yeast seminars - jaguar.biologie.hu-berlin.de
TRANSCRIPT
Towards a holistic understanding of the Eukaryotic cell and a Eukaryotic whole cell model
Marcus Krantz [email protected]
Yeast seminars
The whole cell perspective
First whole cell model available - Mycoplasma genitalium - 525 genes - 28 Submodels Karr et al.; A Whole-Cell Computational Model Predicts Phenotype from Genotype; Cell, Volume 150, Issue 2, 20 July 2012, Pages 389–401
The whole cell perspective
Defining a yeast whole cell model? - Eukaryote - 10x as many genes How could we do something similar in yeast? Which processes should be considered? How are they built? How do they work together?
The whole cell perspective
CDC
Growth
Growth & Proliferation: - Metabolism
- Catabolism - Anabolism
- Growth - Division
The whole cell perspective
Transcription
Replication
Ribosome assembly
CDC
Growth
Protein Synthesis
Growth & Proliferation: - Metabolism
- Catabolism - Anabolism
- Growth - Division
The central dogma: - DNA -> RNA -> Protein
The whole cell perspective
Transcription
Replication
Ribosome assembly
CDC
Growth
Protein Synthesis
Growth & Proliferation: - Metabolism
- Catabolism - Anabolism
- Growth - Division
The central dogma: - DNA -> RNA -> Protein
The whole cell perspective
Transcription
Replication
Ribosome assembly
CDC
Growth
Protein Synthesis
Sensing & Signalling
Growth & Proliferation: - Metabolism
- Catabolism - Anabolism
- Growth - Division
The central dogma: - DNA -> RNA -> Protein
Signalling: - Available nutrients - Pheromones & Stress
The whole cell perspective
Transcription
Replication
Ribosome assembly
Morphology
Life cycle
CDC
Mitochondria
Peroxisomes
Vacuoles
Growth
Protein Synthesis
Sensing & Signalling
Growth & Proliferation: - Metabolism
- Catabolism - Anabolism
- Growth - Division
The central dogma: - DNA -> RNA -> Protein
Signalling: - Available nutrients - Pheromones & Stress
Life decisions: - Sporulation & Mating
Cell structure: - Compartments - Morphology
Yeast seminars
Learning objectives Reading and extracting information from review papers. Interpreting the information in context of the whole cell. Presenting and discussing the information with your peers.
Form Literature seminars
Perspective Whole cell modelling
Yeast seminars
The system: - baker’s yeast; Saccharomyces cerevisiae - the premier Eukaryotic model - well charactersied - outstanding knowledge
The resources: - Genetic Techniques for Biological Research
(Michels, CA; 2002) - Genetics’ yeast book - set of comprehensive review articles - one review per topic - use of additional material encouraged
Yeast seminars
Transcription
Replication
Ribosome assembly
Morphology
Life cycle
CDC
3 Cellular building blocks
6 The cell division cycle
7 Morphology
2 Quality control
1 Transcription 9 Organelles
10 Nuclear function
4 Nutritional control
5 The life cycle
Mitochondria
Peroxisomes
Vacuoles
Growth
Protein Synthesis
Sensing & Signalling
8 Signalling
Yeast seminars
The presentations: - 25-30 minutes + discussion - based on a (given) review paper; use of
additional material is optional - split between: - overview; cellular context - mechanistic detail of (parts) of module - discuss: - function in whole cell perspective - connection to other modules (topics) - how could the module be modelled?
Integrated with other modules?
