Life and death of cells part I & II

1. Cell biology 2014 (revised 4/2 -14) Lecture 6 & 7: Life and death of cells part I & II All diseases involve changes of cells All cells come from cells Cells of all life forms have a common ancestor Rudolf Virchow, Die Cellularpathologie (1858)

2. Cell cycle control in multi-cellular eukaryotes somatic mutations & chromosomal instability Controlled and co- ordinated divisions Uncontrolled divisions  Tumor (clonalorigin!) 1013 Minimal length of DNA replicated by (cells of) a human Human diploid genome: ~6 x109 bp (cost of MD education: ~6 x109 Skr/year) (length of 1 bp) x (number of bp per cell) (0.34 nm) x (6 × 109) = 2 m, 1013 cells  2 x 1013 m 0.5 x1013 m Earth Uranus Pluto Sun Mars

3. “Simple” model systems for studies of eukaryotes Budding yeast, S. cerevisiae Polarized (bud & shmoo) Primordial eukaryote (polarity & sex) Fission yeast, S pombe Symmetric cell division • Yeast model systems: • Unicellular eukaryotic organism (autonomous cells) • Sexually active (mating & sporulation) • Short generation time • Haploid:  phenotype of recessive • (loss-of-function) mutations vs.

4. Principles of conditional mutants Wild type protein Mutant protein Functional (i.e. nophenotype) 26oC Non-functional (i.e. phenotype) 36oC The mutant gene product is temperature sensitive (Ts) OK at 26oC, butunfolds at 36oC (i.e. the function of gene product can be switched off)

5. Missing! Temperature sensitive yeast mutants = Ts clone 26o C 36o C Cell cycleTs mutant or not? 26o C 36o C or (if lucky!) House keeping gene mutated Cell cycle control gene mutated

6. Identification of cell division control (cdc) genes ZZZ Gene library (i.e., wild type genes cloned into bacterial plasmids) ZZZ ZZZ + 36o C OK OK OK Yeast cell cycle Ts mutant Mutated gene: (OK at 26oC but not 36oC) ZZZ Complementation by :

7. Cell cycle regulators are evolutionary conserved Budding yeast with a temperature sensitive mutation inan essential cell division control (cdc) gene 36o C 36o C cDNA library (copies of all human mRNA’s)  identification of a human “ortholog” The cell cycle control machinery is highly conserved in eukaryots Nobel prize in medicine 2001!

8. Three distinct cell cycle regulated events DNA replication Increased size Nuclear division followed by cytoplasmic division

9. S G2 GO G1 Interphase and mitosis Quiescent or post-mitotic Condensed chromatin ”thread-like” Interphase (“between-phase(s)”) (90100% of a cell population) S (DNA-Synthesis) Mitosis (Cell division phase) (010% of a cell population) G1 (Gap 1) G2 (Gap 2) Chromosome segregation Cell division Cell cycle exit  “G0” Chromatin “attract dyes” (Greek: mitos= thread, khrōma = color, soma = body)

10. 2. 3. 2. 1. 3. 1. Key events and checkpoints of the cell cycle G1/S checkpoint (“Start”) Sense: Surroundings Cell size Block: DNA replication M G1 G2/M checkpoint Sense: DNA replication status DNA damage Cell size G2 S Block: Mitotic entry Spindle assembly checkpoint Sense: Chromosome attachment to the mitotic spindle Block: Chromosome separation and cytoplasmic division

11. P P P Ub Ub Ub Cell cycle regulation of proteins Amount of protein Activity of protein - Control of protein expression - Binding partners DNA Transcription + = mRNA Translation Protein + = - Control of protein turnover • Phosphorylation 26S Proteosome

12. Cyclin dependent kinases (Cdk) – the controllers Cdk Cyclin H O Serine or threonine P Phosphatase Kinase - O Kinase motif - P O O P Inhibitory domain (T-loop) O Both the cyclin and are required to activate the Cdk P Serine or threonine Cyclin  substrate specificity

