽  
Single Cell ◽  
Host Cell ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Inflammasome Activation ◽  
Content Type ◽  
Host Cell Death

ABSTRACT The yeast Candida albicans causes human infections that have mortality rates approaching 50%. The key to developing improved therapeutics is to understand the host-pathogen interface. A critical interaction is that with macrophages: intracellular Candida triggers the NLRP3/caspase-1 inflammasome for escape through lytic host cell death, but this also activates antifungal responses. To better understand how the inflammasome response to Candida is fine-tuned, we established live-cell imaging of inflammasome activation at single-cell resolution, coupled with analysis of the fungal ERMES complex, a mitochondrial regulator that lacks human homologs. We show that ERMES mediates Candida escape via inflammasome-dependent processes, and our data suggest that inflammasome activation is controlled by the level of hyphal growth and exposure of cell wall components as a proxy for severity of danger. Our study provides the most detailed dynamic analysis of inflammasome responses to a fungal pathogen so far and establishes promising pathogen- and host-derived therapeutic strategies. The pathogenic yeast Candida albicans escapes macrophages by triggering NLRP3 inflammasome-dependent host cell death (pyroptosis). Pyroptosis is inflammatory and must be tightly regulated by host and microbe, but the mechanism is incompletely defined. We characterized the C. albicans endoplasmic reticulum (ER)-mitochondrion tether ERMES and show that the ERMES mmm1 mutant is severely crippled in killing macrophages despite hyphal formation and normal phagocytosis and survival. To understand dynamic inflammasome responses to Candida with high spatiotemporal resolution, we established live-cell imaging for parallel detection of inflammasome activation and pyroptosis at the single-cell level. This showed that the inflammasome response to mmm1 mutant hyphae is delayed by 10 h, after which an exacerbated activation occurs. The NLRP3 inhibitor MCC950 inhibited inflammasome activation and pyroptosis by C. albicans, including exacerbated inflammasome activation by the mmm1 mutant. At the cell biology level, inactivation of ERMES led to a rapid collapse of mitochondrial tubular morphology, slow growth and hyphal elongation at host temperature, and reduced exposed 1,3-β-glucan in hyphal populations. Our data suggest that inflammasome activation by C. albicans requires a signal threshold dependent on hyphal elongation and cell wall remodeling, which could fine-tune the response relative to the level of danger posed by C. albicans. The phenotypes of the ERMES mutant and the lack of conservation in animals suggest that ERMES is a promising antifungal drug target. Our data further indicate that NLRP3 inhibition by MCC950 could modulate C. albicans-induced inflammation. IMPORTANCE The yeast Candida albicans causes human infections that have mortality rates approaching 50%. The key to developing improved therapeutics is to understand the host-pathogen interface. A critical interaction is that with macrophages: intracellular Candida triggers the NLRP3/caspase-1 inflammasome for escape through lytic host cell death, but this also activates antifungal responses. To better understand how the inflammasome response to Candida is fine-tuned, we established live-cell imaging of inflammasome activation at single-cell resolution, coupled with analysis of the fungal ERMES complex, a mitochondrial regulator that lacks human homologs. We show that ERMES mediates Candida escape via inflammasome-dependent processes, and our data suggest that inflammasome activation is controlled by the level of hyphal growth and exposure of cell wall components as a proxy for severity of danger. Our study provides the most detailed dynamic analysis of inflammasome responses to a fungal pathogen so far and establishes promising pathogen- and host-derived therapeutic strategies.

scholarly journals Competence-Stimulating-Peptide-Dependent Localized Cell Death and Extracellular DNA Production in Streptococcus mutans Biofilms

2020 ◽  
Vol 86 (23) ◽  
Author(s):  
Ryo Nagasawa ◽  
Tatsuya Yamamoto ◽  
Andrew S. Utada ◽  
Nobuhiko Nomura ◽  
Nozomu Obana
Keyword(s):  
Cell Death ◽  
Single Cell ◽  
Streptococcus Mutans ◽  
Structural Stability ◽  
Biofilm Formation ◽  
Extracellular Polymeric Substances ◽  
Imaging Techniques ◽  
Extracellular Dna ◽  
Content Type ◽  
Cariogenic Bacterium

