Cell cycle control in multi-cellular eukaryotes. Lecture 6 & 7: Principles of conditional mutants. Simple model systems for studies of eukaryotes

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1 Cell biology 2017 Lecture 6 & 7: Cell cycle control in multi-cellular eukaryotes ~10 13 somatic mutations & chromosomal instability Controlled and coordinated divisions Uncontrolled divisions Tumor (clonal origin!) All cells come from cells Cells of all life forms have a common ancestor All diseases involve changes of cells Rudolf Virchow, Die Cellularpathologie (1858) 1 inimal length of DNA replicated by (cells of) a human Human diploid genome: ~6 x10 9 bp (cost of D education: ~6 x10 9 kr/year) (length of 1 bp) x (number of bp per cell) (0.34 nm) x ( ) = 2 m, ~10 13 nucleated cells 2 x m 0.5 x10 13 m un Earth Uranus luto ars 2 imple model systems for studies of eukaryotes rimordial eukaryote (polarity & sex) Budding yeast,. cerevisiae olarized (bud & shmoo) Fission yeast, pombe ymmetric cell division 26 o C rinciples of conditional mutants Wild type protein utant protein Functional (i.e. no phenotype) Non-functional (i.e. phenotype) Yeast model systems: - Unicellular eukaryotic organism (autonomous cells) - hort generation time - Haploid: phenotype of recessive (loss-of-function) mutations - exually active (mating diploid sporulation) vs. 3 The mutant gene product is temperature sensitive (Ts) OK at 26 o C, but unfolds at (i.e. the function of gene product can be switched off) 4 Temperature sensitive yeast mutants 5 Identification of cell division control (cdc) genes 6 = Ts clone Cell cycle Ts mutant or not? 26 o C issing! Gene library (i.e., wild type genes cloned into bacterial plasmids) OK ZZZ ZZZ ZZZ 26 o C or (if lucky!) Yeast cell cycle Ts mutant utated gene: (OK at 26 o C but not ) ZZZ OK House keeping gene mutated Cell cycle control gene mutated OK Complementation by : 1

2 Cell cycle regulators are evolutionary conserved Budding yeast with a temperature sensitive mutation in an essential cell division control gene, termed cdc 7 Three distinct cell cycle regulated events DNA replication cdna library (copies of all human mrna s) identification of the human cdc2 orthologue (i.e. CDK1) The cell cycle control machinery involves probably the most conserved proteins among eukaryotes (Nobel prize in medicine 2001) Increased size Nuclear division followed by cytoplasmic division 8 Interphase and mitosis Key events and checkpoints of the cell cycle itosis (Cell division phase) (0 10% of a cell population) Chromosome segregation Cell division Condensed chromatin thread-like Interphase ( between-phase(s) ) (90 100% of a cell population) (DNA-ynthesis) (Gap 1) (Gap 2) Cell cycle exit G0 (Greek: mitos= thread, khrōma = color, soma = body) Chromatin attract dyes Quiescent or post-mitotic GO 9 / checkpoint ( tart ) ense: urroundings Cell size Block: DNA replication / checkpoint ense: DNA replication status DNA damage Cell size Block: itotic entry pindle assembly checkpoint ense: Chromosome attachment to the mitotic spindle Block: Chromosome separation and cytoplasmic division 10 Cell cycle regulation of proteins Amount of protein - Control of protein expression Activity of protein - Binding partners DNA mrna Transcription Translation = rotein = - Control of protein turnover Cyclin dependent kinases () the controllers On-Off switch : H O erine or threonine hosphatase Cyclin 26 roteosome - hosphorylation 11 O O - O - O erine or threonine motif Inhibitory domain (T-loop) Both the cyclin and are required to activate the Cyclin substrate specificity 12 2

3 s are stable while cyclin levels are cyclic echanisms for interphase cyclin degradation 4/6 2 High intrinsic turnover of cyclin X 1 Cyclin: (expression & degradation) (D) / (E) (A) Interphase cyclins HC (B) itotic cyclin 13 -dependent ubiquitination of / cyclin /-cyclin mediated ubiquitination of cyclin / / 14 /cyclin control of progression and transitions Active: X Transitions: / External signals (mitogens) cyclin expression Hedgehog Wnt XGF GT Ras GEF H2 Receptor osine I-3 K (etc) All mitogen signal converge on yc -pathway 16 G0 15 yc myc gene yc = DNA cyclin gene phosphorylation by / cyclin activation repressor ositive feedback loop / activator Transcription of regulated genes = DNA binding protein / hosphorylation by - cyclin dissociates from Enhancement by newly formed -/ cyclin 17 Transcriptional control of phase components Non-dividing cell: -phase genes Dominant repression by the / complex / -phase genes roliferating cell: mediated transcription of phase genes 18 3

