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Characterization of Differentiated Quiescent and

Characterization of Differentiated Quiescent and Nonquiescent Cells in Yeast Stationary-Phase Cultures How Yeast Cells Plan to Live Forever: The Paradox of Increasing Diversity in Stationary Phase. AD Aragon, et al 2008. John Trotter. Overview.

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Characterization of Differentiated Quiescent and

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  1. Characterization of Differentiated Quiescent and Nonquiescent Cells in Yeast Stationary-Phase Cultures How Yeast Cells Plan to Live Forever: The Paradox of Increasing Diversity in Stationary Phase AD Aragon, et al 2008 John Trotter

  2. Overview • Cellular differentiation in yeast after glucose exhaustion • Characterization of these cells • Mutant analysis • Microarrays • Identification of protease-labile mRNAs, • Protein localization • New models in a tractable system for • G0→ G1 transition • Chronological aging • Apoptosis • The viable but unculturable state • Stem cell biogenesis?

  3. Other important collaborations • Chemical engineering • Surfaces and microdevices for analysis • high throughput analyses • Computational Biology • Predicting networks, including interacting proteins • Insight in to data needs and integration • Understanding motif function, evolution, and systems • Statistics • critical for understanding the value of data • approaches for estimating distributions of genes, patterns of expression changes, and relationships • Experimental design!

  4. G0 G1 M S G2 • Most cells on earth are quiescent • Neurons, egg cells, stem cells, unculturable microbes • Cells are unbudded, cell-cycle arrest • In eukaryotes, this state is called G0 • Biogenesis may be associated with an asymmetric cell division and apoptosis • Typically a highly regulated state • Cells may localize mRNA and be stress resistant • Cells are long lived and able to reproduce • Quiescence plays an essential role in health, disease, and environment • There are probably distinct quiescent states

  5. Growth of cultures to SP No more carbon! Requires further down regulation of PKA Diauxic shift Can live 100s of years! Cells/ml StationaryPhase Post-diauxic phase Time (days) Fermentation → Respiration.Requires SNF1, PKC, and down reg of PKA and TOR

  6. Dense, quiescent cells form after glucose but before carbon exhaustion Diauxic shift Stationary phase NQ Q Allen et al, JCellBiol 2006

  7. c e g f h d Q and NQ cells differ morphologically NQ Q

  8. a Quiescent Non-quiescent 80 % budded 60 40 20 0 0 1 2 3 4 time (hrs) Budding index Q cell characteristics • Denser • Morphologically distinct • daughters (91%) • more stress resistant • Synchronous • G0→ G1 transition

  9. NQ cell characteristics: viable but unculturable Metabolic activity Colony-forming capacity *** *** * control *** * * 100 100 Quiescent Non-quiescent 80 80 60 60 % CFU % FUN1 pos 40 40 20 20 0 0 7 14 21 7 14 21 days after inoculation days after inoculation NQ cells also show high ROS and about 50% are necrotic or apoptotic by day 14

  10. a Q NQ Non-quiescent cells are heterogeneous and ½ are apoptotic or necrotic by 21 days b Q NQ Q NQ Q NQ Apoptosis and Necrosis Reactive Oxygen Species The NQ fraction contains at least 2 populations of cells

  11. G0 G1 apoptosis, necrosis, senescence M S G2 Models for entry into stationary phase and G0in yeast Nutrients

  12. Questions • What genes are required for viability and/or reproductive capacity in Q and NQ cells? (mutant analysis) • What mRNAs differentiate Q and NQ cells? (microarray analysis) • What protease-labile mRNAs, i.e. localized mRNAs, are present in Q or NQ cells? • ~ 90 mutants: Q and NQ cell fractions • Six strains known that don’t form Q cells • sdh4, kgd1, atp1, qcr7, rip1, and cor1 • Viability determined with FungaLight, FACScan • CFU done using FACS (MO-FLO)

  13. Yeast deletion set • ~6000 strains of yeast, each with one gene knocked out • Next week we will talk about how you look at all of these strains in a mixture • Meanwhile, just know that for $1500 you can purchase the entire deletion set to begin to study, on a genomic level- what genes are required for any particular process.