ABSTRACT Extracellular DNA (eDNA) is a biofilm component that contributes to the formation and structural stability of biofilms. Streptococcus mutans, a major cariogenic bacterium, induces eDNA-dependent biofilm formation under specific conditions. Since cell death can result in the release and accumulation of DNA, the dead cells in biofilms are a source of eDNA. However, it remains unknown how eDNA is released from dead cells and is localized within S. mutans biofilms. We focused on cell death induced by the extracellular signaling peptide called competence-stimulating peptide (CSP). We demonstrate that nucleic acid release into the extracellular environment occurs in a subpopulation of dead cells. eDNA production induced by CSP was highly dependent on the lytF gene, which encodes an autolysin. Although lytF expression was induced bimodally by CSP, lytF-expressing cells further divided into surviving cells and eDNA-producing dead cells. Moreover, we found that lytF-expressing cells were abundant near the bottom of the biofilm, even when all cells in the biofilm received the CSP signal. Dead cells and eDNA were also abundantly present near the bottom of the biofilm. The number of lytF-expressing cells in biofilms was significantly higher than that in planktonic cultures, which suggests that adhesion to the substratum surface is important for the induction of lytF expression. The deletion of lytF resulted in reduced adherence to a polystyrene surface. These results suggest that lytF expression and eDNA production induced near the bottom of the biofilm contribute to a firmly attached and structurally stable biofilm. IMPORTANCE Bacterial communities encased by self-produced extracellular polymeric substances (EPSs), known as biofilms, have a wide influence on human health and environmental problems. The importance of biofilm research has increased, as biofilms are the preferred bacterial lifestyle in nature. Furthermore, in recent years it has been noted that the contribution of phenotypic heterogeneity within biofilms requires analysis at the single-cell or subpopulation level to understand bacterial life strategies. In Streptococcus mutans, a cariogenic bacterium, extracellular DNA (eDNA) contributes to biofilm formation. However, it remains unclear how and where the cells produce eDNA within the biofilm. We focused on LytF, an autolysin that is induced by extracellular peptide signals. We used single-cell level imaging techniques to analyze lytF expression in the biofilm population. Here, we show that S. mutans generates eDNA by inducing lytF expression near the bottom of the biofilm, thereby enhancing biofilm adhesion and structural stability.

scholarly journals Live-Cell Imaging Tool Optimization To Study Gene Expression Levels and Dynamics in Single Cells of Bacillus cereus

2013 ◽  
Vol 79 (18) ◽  
pp. 5643-5651 ◽  
Author(s):  
Robyn T. Eijlander ◽  
Oscar P. Kuipers
Keyword(s):  
Gene Expression ◽  
Fluorescence Microscopy ◽  
Single Cell ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Single Cell Level ◽  
Cell Level ◽  
Content Type ◽  
Over Time ◽  
Gene Expression Levels

ABSTRACTSingle-cell methods are a powerful application in microbial research to study the molecular mechanism underlying phenotypic heterogeneity and cell-to-cell variability. Here, we describe the optimization and application of single-cell time-lapse fluorescence microscopy for the food spoilage bacteriumBacillus cereusspecifically. This technique is useful to study cellular development and adaptation, gene expression, protein localization, protein mobility, and cell-to-cell communication over time at the single-cell level. By adjusting existing protocols, we have enabled the visualization of growth and development of singleB. cereuscells within a microcolony over time. Additionally, several different fluorescent reporter proteins were tested in order to select the most suitable green fluorescent protein (GFP) and red fluorescent protein (RFP) candidates for visualization of growth stage- and cell compartment-specific gene expression inB. cereus. With a case study concerningcotDexpression during sporulation, we demonstrate the applicability of time-lapse fluorescence microscopy. It enables the assessment of gene expression levels, dynamics, and heterogeneity at the single-cell level. We show thatcotDis not heterogeneously expressed among cells of a subpopulation. Furthermore, we discourage using plasmid-based reporter fusions for such studies, due to an introduced heterogeneity through copy number differences. This stresses the importance of using single-copy integrated reporter fusions for single-cell studies.

scholarly journals Molecular Characterization of Propolis-Induced Cell Death in Saccharomyces cerevisiae