4 The stability of the genome: two challenges -phase: 46 x ~4 cm DNA has to be replicated once (but only once!) -phase: sorting of 2 x 46 sister chromatids (no errors!) Licensing of DNA for a single round of replication Early -phase (6 h) ~4 cm DNA= 130 x10 6 bp ~ 100 nt/s ~300 h/ 4 cm Note pre replicative complexes are formed after mitosis independently of progression into a new phase is recruited to s Formation of a pre RC (i.e., licensing of DNA): dependent loading of cm proteins onto DNA Late Firing of pre-rc during -phase hosphorylates & initiator proteins Firing of the first point of no return! DNA strand separation by helicase activity of cm proteins 21 A fool-proof system for prevention of re-replication 4. hosphorylated dissociates from hosphorylated is recognized by CF Ubiquitylation of by CF (an E3-ligase) Degradation of by the proteosome CF 4. roteosome 22 levels revention of DNA licensing until the next -phase Degradation of free : free is available 23 ummary: cell cycle entry and DNA replication External signal (mitogen) Activation of the -pathway! Growth factor yc ositive feedback loop / 24 / = dominant repressor of 4

5 : The guardian against tumors 25 Target genes of dependent transcription dm2 AT ARF gene 4. p21 DNA repair proteins Bax UA is constitutively expressed but is normally Cell cycle block DNA repair Apoptosis 4. degraded through ubiquitination by dm2 (E3-ligase) DNA damage ( AT ) or unbalanced/excessive proliferation signaling ( ARF ) inhibits dm2 stabilization of Transcriptional activation of responsive genes (AT: ataxia telangiectasia-mutated, ARF: Alternative Reading Frame) Loss of function Genetic instability I m gonna live forever 26 Two distinct CdK Inhibitor (CKI) families pecificity:, / and p21 Cyclin only 4/6 Cyclin Inhibits: Catalytic activity Cyclin association (Cip/Kip family) (Ink4 family) olecular_models: In both cases, the activity is abolished 27 urvival I3K KB/Akt Apoptosis & pathways cell cycle and survival Growth factor AT itogen signal p21 / block yc ARF / Ink4 pathway detects: Unbalanced/ excessive proliferation signals () DNA damage Cell cycle block Apoptosis 28 Three distinct cell cycle regulated events Cell division and cell growth 30 DNA replication ize growth Oocytes (egg precursors) grow without dividing Nuclear division followed by cytoplasmic division 29 Fertilized eggs replicate and divide without growing (cleavage phase, which is prior to gastrulation) 5

6 I-3 kinase signals increased protein synthesis.. RTK Ribosomes H2 I-3 K Translation initiation factor 3 3 KB/ DK1 KB/Akt mrna KB/Akt = roteins Increased cell size 31 Divergent (cooperating) RTK receptor signals itogen signaling (AK/-pathway) GT Ras / transition GEF H2 RTK Growth factor signaling (I-3 -pathway) H2 I-3 K Translation initiation factor Increased cell size TEN 3 3 KB/Akt BH3-only urvival 32 ummary: -, - and -phase (interphase) DNA replication itogen signaling: GT Ras The end of the cell cycle Cell division = nuclear division cytoplasmic division -phase itosis Cytokinesis Cell growth Growth factor & survival signaling: H2 I-3 K BH3-only Translation Initiation factor = (family of proapoptotic proteins) Increased cell size 33 orting of 2 x 46 sister chromatids Defines the division plane/symmetry rotein sorting (if asymmetric division) Topics of case 13: The cytoskeleton 34 rogression of cell division: two points of No return Interphase () rophase Telophase/ cytokinesis Anaphase Transition points Checkpoint control etaphase rometaphase Checkpoint control of /-cyclin activity / checkpoint control and regulation of Cdc25 activity: DNA replication stall DNA damage Insufficient cell size Cdc25 Inhibitory sites: Activating site: wee1 CAK Cdc25 36 Dual feedback loops activity burst! Inactive Inactive Inactive Active Video: 17.4 Animal_cell_division 35 6