  14. Viability and reproductive capacity (CFU) of Q and NQ mutant cells

  15. Questions • What genes are required for viability and reproductive capacity in Q and NQ cells? • What mRNAs differentiate Q and NQ cells? • Do Q or NQ cells localize mRNAs? • ~ 400 microarrays of mutants • abundant mRNAs identified with p < 10-5 • > 600 mRNAs identified for Q and NQ cells, 1300 total

  16. Q and NQ genes do not cluster during the cell-cycle This study Spellman’s cell cycle dataset We conclude that Q cells are not in a G1 arrest

  17. Q mRNAs – GO processes: membranes and stress

  18. Q cells: Signaling pathways that must be activated or inhibited for survival are present SNF: SNF1, SAK1, SIP1, etc. PKA: BCY1, TPK1, TPK2, RIM15, YAK1, IRA1, etc. TOR: TOR1, TOR2, PPH22, RHO1 etc. • mRNAs for key pro-growth kinases are present • Inhibitors are also present • Q cells are poised to respond to growth signals

  19. Pathways involved in regulating stem cell proliferation are conserved in yeast and most mRNAs for these are present in Quiescent cells. VPS34 PHO85 TOS3 TEF1nq USO1 PKC1 SNF1 HCM1 TOR

  20. NQ mRNAs - GO process GO term p-value This is consistent with chromatin instability and apoptosis

  21. Q cells: Computational prediction:More proteins may interact 0.9 Predicted True Interactions GRID (35059) Known interactions p-val Number p-val Number Gene set: 380 0 Wild Type Q (37950) 183 1.70E-38 1 1 Wild Type NQ (62481) 149 6.22E-07 637 0 Parental Q (102378) 244 2.53E-10 11 1 Parental NQ (94395) 226 5.47E-19 737 0 Mutants Q (232903) 555 7.70E-22 12 0.8 Mutants NQ (190653) 332 0.023

  22. Questions • What genes are required for viability and reproductive capacity in Q and NQ cells? • What mRNAs differentiate Q and NQ cells? • Do Q or NQ cells localize mRNAs? • RNA localization is also characteristic of quiescent cells, e.g. neurons, stem cells, and egg • Localization is likely due to interaction of mRNAs with p-bodies or stress granules

  23. Cells in SP cultures contain protease-labile mRNAs Stationary Phase Exponential Aragon, AD Genome Biol 2006

  24. Transcript release is stress specific

  25. Protease-labile mRNAsare predominantly in Q cells SP culture 1570 NQ cells 223 Q cells 1957 Sample mRNAs GO term # mRNAs p-value

  26. Summary • NQ - mothers and some daughters • maintain viability but rapidly lose reproductive capacity • become apoptotic and necrotic • abundant mRNAs are involved in DNA transposition/recombination • contain few signaling mRNAs • not capable of significant response to environmental conditions • Quiescent cells • daughters, synchronous (G0), more stress tolerant • less sensitive to perturbations than NQ cells • maintain a high reproductive capacity • contain mRNAs for many key signal transduction proteins • contain > 1000 protease-labile mRNAs, probably in p-bodies • Primed to respond to environmental signals in a variety of ways, including by apoptosis

  27. Final Questions Do apoptotic NQ cells provide a mechanism for population diversity, speciation, and cancer? What are the evolutionary consequences? How asymmetric is this and what regulates it? Is this a common process for producing Q cells? What is communication occurs between Q and NQ cells? Answering these questions will require new technologies, analyses, and datasets! What genes are required for Q cell survival? Are these cells able to produce other Q cells? Is this a primitive stem cell?

  28. Current experiments • What proteins are sequestering mRNAs in quiescent cells? • Fluorescent microscopy • Proteomics – MS/MS and TAP-tag pull downs • What proteins are abundant in Q and NQ cells? • GFP-fusion library and highthroughput flow cytometry • Need high throughput microscopy • Prediction of condition specific networks • Machine learning approaches, graph theory • Statistical weighting of array data

  29. Collaborators • Sandia National Laboratories • George Davidson • Dalhousie University • Peter Wentzell • Washington University • John Heuser • University of Graz • Frank Madeo, Sabrina Bűttner • Sepp Kohlwein • University of Gronningham • Marten Veenhuis • UNM • Terran Lane (CS) • Mary Ann Osley • Fred Hutchinson CRC • Linda Breeden

  30. Conclusions • Quiescent cells contain most of the protease-labile (localized) mRNAs • The most significant GO groups are for categories of genes abundant in NQ cells • Other protease-labile mRNAs encode stress proteins, cellular homeostasis, amino acid biosynthesis, and proteasome function • Provides strong evidence that Q cells are poised to respond to a number of different environmental situations without requiring transcription

  31. Conclusions • Quiescent cells are prepared to preserve membrane intactness, deal with oxidative stress, and respond to environmental stimuli • The large number of mRNAs encoding signal transduction genes in Q cells suggests they are capable of mounting a response to a variety of stimulii • Based on mRNA abundance, Q cells are in a regulated, cell-cycle arrest while NQ cells do not appear to be capable of arresting growth and metabolism

  32. Most protease-labile mRNAs are in Quiescent Cells Stat-Phase Culture (1570) Quiescent Cells (1957) 1208 207 573 130 25 46 Non-quiescent Cells (223) 22

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