13. Cdk’s are stable while cyclin levels are “cyclic” Cdk 4/6 Cdk 2 Cdk 1 Diffuse border M G1 S G2 M G1 Cyclin: (expression) G1 (D) S (A) G1/S (E) M (B) Interphase cyclins Mitotic cyclin v-SNARE v-SNARE Ci NFAT Ras Src v-SNARE R7 cGMP Hsp70 MHC The cell can not reside in two cell cycle phases simultaneously The cell can not reside in two cell cycle phases simultaneously Wnt cGMP cGMP Notch COPII ACTH Retinoic acid RelA v-SNARE Clathrin v-SNARE

14. P P P P P P 1. 2. 3. Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub Mechanisms for interphase cyclin degradation Cdk Cdk G1 High intrinsic turnover of G1 cyclin + Kinase X -dependent ubiquitination of G1/S cyclin P Cdk Cdk Cdk G1/S G1/S + Cdk M Cdk/M-cyclin mediated  ubiquitination of S cyclin P Cdk Cdk Cdk S S +

15. Cdk Cdk G1 G1/S Cdk/cyclin control of progression and transitions Active: Cdk Cdk X S Cdk S Cdk M Transitions: M G1 S G2 M G1 G0

16. Wnt XGF External signals (mitogens)G1 cyclin expression Hedgehog Receptor Tyrosine Kinase GTP Ras = DNA Myc Myc G1 myc gene G1 cyclin gene

17. P P Cdk Cdk G1/S G1 2. 1. 2. 3. 3. 1. Rb Rb phosphorylation by Cdk/G1 cyclin  E2F activation Positive feedback loop G1/S DNA replisome E2F E2F Rb S activator repressor = DNA binding protein Phosphorylation by Cdk-G1 cyclin dissociates Rb from E2F Transcription of E2F regulated genes Enhancement by newly formed Cdk-G1/S cyclin

18. P P S-phase components DNA replisome E2F Rb Transcriptional control of S phase components E2F E2F E2F E2F Rb S-phase components Non-dividing cell: Dominantrepressionby the E2F/Rb complex G1/S S Proliferating cell: E2F mediated transcription of “S phase genes” Rb is mutated in ~40% of human tumors constitutive production of S-phase components

19. The stability of the genome: two levels of threat S-phase: 46 x ~4 cm DNA has to be replicated once (but only once!) M-phase: sorting of 2 x 46 sister chromatids (no errors!) G2 S-phase (6 h) ~4 cm DNA= 130 x106 bp ~ 250 nt/s  144 h/ 4 cm G1 G1

20. ORC ORC ORC Cdc6 Cdc6 Cdc6 ORC ORC ORC Cdc6 Cdc6 Cdc6 Mcm Mcm Mcm ORC ORC ORC Mcm Mcm Mcm Licensing of DNA for a single round of replication Early G1 Cdc6 is recruited to ORCs Formation of a pre-RC (i.e., licensing of DNA): Cdc6 dependent loading of Mcm proteins onto DNA Note - pre-replicative complexes are formed after mitosis independently of progression into a new S-phase

21. P P P Cdk S DNA replisome Firing of pre-RC during S-phase DNA replisome Cdc6 Mcm Late G1 ORC Mcm Firing of the first ORC point of no return! Phosphorylates Cdc6 and Mcm Cdc6 ORC DNA replisome S Mcm Mcm ORC DNA strand separation by helicase activity of Mcm proteins

22. P P P P 1. 4. 1. 2. 4. 3. 2. 3. DNA replisome Ub Ub Ub A fool-proof system for prevention of re-replication ORC DNA replisome Mcm Mcm ORC Phosphorylated Cdc6 dissociates from ORC Cdc6 Phosphorylated Cdc6 is recognized by SCF S C F Cdc6 Ubiquitylation of Cdc6 by SCF (an E3-ligase) Proteosomal degradation of Cdc6 Proteosome

23. P P P Cdk Cdk M S Ub Ub Ub Ub Ub Ub Ub Ub Ub Cdk S Prevention of DNA licensing until the next G1-phase Degradation of “free” Cdc6 G1: “free” Cdc6 is available Cdc6 Cdc6 Cdc6 Cdc6 Cdc6 Cdc6 Cdc6 Cdc6 Mcm ORC S G2 M G1 Cdc6 levels

