Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download BC4 – The Cell Cycle
Document related concepts
Transcript
BC4 – The Cell Cycle Boris Pfander 1 Max Planck Ins9tute of Biochemistry [email protected] +49-‐89-‐8578-‐3050 www.biochem.mpg.de/pfander Topics Overview on the Cell Cycle The discovery of CDK – the master regulator of the cell cycle S-‐phase and DNA replica9on Checkpoints that sense DNA damage M-‐phase – mitosis and cytokinesis Chromosome condensa9on & cohesion 2 Relevant Literature Bruce Alberts, Alexander Johnson, Julian Lewis, Mar9n Raff, Keith Roberts, Peter Walter Molecular Biology of the Cell, 5th Edi9on Chapters 17, 18, 5 (p281-‐95) Harvey Lodish, Arnold Berk, Paul Matsudaira, Chris A. Kaiser, Monty Krieger, Ma`hew P. Sco`, Lawrence Zipursky, James Darnell Molecular Cell Biology, 5th Edi9on Chapter 21 3 David O. Morgan The Cell Cycle: Principles of control, 1st Edi9on (out of print) Figures online at Oxford University Press h`p://global.oup.com/uk/orc/biosciences/biochem/morgan/resources/ figures/ Why study the Cell Cycle? 4 Why study the Cell Cycle? All organism require cell reproduc9on by cell division. à A fundamental requirement of (eukaryo9c) life! Misregulated cell reproduc9on gives rise to Cancer. à medical relevance! 5 Cell Division -‐ giving rise to an organism Development of a zebra fish embryo 6 http://www.embl-heidelberg.de/digitalembryo/ How to duplicate a (complex) cell? The main task of the Cell Cycle is to accurately copy the cellular blueprint (DNA/chromosomes) and distribute it to the daughter cells. The cell cycle is ordered and consists of specific steps/phases Cells have a cell cycle control system, which orders and coordinates these steps 7 The eukaryo9c cell cycle S-‐phase: Duplica9on of DNA à Sister Chroma9ds M-‐phase: Distribu9on of sister chroma9ds to the 2 daughter cells; cell dividion 8 Mitosis 9 Mitosis Microtubules DNA Centrosomes 10 The eukaryo9c cell cycle Intra-‐S DNA damage checkpoint G1 DNA damage checkpoint G1: decision if cells enters a new cell cycle; coordina9on of cell cycle and cell growth G2/M DNA damage checkpoint Mito9c checkpoint = Spindle assembly checkpoint 11 Essen9al Terminology S-‐phase synthesis phase M-‐phase mitosis and cytokinesis G-‐phases for "gaps" between S and M phases, G1, G2, G0; Interphase period between mitoses Mitosis nuclear division (prophase, metaphase, anaphase, telophase) Cytokinesis cellular division 12 Cell cycle phases vary between different cells and organisms 13 The cell cycle was described already in the 19th century About 1880: very detailed cytological descrip4on of cell cycle by Walther Flemming (see Nat Rev Mol Cell Biol 2, 72-‐5) Introduced terms: mitosis, chroma4n, midbody, centriole 14 ? How is the cell cycle organized at a molecular level? 15 Model Organisms for Cell Cycle Research Budding yeast -‐ Simple, single cellular eukaryotes -‐ Easy to culture -‐ Can live as haploid (easy gene9c manipula9on) -‐ Can be used for gene9c screens -‐ Morphology is indica9ve of cell cycle phase Fission yeast 16 Model Organisms for Cell Cycle Research Early Embryo Systems (e.g. Xenopus laevis) -‐ Big Cells (suscep9ble to micro-‐injec9on) -‐ High content of nuclear proteins (biochemical systems) -‐ Simplified cell cycle 17 Model Organisms for Cell Cycle Research Human/Mammalian cell culture cells -‐ Show conserva9on of principles in human cells 18 ? How is the cell cycle organized at a molecular level? 