scholarly journals An aging-independent replicative lifespan in a symmetrically dividing eukaryote

2016 ◽  
Author(s):  
Eric C. Spivey ◽  
Stephen K. Jones ◽  
James R. Rybarski ◽  
Fatema A. Saifuddin ◽  
Ilya J. Finkelstein
Keyword(s):  
Fission Yeast ◽  
Molecular Mechanisms ◽  
Single Cell Analysis ◽  
Eukaryotic Cell ◽  
Culture Conditions ◽  
Cellular Aging ◽  
Yeast Cells ◽  
Replicative Lifespan ◽  
A Cell ◽  
Defined Culture

AbstractThe replicative lifespan (RLS) of a cell—defined as the number of generations a cell divides before death—has informed our understanding of the molecular mechanisms of cellular aging. Nearly all RLS studies have been performed on budding yeast and little is known about the mechanisms of aging and longevity in symmetrically dividing eukaryotic cells. Here, we describe a multiplexed fission yeast (Schizosaccharomyces pombe) lifespan micro-dissector (FYLM); a microfluidic platform for performing automated micro-dissection and high-content single-cell analysis in well-defined culture conditions. Using the FYLM, we directly observe continuous and robust replication of hundreds of individual fission yeast cells for over seventy-five cell divisions. Surprisingly, cells die without any classic hallmarks of cellular aging such as changes in cell morphology, increased doubling time, or reduced sibling health. Genetic perturbations and longevity-enhancing drugs can further extend the replicative lifespan (RLS) via an aging-independent mechanism. We conclude that despite occasional sudden death of individual cells, fission yeast does not age. These results highlight that cellular aging and replicative lifespan can be uncoupled in a eukaryotic cell.

scholarly journals An aging-independent replicative lifespan in a symmetrically dividing eukaryote

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Eric C Spivey ◽  
Stephen K Jones ◽  
James R Rybarski ◽  
Fatema A Saifuddin ◽  
Ilya J Finkelstein
Keyword(s):  
Fission Yeast ◽  
Eukaryotic Cell ◽  
Quantitative Model ◽  
Cellular Aging ◽  
Yeast Cells ◽  
Replicative Lifespan ◽  
Processing Pipeline ◽  
Independent Mechanism ◽  
Genetic Perturbations ◽  
A Cell

The replicative lifespan (RLS) of a cell—defined as the number of cell divisions before death—has informed our understanding of the mechanisms of cellular aging. However, little is known about aging and longevity in symmetrically dividing eukaryotic cells because most prior studies have used budding yeast for RLS studies. Here, we describe a multiplexed fission yeast lifespan micro-dissector (multFYLM) and an associated image processing pipeline for performing high-throughput and automated single-cell micro-dissection. Using the multFYLM, we observe continuous replication of hundreds of individual fission yeast cells for over seventy-five generations. Surprisingly, cells die without the classic hallmarks of cellular aging, such as progressive changes in size, doubling time, or sibling health. Genetic perturbations and drugs can extend the RLS via an aging-independent mechanism. Using a quantitative model to analyze these results, we conclude that fission yeast does not age and that cellular aging and replicative lifespan can be uncoupled in a eukaryotic cell.

scholarly journals High-throughput analysis of yeast replicative aging using a microfluidic system

2015 ◽  
Vol 112 (30) ◽  
pp. 9364-9369 ◽  
Author(s):  
Myeong Chan Jo ◽  
Wei Liu ◽  
Liang Gu ◽  
Weiwei Dang ◽  
Lidong Qin
Keyword(s):  
High Resolution ◽  
High Throughput ◽  
Large Scale ◽  
Molecular Mechanisms ◽  
Single Cell Analysis ◽  
Retention Rate ◽  
Yeast Cells ◽  
High Throughput Analysis ◽  
Replicative Lifespan

Saccharomyces cerevisiaehas been an important model for studying the molecular mechanisms of aging in eukaryotic cells. However, the laborious and low-throughput methods of current yeast replicative lifespan assays limit their usefulness as a broad genetic screening platform for research on aging. We address this limitation by developing an efficient, high-throughput microfluidic single-cell analysis chip in combination with high-resolution time-lapse microscopy. This innovative design enables, to our knowledge for the first time, the determination of the yeast replicative lifespan in a high-throughput manner. Morphological and phenotypical changes during aging can also be monitored automatically with a much higher throughput than previous microfluidic designs. We demonstrate highly efficient trapping and retention of mother cells, determination of the replicative lifespan, and tracking of yeast cells throughout their entire lifespan. Using the high-resolution and large-scale data generated from the high-throughput yeast aging analysis (HYAA) chips, we investigated particular longevity-related changes in cell morphology and characteristics, including critical cell size, terminal morphology, and protein subcellular localization. In addition, because of the significantly improved retention rate of yeast mother cell, the HYAA-Chip was capable of demonstrating replicative lifespan extension by calorie restriction.