2010 ◽  
Vol 10 (3) ◽  
pp. 398-411 ◽  
Author(s):  
Patrícia Alves de Castro ◽  
Marcela Savoldi ◽  
Diego Bonatto ◽  
Mário Henrique Barros ◽  
Maria Helena S. Goldman ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Death ◽  
Rna Polymerase Ii ◽  
Cell Biology ◽  
Cellular Response ◽  
Protein Targeting ◽  
Model Organism ◽  
Regulation Of Transcription ◽  
Content Type ◽  
Vacuolar Acidification

ABSTRACTPropolis, a natural product of plant resins, is used by the bees to seal holes in their honeycombs and protect the hive entrance. However, propolis has also been used in folk medicine for centuries. Here, we apply the power ofSaccharomyces cerevisiaeas a model organism for studies of genetics, cell biology, and genomics to determine how propolis affects fungi at the cellular level. Propolis is able to induce an apoptosis cell death response. However, increased exposure to propolis provides a corresponding increase in the necrosis response. We showed that cytochromecbut not endonuclease G (Nuc1p) is involved in propolis-mediated cell death inS. cerevisiae. We also observed that the metacaspaseYCA1gene is important for propolis-mediated cell death. To elucidate the gene functions that may be required for propolis sensitivity in eukaryotes, the full collection of about 4,800 haploidS. cerevisiaedeletion strains was screened for propolis sensitivity. We were able to identify 138 deletion strains that have different degrees of propolis sensitivity compared to the corresponding wild-type strains. Systems biology revealed enrichment for genes involved in the mitochondrial electron transport chain, vacuolar acidification, negative regulation of transcription from RNA polymerase II promoter, regulation of macroautophagy associated with protein targeting to vacuoles, and cellular response to starvation. Validation studies indicated that propolis sensitivity is dependent on the mitochondrial function and that vacuolar acidification and autophagy are important for yeast cell death caused by propolis.

Neuroglobin and Estrogen Receptors: A New Pathway of Cell Survival and Cell Death Balance

2015 ◽  
Vol 14 (2) ◽  
pp. 91-99 ◽  
Author(s):  
Maria T. Nuzzo ◽  
Marco Fiocchetti ◽  
Paolo Ascenzi ◽  
Maria Marino
Keyword(s):  
Cell Death ◽  
Estrogen Receptors ◽  
Cell Survival

scholarly journals Translation machinery reprogramming in programmed cell death in Saccharomyces cerevisiae

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Francesco Monticolo ◽  
Emanuela Palomba ◽  
Maria Luisa Chiusano
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Death ◽  
Acetic Acid ◽  
Programmed Cell Death ◽  
Differential Expression ◽  
Ribosomal Proteins ◽  
Relative Proportion ◽  
Duplication Event ◽  
Genome Duplication Event ◽  
Cell Death Pathway

AbstractProgrammed cell death involves complex molecular pathways in both eukaryotes and prokaryotes. In Escherichia coli, the toxin–antitoxin system (TA-system) has been described as a programmed cell death pathway in which mRNA and ribosome organizations are modified, favoring the production of specific death-related proteins, but also of a minor portion of survival proteins, determining the destiny of the cell population. In the eukaryote Saccharomyces cerevisiae, the ribosome was shown to change its stoichiometry in terms of ribosomal protein content during stress response, affecting the relative proportion between ohnologs, i.e., the couple of paralogs derived by a whole genome duplication event. Here, we confirm the differential expression of ribosomal proteins in yeast also during programmed cell death induced by acetic acid, and we highlight that also in this case pairs of ohnologs are involved. We also show that there are different trends in cytosolic and mitochondrial ribosomal proteins gene expression during the process. Moreover, we show that the exposure to acetic acid induces the differential expression of further genes coding for products related to translation processes and to rRNA post-transcriptional maturation, involving mRNA decapping, affecting translation accuracy, and snoRNA synthesis. Our results suggest that the reprogramming of the overall translation apparatus, including the cytosolic ribosome reorganization, are relevant events in yeast programmed cell death induced by acetic acid.

Sign in / Sign up

Export Citation Format

Share Document