24. P P Cdk Cdk S G1/S Cell cycle entry and DNA replication Externalsignal  Activationof the Rb pathway! Cdk G1 G1 Myc Growth factor Positive feedback loop Rb Rb/E2F = dominant repressor of E2F E2F Cdc6 ORC Mcm DNA replisome

25. Mdm2 4. 3. 1. 3. 2. 3. 4. 1. 2. Ub Ub Ub p53: The guard against tumors p53 p53 p53 p53 p53 ATM p53 gene ARF p53 is constitutively expressed but is normally… degraded through ubiquitination by Mdm2 (E3-ligase) DNA damage ( ) or unbalanced/excessive proliferation signaling ( ) inhibits Mdm2  stabilization of p53 ATM ARF Transcriptional activation of p53 responsive genes (ATM: ataxia telangiectasia-mutated, ARF: Alternative Reading Frame)

26. p21 DNA repair proteins Target genes of p53 dependent transcription p53 PUMA Bax Cell cycle block DNA repair Apoptosis Loss of p53 function I´m gonna live forever Genetic instability

27. p16 Two distinct CdKInhibitor (CKI) families Specificity: G1, G1/S and S G1 only p21 Cdk G1 Cyclin Cyclin Cdk 4/6 Inhibits: Catalytic activity Cyclin association (Cip/Kip family) (Ink4 family) In both cases, the Cdk activity is abolished Molecular_models: 17.1-Cdk2

28. Rb & p53 pathways cell cycle and apoptosis P Cdk Cdk Cdk G1 S G1/S p21 1. 2. 1. DNA replisome Myc p16 Growth factor Ras PI3K • p53 pathway detects: • 1. Unbalanced/ excessive • proliferation signals (+++) • 2. DNA damage • Cell cycle block • Apoptosis Survival signals +++ ARF PKB/Akt Rb ATM +++ p53 E2F Cdc6 ORC Apoptosis G1/S block

29. Three distinct cell cycle regulated events DNA replication Size growth Nuclear division followed by cytoplasmic division

30. Cell division and cell growth Oocytes grow without dividing Fertilized eggs replicate and divide without growing

31. P P P P P P P P P P PI3-kinase signaling regulates protein synthesis RTK P.M. 3 3 PKB/ Akt PI-3 K PKB/Akt PDK1 PKB/Akt Proteins Translation initiation factor Cellular size growth mRNA = + + Ribosomes

32. P P P P P P P P P P Ras Divergent (cooperating) RTK receptor signals RTK ”Mitogen” signaling (Rb-pathway) ”Growth factor” signaling PTEN G1 3 3 G1 G1 PI-3 K Cell cycle entry PKB/Akt Increased cell size Bad Survival

33. DNA replication Sizegrowth Summary: G1-, S- and G2-phase (interphase) Translation Initiation factor ”Mitogen” signaling: G1 + = G1 G1 DNA replisome G1 G1 G1 Ras G1 G1 G1 G1 Bad ”Growth factor” signaling: PI-3 K

34. The end of the cell cycle Cell division = nuclear division + cytoplasmic division M-phase Mitosis Cytokinesis Sorting of 2 x 46 sister chromatids Defines the division plane/symmetry Protein sorting (if asymmetric division) Topics of case 13: The cytoskeleton

35. Progression of cell division: two points of “No return” Interphase (G2) Prophase Telophase/ cytokinesis Prometaphase Transition points  Checkpoint control Anaphase Metaphase Video: 17.4 Animal_cell_division

36. P P P G2  M transition Event Cause Consequence Change in MT dynamics Mitotic spindle formation MAPs DNA packaging Chromosome condensation Condensin Breakdown of nuclear envelope Disassembly of nuclear lamina Nuclear lamins

37. P P P Cdk Cdk Cdk Cdk M M M M P P P Checkpoint control of Cdk/M-cyclin activity G2/M checkpoint control and regulation of Cdc25 activity: DNA replication stall DNA damage Cdc25 Insufficient cell size Dual feedback loops  activity burst! P P Inhibitory sites: Activating site: CAK wee1 Cdc25 P Cdk M + Inactive Inactive Inactive Active G2 G2M