Three proteins required for early steps in the protein secretory pathway also affect nuclear envelope structure and cell cycle progression in fission yeast

2002 ◽  
Vol 115 (2) ◽  
pp. 421-431
Author(s):  
Anna Matynia ◽  
Sandra S. Salus ◽  
Shelley Sazer
Keyword(s):  
Cell Cycle ◽  
Nuclear Envelope ◽  
Fission Yeast ◽  
Cell Cycle Progression ◽  
Secretory Pathway ◽  
Yeast Cells ◽  
Ran Gtpase ◽  
A Cell ◽  
Cell Cycle Block ◽  
Er Membrane

The Ran GTPase is an essential protein that has multiple functions in eukaryotic cells. Fission yeast cells in which Ran is misregulated arrest after mitosis with condensed, unreplicated chromosomes and abnormal nuclear envelopes. The fission yeast sns mutants arrest with a similar cell cycle block and interact genetically with the Ran system. sns-A10, sns-B2 and sns-B9 have mutations in the fission yeast homologues of S. cerevisiae Sar1p, Sec31p and Sec53p, respectively, which are required for the early steps of the protein secretory pathway. The three sns mutants accumulate a normally secreted protein in the endoplasmic reticulum (ER), have an increased amount of ER membrane, and the ER/nuclear envelope lumen is dilated. Neither a post-ER block in the secretory pathway, nor ER proliferation caused by overexpression of an integral ER membrane protein, results in a cell cycle-specific defect. Therefore, the arrest seen in sns-A10, sns-B2 and sns-B9 is most likely due to nuclear envelope defects that render the cells unable to re-establish the interphase organization of the nucleus after mitosis. As a consequence, these mutants are unable to decondense their chromosomes or to initiate of the next round of DNA replication.

scholarly journals Hydrogen peroxide induced loss of heterozygosity correlates with replicative lifespan and mitotic asymmetry inSaccharomyces cerevisiae

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2671 ◽  
Author(s):  
Emine Güven ◽  
Lindsay A. Parnell ◽  
Erin D. Jackson ◽  
Meighan C. Parker ◽  
Nilin Gupta ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Genomic Instability ◽  
Loss Of Heterozygosity ◽  
Population Level ◽  
Cellular Aging ◽  
Yeast Cells ◽  
Replicative Lifespan ◽  
Chronological Aging ◽  
Transition Points

Cellular aging inSaccharomyces cerevisiaecan lead to genomic instability and impaired mitotic asymmetry. To investigate the role of oxidative stress in cellular aging, we examined the effect of exogenous hydrogen peroxide on genomic instability and mitotic asymmetry in a collection of yeast strains with diverse backgrounds. We treated yeast cells with hydrogen peroxide and monitored the changes of viability and the frequencies of loss of heterozygosity (LOH) in response to hydrogen peroxide doses. The mid-transition points of viability and LOH were quantified using sigmoid mathematical functions. We found that the increase of hydrogen peroxide dependent genomic instability often occurs before a drop in viability. We previously observed that elevation of genomic instability generally lags behind the drop in viability during chronological aging. Hence, onset of genomic instability induced by exogenous hydrogen peroxide treatment is opposite to that induced by endogenous oxidative stress during chronological aging, with regards to the midpoint of viability. This contrast argues that the effect of endogenous oxidative stress on genome integrity is well suppressed up to the dying-off phase during chronological aging. We found that the leadoff of exogenous hydrogen peroxide induced genomic instability to viability significantly correlated with replicative lifespan (RLS), indicating that yeast cells’ ability to counter oxidative stress contributes to their replicative longevity. Surprisingly, this leadoff is positively correlated with an inverse measure of endogenous mitotic asymmetry, indicating a trade-off between mitotic asymmetry and cell’s ability to fend off hydrogen peroxide induced oxidative stress. Overall, our results demonstrate strong associations of oxidative stress to genomic instability and mitotic asymmetry at the population level of budding yeast.

scholarly journals MET18 Deficiency Increases the Sensitivity of Yeast to Oxidative Stress and Shortens Replicative Lifespan by Inhibiting Catalase Activity

2017 ◽  
Vol 2017 ◽  
pp. 1-8 ◽  
Author(s):  
Ya-qin Chen ◽  
Xin-guang Liu ◽  
Wei Zhao ◽  
Hongjing Cui ◽  
Jie Ruan ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Catalase Activity ◽  
Molecular Mechanisms ◽  
Cumene Hydroperoxide ◽  
Yeast Cells ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Replicative Capacity ◽  
Replicative Lifespan ◽  
Increased Sensitivity