7 transition by /-cyclin activation 37 Chromosome segregation Anaphase initiation 38 Event Chromosome condensation Breakdown of nuclear envelope itotic spindle formation Cause Condensin Nuclear lamins As Consequence DNA packaging Disassembly of nuclear lamina earch & capture of chromosomes Attached during -phase Cohesin eparase ecurin ister chromatid separation (anaphase) Checkpoint control of metaphase-anaphase transition itotic exit and initiation of cytokinesis Cdc20 Active AC/C AC/C Inactive Cdc20 Active 1 unattached kinetochore (spindle assembly checkpoint) Checkpoint Inactivation of orting of 2 x 46 chromosomes etaphase Anaphase 39 itotic exit and cytokinesis / activity block initiation of cytokinesis! 40 olecular events during mitotic exit Active AC/C cyclin degradation inactive mitotic exit AC/C Cdc20 41 Checkpoints function: block of premature transitions pindle assembly checkpoint AC/C pindle attachment to chromosomes Cdc20 Constitutively active phosphatases drives the cell out of mitosis As hosphatase As Nuclear lamins Nuclear lamins itotic exit Condensin Condensin / checkpoint DNA status and cell size Cdc25 itosis vs meiosis: Animation: 22-meiosis Checkpoints verify that the cell is ready to enter the next cell cycle phase / checkpoint The surroundings itogen 42 7

8 Cell death in multi-cellular eukaryots Cell death Importance of apoptosis Development Homeostasis Immune survelliance The word apoptosis is greek for fallen leaves x x Necrosis Apoptosis - urder - Results in inflammation (and vice versa) - uicide - Death for the benefit of the organism (altruism) - No inflammatory response Elimination of superfluous cells between the developing fingers Apoptosis and cell proliferation must balance each other Killing of virus infected cells (tumor cells?!) Normal cell Cellular changes during apoptosis Different stages of apoptosis Apoptosis a protease cascade Initiator caspase monomers Apoptotic signal multimers 46 Active initiator caspase Cell shrinkage embrane ruffling DNA condensation DNA fragmentation hagocytic cells recognizes phosphatidyl serine ( ) which becomes exposed on the surface during apoptosis Video: 18.1 Apoptosis Burp! 45 Cytoskeleton DNA Other things A t = Active effector caspase Inactive effector caspase Control of apoptosis Apoptosis is controlled by the balance of ro- and Anti-apoptotic regulatory proteins External and internal signals 47 The intrinsic apoptotic pathway roapoptotic Antiapoptotic BH4 BH3 BH1 BH2 BH3 BH1 BH2 48 ro-apoptotic Anti-apoptotic Cell survival Apoptosis Apoptosis is initiated by either of two distinct pathways: Extrinsic (receptor-mediated) and Intrinsic (mitochondrial) BH123 ore former (similar to Bcl-2, but lack BH4) Bcl-2 Inhibitor of pore formation (defines BH1-4) The Bcl-2 protein sequence defines each of the four Bcl-2 Homology domains (see Albert et al: Fig. 18-8) ore formation in the outer mitochondrion membrane release 8

9 Cytochrome C in the cytosol triggers apoptosis Apoptosome Apaf1 : Aoptosis Activating Factor Caspase 9 Apaf1 Apaf1 Caspase 9 49 Regulation of the intrinsic apoptotic pathway roapoptotic Antiapoptotic pathway: ro-apoptotic downstream mediators 50 Target proteins Caspase 3 Caspase 9 BH123 (family) Bcl-2 Bax Caspase 3 BH3-only (family) UA a g t r t i s T r e p o e n BH123: Bax and Bak. At least one of these is required for Cyt C release AT ARF Intrinsic apoptosis ummary on apoptosis Extrinsic apoptosis Distinct signals elicited by a single ligand Ligand 52 urvival signals Bcl-2 BH123 BH3-only Death signals Death receptor Ligand domain domain domain domain Other signals Initiator caspase Initiator caspase - DNA - Cytoskeleton - Other things Effector caspases A t 51 cyclin Increased cell size One specific receptor tyrosine kinase may transduce several distinct signals, which at least in part differ between cell types Recommended reading: Alberts et al. 5 th or 6 th edition, Chapter 17 & 18. Only topics covered by lectures are relevant. Focus on pictures and use the text book for clarifications of the lecture content 53 The art of making knowledge useful Johan Bergkvist vt