38. Chromosome segregation Ub Ub Ub Ub Ub Ub Anaphase initiation + Attached during S-phase Sister chromatid separation (anaphase) + Securin Cohesin Separase

39. Ub Ub Ub Checkpoint control of metaphase-anaphase transition Cdc20 APC/C APC/C Cdc20 Inactive Active +  1 unattached kinetochore (spindle assembly checkpoint) Sorting of 2 x 46 chromosomes Metaphase Anaphase

40. P Cdk M Ub Ub Ub Mitotic exit and initiation of cytokinesis Checkpoint Active Cdk M Inactivation of Mitotic exit and cytokinesis Cdk/M activity block initiation of cytokinesis!

41. P P P P P Cdk Cdk M M Ub Ub Ub Ub Ub Ub Ub Ub Ub Molecular events during mitotic exit Active APC/C  cyclin degradation  inactive Cdk  mitotic exit APC/C Cdc20 + Cdk + Constitutively active phosphatases drives the cell out of mitosis MAPs MAPs Phosphatase Mitotic exit Nuclear lamins Condensin Condensin Nuclear lamins Mitosisversusmeiosis: Animation: 21.2-meiosis

42. P P P Cdk Cdk M M P Checkpoints function: block of premature transitions Spindle assembly checkpoint Spindle attachment to chromosomes APC/C Cdc20 G1/S checkpoint The surroundings M G1 G2/M checkpoint DNA status and cell size Mitogen G2 S E2F Cdc25 Checkpoints verify that all processes at each cell cycle phase have been completed before transition into the next P DNA replisome Mcm

43. Cell death in multi-cellular eukaryots Cell death The word apoptosis is greek for fallen leaves Necrosis Apoptosis - ”Suicide” - ”Murder” • Death for the benefit • of the organism • Associated with • inflammation • (and vice versa) • No inflammatory response

44. Importance of apoptosis Immune survelliance Homeostasis Development x x Elimination of superfluous cells between the developing fingers Killing of virus infected cells Apoptosis and cell proliferation must balance each other

45. Cellular changes during apoptosis Different stages of apoptosis Normal cell Cell shrinkage Membrane ruffling DNA condensation DNA fragmentation Phagocytic cells recognizes phosphatidyl serine ( ) which becomes exposed on the surface during apoptosis Burp! Video: 18.1 Apoptosis

46. Apoptosis – a protease cascade Initiator caspase monomers Apoptotic signal multimers Active initiator caspase Cytoskeleton DNA Other things Inactive effector caspase Active effector caspase o A s C = D y t k N

47. Control of apoptosis • Two pathways for initiation of apoptosis: • Extrinsic(receptor-mediated) and Intrinsic (mitochondrial) • Apoptosis is controlled by the balance of • Pro- and Anti-apoptotic regulatory proteins External and internal signals Anti-apoptotic Pro-apoptotic Cell survival Apoptosis

48. The intrinsic apoptotic pathway Anti- apoptotic Pro- apoptotic Cyt. C Cyt. C Bcl-2 BH123 Cyt. C Cyt. C Pore former Inhibitor of pore formation Cyt. C Pore formation in the outer mitochondrion membrane release Note - Bcl2 has all four BH domains (Albert et al: Fig. 18-9) Cyt. C

49. Cytochrome C in the cytosol triggers apoptosis Apoptosome Apaf1 : APoptosisActivating Factor Caspase 9 Cyt. C Cyt. C Apaf1 Apaf1 Caspase 9 Cyt. C Caspase 3 Caspase 9 Target proteins a g t r t i s T r e p o e n Caspase 3

50. Regulation of the intrinsic apoptotic pathway Anti- apoptotic Pro- apoptotic p53 p53 pathway: Pro-apoptotic downstream mediators Bcl-2 BH123 (family) Bax PUMA BH3-only (family) ATM ARF Note - Bcl2 has all four BH domains  per definition! (Albert et al: Fig. 18-9)