Yeast MET18, a subunit of the cytosolic iron-sulfur (Fe/S) protein assembly (CIA) machinery which is responsible for the maturation of Fe/S proteins, has been reported to participate in the oxidative stress response. However, the underlying molecular mechanisms remain unclear. In this study, we constructed a MET18/met18Δ heterozygous mutant yeast strain and found that MET18 deficiency in yeast cells impaired oxidative stress resistance as evidenced by increased sensitivity to hydrogen peroxide (H2O2) and cumene hydroperoxide (CHP). Mechanistically, the mRNA levels of catalase A (CTA1) and catalase T (CTT1) as well as the total catalase activity were significantly reduced in MET18-deficient cells. In contrast, overexpression of CTT1 or CTA1 in MET18-deficient cells significantly increased the intracellular catalase activity and enhanced the resistance ability against H2O2 and CHP. In addition, MET18 deficiency diminished the replicative capacity of yeast cells as evidenced by the shortened replicative lifespan, which can be restored by CTT1 overexpression, but not by CTA1, in the MET18-deficient cells. These results suggest that MET18, in a catalase-dependent manner, plays an essential role in enhancing the resistance of yeast cells to oxidative stress and increasing the replicative capacity of yeast cells.

The Wellcome Lecture, 1992. Cell cycle control

1993 ◽  
Vol 341 (1298) ◽  
pp. 449-454 ◽  
Keyword(s):  
Cell Cycle ◽  
Fission Yeast ◽  
Gene Network ◽  
Cell Cycle Control ◽  
Regulatory Gene ◽  
Eukaryotic Cell ◽  
S Phase ◽  
M Phase ◽  
A Cell ◽  
Future Work

Genetic analysis using the fission yeast has provided a powerful methodology to investigate the eukaryotic cell cycle and its control. The onset of M -phase in fission yeast is controlled by a regulatory gene network which activates the p34 cdc2 protein kinase encoded by the cdc 2 + gene. The coupling of M -phase to the completion of S-phase also works through p34 cdc2 . A similar network is operative in vertebrate cells. Future work will focus on the controls regulating onset of S-phase and on the mechanisms by which a cell duplicates itself in space during division.

scholarly journals A physicochemical perspective of aging from single-cell analysis of pH, macromolecular and organellar crowding in yeast

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Sara N Mouton ◽  
David J Thaller ◽  
Matthew M Crane ◽  
Irina L Rempel ◽  
Owen T Terpstra ◽  
...  
Keyword(s):  
Physicochemical Properties ◽  
Single Cell Analysis ◽  
Macromolecular Crowding ◽  
Cellular Aging ◽  
Yeast Cells ◽  
Cell Analysis ◽  
Molecular Processes ◽  
The Stability ◽  
Micrometer Scale

Cellular aging is a multifactorial process that is characterized by a decline in homeostatic capacity, best described at the molecular level. Physicochemical properties such as pH and macromolecular crowding are essential to all molecular processes in cells and require maintenance. Whether a drift in physicochemical properties contributes to the overall decline of homeostasis in aging is not known. Here, we show that the cytosol of yeast cells acidifies modestly in early aging and sharply after senescence. Using a macromolecular crowding sensor optimized for long-term FRET measurements, we show that crowding is rather stable and that the stability of crowding is a stronger predictor for lifespan than the absolute crowding levels. Additionally, in aged cells, we observe drastic changes in organellar volume, leading to crowding on the micrometer scale, which we term organellar crowding. Our measurements provide an initial framework of physicochemical parameters of replicatively aged yeast cells.

scholarly journals Size-dependent increase in RNA Polymerase II initiation rates mediates gene expression scaling with cell size

2019 ◽  
Author(s):  
Xi-Ming Sun ◽  
Anthony Bowman ◽  
Miles Priestman ◽  
Francois Bertaux ◽  
Amalia Martinez-Segura ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Fission Yeast ◽  
Rna Polymerase Ii ◽  
Cell Size ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Single Cells ◽  
Yeast Cells ◽  
Limiting Factor