Theme Topic Transcription Transcriptional regulation
Chromatin dynamics Quality control RNA Degradation
The ubiquitin-proteasome system Cellular building blocks Amino acid, nucleotide, and phosphate metabolism
Metabolism & Regulation of Glycerolipids Nutritional control Nutritional Control of Growth and Development
Target of Rapamycin (TOR) The life cycle Sporulation
Mating The cell division cycle Cdk1-controlled targets and processes
Mitotic Exit Morphology Morphogenesis and the Cell Cycle
Cell Polarization and Cytokinesis Signalling Response to hyperosmotic stress
Regulation of Cell Wall Biogenesis Organelles Mitochondrial assembly
Lipid Droplets and Peroxisomes Nuclear function The Yeast Nuclear Pore Complex
Structure and function in the nucleus
Yeast seminars
Group divisions and topics: - two topics per session - each topic covered by a pair of students Questions: - which processes should be considered in a
whole cell model? - in what priority? - core? - peripheral? - optional? - connection between processes/modules?
Theme Topic Transcription Transcriptional regulation
Chromatin dynamics Quality control RNA Degradation
The ubiquitin-proteasome system Cellular building blocks Amino acid, nucleotide, and phosphate metabolism
Metabolism & Regulation of Glycerolipids Nutritional control Nutritional Control of Growth and Development
Target of Rapamycin (TOR) The life cycle Sporulation
Mating The cell division cycle Cdk1-controlled targets and processes
Mitotic Exit Morphology Morphogenesis and the Cell Cycle
Cell Polarization and Cytokinesis Signalling Response to hyperosmotic stress
Regulation of Cell Wall Biogenesis Organelles Mitochondrial assembly
Lipid Droplets and Peroxisomes Nuclear function The Yeast Nuclear Pore Complex
Structure and function in the nucleus
The course
Date Theme Topic PMID 13/4 Lecture I Introduction
Yeast as a model system N/A
20/4 Lecture II Overview of yeast cell biology N/A
27/4 Transcription Transcriptional regulation Chromatin dynamics
22084422 21646431
4/5 Quality control RNA Degradation The ubiquitin-proteasome system
22785621 23028185
11/5 Cellular building blocks Amino acid, nucleotide, and phosphate metabolism Metabolism & Regulation of Glycerolipids
22419079 22345606
18/5 Nutritional control Nutritional Control of Growth and Development Target of Rapamycin (TOR)
22964838 22174183
1/6 The life cycle Sporulation Mating
22084423 20066086
8/6 The cell division cycle Cdk1-controlled targets and processes Mitotic Exit
20465793 23212898
15/6 Morphology Morphogenesis and the Cell Cycle Cell Polarization and Cytokinesis
22219508 22701052
22/6 Signalling Response to hyperosmotic stress Regulation of Cell Wall Biogenesis
23028184 22174182
29/6 Organelles Mitochondrial assembly Lipid Droplets and Peroxisomes
23212899 23275493
6/7 Nuclear function The Yeast Nuclear Pore Complex Structure and function in the nucleus
22419078 22964839
13/7 Conclusion Summary, discussion & evaluation N/A
Why yeast?
Food Baking, Winemaking; - Fermentation (Pasteur 1857) Brewing (Carlsberg); - Pure cultures (1883)
Biotechnology Biofulels; - ethanol, etc.. Biopharma; - Interferon (1981) - Vaccine (HBAg) - Insulin (50%) Drug screening
Model organism Cultivation; easy, fast, safe Genetic manipulation; - Crossing, ploidity - Transformation, deletion Functional genomics; - Sequenced, collections Systems biology; - Knowledge resources
Basic facts – what is yeast?
I. One of the oldest domesticated organisms – and one of the economically most important.
II. Brewers’ or Bakers’ Yeast: Ferments sugar to ethanol + CO2.
III. Favourite model system, as it is: I. Domestic (easy to culture) II. Genetically amenable (via homologous
recombination, both haploid and diploid life cycle)