19 The cell cycle is ordered A`ributes of the cell cycle system: Direc9onal (i.e. no “reverse reac9on”) Ordered à no phase is ever ler out à G1-‐S-‐G2-‐M order is always kept 20 What are the underlying principles of the order? S-‐Phase M-‐Phase S-‐Phase M-‐Phase “Timer”: Every Reac9on/Phase occurs in a temporal order Suggested by experiments in early embryos 21 “Biochemical Pathway”: Reac9on/Phases depend on the previous one Suggested by experiments using yeast mutants The cell cycle – 9mer and checkpoints Intra-‐S DNA damage checkpoint G1 DNA damage checkpoint G1: decision if cells enters a new cell cycle; coordina9on of cell cycle and cell growth G2/M DNA damage checkpoint Mito9c checkpoint = Spindle assembly checkpoint 22 ? What is the nature of the cell cycle 9mer? 23 The cell cycle 9mer is… Cyclin-‐CDK Yeast genetics Hartwell – budding yeast 60s-80s Nurse – fission yeast 70s-80s Biochemistry Hunt – 70s-80s 24 http://www.nobelprize.org/nobel_prizes/medicine/laureates/2001/ The cell cycle 9mer is… Cyclin-‐CDK 25 http://www.nobelprize.org/nobel_prizes/medicine/laureates/2001/ Cell fusion experiments Rao & Johnson, 1970 fusion of M phase cell with interphase cell à chromosome condensation in interphase cell fusion of S phase cell with G1 cell à replication of G1 nucleus Cells contain diffusable factors (proteins?) that promote cell cycle progression. 26 The screen for CDC genes in budding yeast G1 M S Budding yeast: Morphological changes during the cell cycle 27 The screen for CDC genes in budding yeast The CDC (cell division cycle) screen (Leland Hartwell, 1970-‐1973) Assump9ons: Cell cycle genes are essen9al Cell cycle mutants arrest at a specific point in the cell cycle à Forward gene9cs screen for temperature sensi9ve mutants that arrest with a terminal phenotype (indica9ve of a defect in a specific cell cycle stage) 28 Temperature sensi9ve mutants conformation of wild type protein: conformation of mutant protein at permissive vs restrictive T: * 29 * Iden9fying the mutant gene by complementa9on interesting mutant "A*" problem: Which gene is mutated? * re-isolation of plasmid and sequencing of the gene 30 The screen for CDC genes in budding yeast Assump9ons: Cell cycle genes are essen9al Cell cycle mutants arrest at a specific point in the cell cycle à Forward gene9cs screen for temperature sensi9ve mutants that arrest with a terminal phenotype (indica9ve of a defect in a specific cell cycle stage) Asynchronous cells Arrested cells 31 cdc mutants Metaphase arrest; Defect in separa9ng chromosomes Telophase arrest; Defect in cytokinesis 32 Cdc28 – A kinase important for entry into S-‐phase The cdc28-‐ts mutant showed the earliest possible arrest point (G1); no entry into the cell cycle à “START” Cdc28 = Cdk of budding yeast 33 A CDC screen in fission yeast The CDC/WEE screen (Paul Nurse, 1975) Fission yeast: Grows by elonga9ng Divides centrally à “Cell cycle morphology” not as clear as budding yeast, but cell size can be determined very easily Can cell growth & cell cycle become uncoupled? 34 CDC2/WEE2 -‐ the pacemaker of the cell cycle cdc2(big) wee2(small) The muta9ons in cdc2-‐ and wee2-‐ affect the same gene – CDC2: cdc2-‐ leads to reduced ac9vity of CDC2 wee2-‐ leads to enhanced ac9vity of CDC2 35 Cdc2 – the pacemaker of the cell cycle Cdc2 = Cdk of fission yeast 36 Embryonic systems with synchronous cell divisons Problem: in a cell popula9on (e.g. cell culture) cells are dividing asynchronously… à Embryo Systems Xenopus laevis embyro 37 The discovery of Cyclins Sea urchin embryo synchronous cell cycles 35S-‐methionine labeling experiment Cleavage in embryos of the sea urchin Arbacia punctulata consists of eight very rapid divisions that require continual protein synthesis to sustain them. This synthesis is programmed by stored maternal mRNAs, which code for three or four particularly