ABSTRACTCell size varies during the cell cycle and in response to external stimuli. This requires the tight coordination, or “scaling”, of mRNA and protein quantities with the cell volume in order to maintain biomolecules concentrations and cell density. Evidence in cell populations and single cells indicates that scaling relies on the coordination of mRNA transcription rates with cell size. Here we use a combination of single-molecule fluorescence in situ hybridisation (smFISH), time-lapse microscopy and mathematical modelling in single fission yeast cells to uncover the precise molecular mechanisms that control transcription rates scaling with cell size. Linear scaling of mRNA quantities is apparent in single fission yeast cells during a normal cell cycle. Transcription rates of both constitutive and regulated genes scale with cell size without evidence for transcriptional bursting. Modelling and experimental data indicate that scaling relies on the coordination of RNAPII transcription initiation rates with cell size and that RNAPII is a limiting factor. We show using real-time quantitative imaging that size increase is accompanied by a rapid concentration independent recruitment of RNAPII onto chromatin. Finally, we find that in multinucleated cells, scaling is set at the level of single nuclei and not the entire cell, making the nucleus the transcriptional scaling unit. Integrating our observations in a mechanistic model of RNAPII mediated transcription, we propose that scaling of gene expression with cell size is the consequence of competition between genes for limiting RNAPII.

scholarly journals Uncovering Natural Longevity Alleles from Intercrossed Pools of Aging Fission Yeast Cells

2018 ◽  
Author(s):  
David A. Ellis ◽  
Ville Mustonen ◽  
María Rodríguez-López ◽  
Charalampos Rallis ◽  
Michał Malecki ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Genetic Variants ◽  
Genetic Factors ◽  
Cellular Aging ◽  
Yeast Cells ◽  
Untranslated Regions ◽  
Protein Coding ◽  
Chronological Lifespan ◽  
Growth Defects ◽  
Chromosome Ii

ABSTRACTQuantitative traits often show large variation caused by multiple genetic factors. One such trait is the chronological lifespan of non-dividing yeast cells, serving as a model for cellular aging. Screens for genetic factors involved in ageing typically assay mutants of protein-coding genes. To identify natural genetic variants contributing to cellular aging, we exploited two strains of the fission yeast, Schizosaccharomyces pombe, that differ in chronological lifespan. We generated segregant pools from these strains and subjected them to advanced intercrossing over multiple generations to break up linkage groups. We chronologically aged the intercrossed segregant pool, followed by genome sequencing at different times to detect genetic variants that became reproducibly enriched as a function of age. A region on Chromosome II showed strong positive selection during ageing. Based on expected functions, two candidate variants from this region in the long-lived strain were most promising to be causal: small insertions and deletions in the 5’-untranslated regions of ppk31 and SPBC409.08. Ppk31 is an orthologue of Rim15, a conserved kinase controlling cell proliferation in response to nutrients, while SPBC409.08 is a predicted spermine transmembrane transporter. Both Rim15 and the spermine-precursor, spermidine, are implicated in ageing as they are involved in autophagy-dependent lifespan extension. Single and double allele replacement suggests that both variants, alone or combined, have subtle effects on cellular longevity. Furthermore, deletion mutants of both ppk31 and SPBC409.08 rescued growth defects caused by spermidine. We propose that Ppk31 and SPBC409.08 may function together to modulate lifespan, thus linking Rim15/Ppk31 with spermidine metabolism.

scholarly journals The puc1 Cyclin Regulates the G1 Phase of the Fission Yeast Cell Cycle in Response to Cell Size

2000 ◽  
Vol 11 (2) ◽  
pp. 543-554 ◽  
Author(s):  
Cristina Martı́n-Castellanos ◽  
Miguel A. Blanco ◽  
José M. de Prada ◽  
Sergio Moreno
Keyword(s):  
Cell Cycle ◽  
Fission Yeast ◽  
Cell Size ◽  
Yeast Cells ◽  
G1 Phase ◽  
Close Relative ◽  
Cell Cycle Distribution ◽  
Cdk Inhibitor ◽  
Wild Type ◽  
A Cell

Eukaryotic cells coordinate cell size with cell division by regulating the length of the G1 and G2 phases of the cell cycle. In fission yeast, the length of the G1 phase depends on a precise balance between levels of positive (cig1, cig2, puc1, and cdc13 cyclins) and negative (rum1 and ste9-APC) regulators of cdc2. Early in G1, cyclin proteolysis and rum1 inhibition keep the cdc2/cyclin complexes inactive. At the end of G1, the balance is reversed and cdc2/cyclin activity down-regulates both rum1 and the cyclin-degrading activity of the APC. Here we present data showing that the puc1 cyclin, a close relative of the Cln cyclins in budding yeast, plays an important role in regulating the length of G1. Fission yeast cells lacking cig1 and cig2 have a cell cycle distribution similar to that of wild-type cells, with a short G1 and a long G2. However, when thepuc1 + gene is deleted in this genetic background, the length of G1 is extended and these cells undergo S phase with a greater cell size than wild-type cells. This G1 delay is completely abolished in cells lacking rum1. Cdc2/puc1 function may be important to down-regulate the rum1 Cdk inhibitor at the end of G1.

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