III. Unicellular with short generation time (~2h) IV. The most well characterised eukaryote
IV. High gene density 1gene/2kb – almost like bacteria (1gene/1kb).
V. Few introns (~4% of the genes).
VI. 16 chromosomes VII. Budding Yeast VIII. Some trivia:
Sugar Fungus from Beer
Baker’s yeast – Saccharomyces cerevisiae
Use and Habitat
Natural habitat unknown: S. cerevisiae has been isolated from a wide range of natural habitats all over the world. It can e.g. be found on fruits, flowers and other sugar containing substrates. It copes with a wide range of environmental conditions:
• Tolerates temperatures from freezing to about 55°C • Proliferate from 12°C to 40°C; optimal growth at 30°C • Growth is possible from pH 2.8-8.0 • Almost complete drying is tolerated (dry yeast) • Yeast can still grow and ferment at sugar concentrations of 3M (high osmotic pressure) • Yeast can tolerate up to 20% alcohol
Workhorse in the food industry... The main organism in wine and beer production
Enormous fermentation capacity even in the presence of oxygen, tolerance to low pH and high ethanol, lager yeast ferments at 8°C
The yeast used in baking because it produces carbon dioxide from sugar very rapidly
...and in Biotechnology for: Heterologous protein production
because it can be genetically engineered, it is regarded as safe and fermentation technology is highly advanced Drug screening and functional analysis
because it is a eukaryote but can be handled as easily as bacteria
And it is the premier eukaryotic model system because it can be studied by powerful genetics and molecular and cellular biology; many important features of the
eukaryotic cell have first been discovered in yeast. Unmatched technical and theoretical toolbox; unmatched understanding. Basic research - Elucidate fundamental biological features/functions/mechanisms. Technolgoical advancement - improve existing or generate new biotechnological processes
Adapted from S. Hohmann
The premier model system
I. The awesome power of yeast genetics I. Targeted mutations via homologous recombination (confirmed mutant in ~2 weeks) II. Stable growth as haploid or diploid III. Crossing is easy, automated and can be performed en masse
II. The functional genomics paradigm I. First sequenced eukaryotic genome (1996). II. Near complete deletion strain collection in four strain collection (∆, ∆/wt, ∆/∆). III. 85% annotated genes. IV. 17% of genes have orthologues associated with disease in human. V. Genetic interaction networks: 5.4*106 double mutants (20%) VI. Protein-protein interaction networks; Protein kinase-target networks VII. Transcriptional network; TF-promoter interactions VIII. Comparative genomics resources with related fungal species as well as individual isolates of
Saccharomyces from across the world.
III. Toward the systems level understanding I. Unparalleled knowledge resources II. Feasible verifiaction experiments
[email protected] http://upload.wikimedia.org/wikipedia/commons/thumb/1/11/Tree_of_life_SVG.svg/600px-Tree_of_life_SVG.svg.png
The origin of yeast(s)
“Contrary to the commonly held view, yeasts do not represent primitive unicellular eukaryotes but instead have repeatedly emerged from distinct phylogenetic lineages of ‘modern’ fungi”
Dujon, 2010 10-20%
Yeast physiology
Budding yeast – asymmetrical division: Mother (old; large) + Daughter (new; small) Cell volume ~50fL Replicative lifespan; 30-40 generations Eukaryotic:
1. Cell wall: 80-90% polysaccharides; 1-3 β-glucan (straight), 1-6 β-glucan (branched), chitin and mannoproteins 2. Periplasmic space with hydrolytic enzymes. 3. Cell membrane: lipids + proteins. 4. Nucleus w nucleolus (rRNA transcription centre). 5. Vacuole: Hydrolysis, storage, detoxification. 6. Secretory system w. ER, Golgi and vesicles • Peroxisomes; oxidative degradation • Mitochondria • Cytoskeleton
• Actin for polarised secretion/endocytosis
• Microtubuli for nuclear positioning and separation
Membranes allow gradients (potential) = Energy
Regulated uptake
1 2 3
4
5
6
Mitchels 2002
Nutritional requirements
Energy/carbon source - Fermentative carbon sources;
- Glucose/Fructose/Mannose (Glucose repression) - Galactose/Sucrose/Raffinose (Glucose repressed)
- Non-fermentative carbon sources; - Ethanol, Glycerol
Nitrogen source - Ammonium, amino acids - Priority; Nitrogen Catabolite Repression (NCR)
Minerals and vitamins (e.g. YNB) - Phosphate, Sulphate, etc.