abundant proteins whose synthesis is barely if at all detectable in the unfertilized egg. One of these proteins is destroyed every time the cells divide. . . . propose to call these proteins the cyclins. We Tim Hunt, 1983 (Cell 33, 389-‐96) 38 MPF – Mitosis Promo9ng Factor Xenopus laevis 39 Frog eggs are arrested in prophase Injec4on of cytoplasm from M phase cells triggers cell division. à Cytoplasm of M phase cells contains a Matura4on-‐Promo4ng Factor = Mitosis-‐Promo4ng Factor = MPF Masui & Markert 1971 Purifica9on of Mitosis Promo9ng Factor à Triggers mitosis à consists of 2 subunits à has a kinase activity Lohka & Maller, 1988 (PNAS85, 3009-13); MPF = Mitotic cyclin + cyclin dependent kinase Cdk1 40 Can trigger mitosis Xenopus egg extract system mature egg arrested in metaphase of meiosis II crush by centrifugation High concentration of cell cycle proteins à biochemistry No transcription (relies on maternal mRNA) Synchronous cell cycles 41 Proof that MPF is Cyclin B-‐CDK Cyclin B levels and kinase ac4vity of MPF change in parallel in cycling Xenopus egg extracts (correla4on!) 42 Murray & Kirschner, 1989 Proof that MPF is Cyclin B-‐CDK diges4on of maternal mRNAs by RNAse treatment RNase treatment , followed by RNase-‐inhibi4on and addi4on cyclin B mRNA à synthesis of cyclin B necessary and sufficient for mito9c entry 43 Proof that MPF is Cyclin B-‐CDK 3. RNase treatment followed by RNase-‐inhibi4on + mRNA coding for stable cyclin B -‐> exit from mitosis requires degrada9on of cyclin B MPF (= M cyclin + Cdk1) is the master regulator of mitosis 44 Cyclin-‐CDK 45 Cyclin-‐dependent kinases and cyclins Cyclin/CDK together form a protein kinase CDK(s) are constant throughout the cell cycle, Cyclins are oscilla4ng – different cyclin types are produced at different cell-‐cycle stages; several different cyclins One CDK controls budding and fission yeast cell cycle Addi4onal CDKs in metazoans (different combina4ons) CDK cyclin Why does the cell need different cyclins? cyclins direct CDK to different substrates cyclins cause different CDK ac4vity à Different cyclin-‐CDK complex causes the phosphoryla4on of different substrates à Different cyclin-‐CDK complexes are required for different cell cycle transi4ons 46 Cyclin CDK complexes control cell cycle transi9ons 47 Cyclin-‐dependent kinases and cyclins -‐ complexity 48 Cyclin CDK complexes control cell cycle transi9ons 49 Molecular control of the cell and the cell cycle Transcrip9on (expression of specific genes turned on/off) Pos`ransla9onal modifica9ons (PTMs = fast) phosphoryla9on Modifica9on of a protein with a phosphate on serine, threonine or tyrosine (oren at >1 residue in the protein) catalyzed by a kinase (such as cyclin-‐CDKs) reversal catalyzed by a phosphatase Phosphorya9on genererates new binding partners or disrupts exis9ng interac9ons 50 Molecular control of the cell and the cell cycle Transcrip9on (expression of specific genes turned on/off) Pos`ransla9onal modifica9ons (PTMs = fast) ubiqui9na9on Modifica9on of a protein with another (small) protein (ubiqui9n) via ε-‐amino of Lysines (iso-‐pep9de) Ubiqui9na9on can (dependent on chain linkage) lead to binding to other proteins or lead to degrada9on by the proteasome 51 Summary 1. The cell cycle regula4on is well conserved among eukaryotes; 2. The first analysis of cell cycle was possible thanks to the naturally synchronized cells (oocytes, embryos) and cell cycle mutants in yeast; 3. Highly ordered events of cell cycle are regulated by cyclin-‐dependent kinases interac4ng with cyclins; 4. The progression through cell-‐cycle is ordered, unidirec4onal and controlled and by checkpoints; 52 53