Amino acids (auxotrophic strains)
Anaerobic specific requirements: - Sterol (Ergosterol) - Unsaturated fatty acids (Tween 80)
Lindegren 1949
Biotin 2 μg Calcium pantothenate 400 μg Folic acid 2 μg Inositol 2000 μg Niacin 400 μg p-Aminobenzoic acid 200 μg Pyridoxine hydrochloride 400 μg Riboflavin 200 μg Thiamine hydrochloride 400 μg Boric acid 500 μg Copper sulfate 40 μg Potassium iodide 100 μg Ferric chloride 200 μg Manganese sulfate 400 μg Sodium molybdate 200 μg Zinc sulfate 400 μg Potassium phosphate monobasic 1 g Magnesium sulfate 500 mg Sodium chloride 100 mg Calcium chloride 100 mg
YNB
Life cycle
I. Unicellular II. Haploid or Diploid life cycle III. Two ”sexes”, or mating types; a and α.
I. Secrete sex-specific pheromone; a- or α-factor II. Respond to pheromone from opposite sex III. Two haploids (a + α) can mate to produce the a/α diploid IV. Wild type haploids can switch gender (HO+)
IV. Starvation triggers the sexual cycle – sporulation V. Spores are haploid an extremely resistant to stress VI. Spores germinate when conditions improve VII. Starved cells may also differentiate into pseudohyphal growth.
Michels (2002)
Cell Division Cycle
M
Spindle formation
Nuclear migration
Chromosome segragation Nuclear division
Cytokinesis
Growth START
Bud emergence
G2
S
G1
http://mpf.biol.vt.edu/research/budding_yeast_model/pp/intro.php
DNA replication
I. The fundamental mechanism of proliferation. II. Conserved in all Eukaryotic cells. III. Driven by ”Cyclin dependent kinases” (CDKs) and
cyclins, and monitored by check points. IV. Cyclins build up during cell cycle phases until they
trigger a transition and their own destruction
Nomenclature
• All characterised genes has a name of three letters and a number. These often describe the protein or function.
• All ”open reading frames” (ORFs) have a systematic name of three letters, a three digit number, and a final letter.
• References to ORFs and genes are written in italics.
• Upper case indicates wild-type or dominant mutations.
• Lower case indicate deletion or loss of function alleles.
• Protein names are given with an initial capital followed by lower case letters, e.g.; Yak1.
http://www.yeastgenome.org/sgdpub/Saccharomyces_cerevisiae.pdf
YJL141C (YAK1) Y = Yeast J = Chromosome 10 L = Left arm (from centromere) 141 = 141st ORF from centromere C = On the lower (Crick) strand.
Reference sites
• Saccharomyces Genome Database (SGD) – http://www.yeastgenome.org/
• Kyoto Encyclopedia of Genes and Genomes (KEGG) – http://www.genome.jp/kegg/
• Munich Information centre for Protein Sequences (MIPS)
– http://mips.helmholtz-muenchen.de/genre/proj/yeast/
• Phenotypic profiles (Prophecy)
– http://prophecy.lundberg.gu.se/
• Biogrid
– http://thebiogrid.org/
• T-profiler
– http://www.t-profiler.org/
SGD: Spell
Search engine for microarray data Identifies most informative dataset and similarly expressed genes 352 datasets representing 5686 total arrays from 233 published studies
Sequence analysis
• RSAT – http://rsat.ulb.ac.be/rsat/
• Blast – http://www.ebi.ac.uk/Tools/msa/clustalw2/
• Plasmapper – http://wishart.biology.ualberta.ca/PlasMapper/