MUNI SCI Cell cycle RNDr. Jan Skoda, Ph.D. Department of Experimental Biology Bi1700enCell Biology / 06 - Cell cycle (13 Apr 2022) Outline Definition and phases of the cell cycle The cell-cycle control system Molecular regulation of the cell cycle Defects in the cell cycle Models to study the cell cycle Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) The cell cycle Omnis cellula e cellula - Law of cell lineage: each cell (stems) from another cell - Pathologic conditions result from defects in cells 4 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) The cell cycle -Well-organized sequence of events in which the cell replicates by duplicating its content and dividing in two - Biogenesis of cell structures (and organelles) - Their distribution within the cell and to daughter cells - Unicellular organisms (bacteria, yeasts, protists) - new organism; Multicellular organisms: development, growth and tissue renewal/regeneration -A minimum set of spatiotemporally-organized processes that a cell must perform to pass the genetic information to the next generation of cells: replication of genome, essential macromolecules and organelles; proper distribution into daughter cells 5 Bi1700en Cell Biology/06-Cell cycle (13 Apr 2022) III The cell cycle Requires a precise control All organisms -Orchestrates cell growth and division: functional cell size Multicellular organisms -Orchestrates cell division with development/renewal - Homeostasis and a proper function of tissues - Prevents uncontrolled proliferation 6 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) fl The cell cycle: growth and division - Prokaryotic cells: cell growth affects spatial localization of cell cycle determinants Slow growth Replication initiation Division In media that support slow growth, replication initiation and Z-ring assembly occur sequentially (top panel), whereas in media that support fast growth, FtsZ can form the Z-ring a few minutes after cell birth, potentially coinciding with replication initiation (bottom panel). 7 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) - Eukaryotic cells: cell cycle entry regulated by growth factors (restriction point: "point of no return") Growth factors and cell cycle Rita Levi-Montalcini and Stanley Cohen - 1950s discovered nerve growth factor (NGF) and epidermal growth factor (EGF) - Nobel Prize in 1986 for "discoveries which are of fundamental importance for our understanding of the mechanisms which regulate cell and organ growth." Growth factors stimulate the proliferation of cells (and regulate differentiation during development) 8 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) Growth factors and cell cycle - Mammalian cell culture protocols include growth factors in media: blood serum defined growth factors 9 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) fl Phases of the eukaryotic cell cycle Interphase - (GO phase) - Resting state (quiescence) - Functional/metabolically active - Terminally differentiated cells -G1 phase - Metabolic activity, cell growth - Biosynthesis, doubling of organelles - S phase Replication of DNA -G2 phase - Biosynthesis of proteins essential for nuclear division and cytokinesis M ph - Nuclear division (mitosis/meiosis) -Cytokinesis S PHASE (DNA replication) G, PHASE ("G" phase comes from "gap" phase) 10 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) Not all eukaryotic organisms follow the typical eukaryotic cell cycle (A) FISSION YEAST (Schizosaccharomycespombe) cod r~o~p r^icovm START (B) BUDDING YEAST (Saccharomyces cerevisiae) @ START - Budding yeasts: mitotic spindle forms during late S phase (no G2 phase) 11 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) The cell-cycle control system 12 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) Transition through different phases must be well controlled Maintenance of genome integrity - Proper sequence of different phases - DNA replication followed by nuclear division (not replicated twice, prevents gene amplification) - Fully replicated error-free DNA - Nuclear division must not start before DNA replication is completed - Proper segregation of chromatids/chromosomes Homeostasis - In coordination with developmental programs 13 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) fl Cell cycle checkpoints - Regulate the cell cycle progression Three major checkpoints: -LateGI (G1/S): Restriction point (mammalian cells) Start (yeasts) - Late G2 (G2/M) - Metaphase-to-anaphase transition Is environment favorable? G2/M CHECKPOINT ENTER MITOSIS Are all chromosomes attached to the spindle? METAPHASE-TO-ANAPHASE TRANSITION TRIGGER ANAPHASE AND PROCEED TO CYTOKINESIS ENTER CELL CYCLE AND PROCEED TO S PHASE START CHECKPOINT Is environment favorable? 14 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) fl G1/S checkpoint - Restriction point - Favorable environment? - Enough nutrients, nucleotides - Extracellular mitogenic signals (growth factors; homeostasis-related) - DNA undamaged? Gi G1/S checkpoint Restriction point 15 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) YES: - "Point of no return" -> committed to proceed through the cell cycle NO: - Progress delayed - e.g., DNA repair - Or enters GO phase - Terminally differentiated cells - Quiescent cells (adult stem cells) - Senescent (aged) cells -Or undergoes apoptosis fi G2/M and metaphase-to-anaphase G2/M checkpoint - DNA fully replicated, no damage? - Sufficient cell size? (Yeasts) Spindle checkpoint G2/M checkpoint Metaphase-to-anaphase transition (spindle checkpoint) -Chromosomes (chromatids) attached to spindle? -Aligned in the metaphase plate? Delay progression until problems solved / Chronic activation leads to cell death 16 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) Molecular regulation of the cell cycle 17 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) Central regulatory system Cyclins & cyclin-dependent kinases (Cdks) Cyclins - Undergo cycles of synthesis and degradation Regulate Cdk activity - positive regulators: bind to and activate Cdks Cdks - Protein kinases: active Cdks phosphorylate target proteins - Govern progression through the cell cycle Different cyclin-Cdk complexes = different target proteins 18 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) Central regulatory system Cdks constantly present Cyclins synthesized and degraded during cell cycle Regulated by cell signaling and degradation systems G/S-cyclin S-cyclin _^_ start IE cyclin G^S-Cdk S-Cdk ^.Gj/M \^ metaphase-anaphase M-Cdk :m apc/c El 19 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) Cyclin-Cdks complexes specific for each phase CYCLIN-CDK COMPLEX VERTEBRATES CYCLIN CDK PARTNER GrCdk cyclin D* Cdk4, Cdk6 G/S-Cdk cyclin E Cdk2 S-Cdk cyclin A Cdk2, Cdkl** M-Cdk cyclin B Cdk1 o Cyclin B Help progression through G1 (Rb phosphorylation) Progression through Restriction point Mainly promote DNA replication Stimulate entry into the M-phase - progression through G2/M checkpoint Evolutionary conserved - Yeast cyclins/Cdks can be compensated by human homologs G, Phase S Phase G,Phase Mitosis CYCLIN-CDK COMPLEX VERTEBRATES CYCLIN CDK PARTNER BUDDING YEAST CYCLIN CDK PARTNER GrCdk cyclin D* Cdk4, Cdk6 Cln3 Cdkl** G/S-Cdk cyclin E Cdk2 Cln1,2 Cdkl S-Cdk cyclin A Cdk2, Cdkl** Clb5,6 Cdkl M-Cdk cyclin B Cdkl Clb1,2,3,4 Cdkl 20 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) Mitosis promoting factor (MPF): M-Cdk -Cyclin B + Cdk1 (Cdc2) CydinB ik^r.iilcttion (f^ mitosis interphase M-Cdk activity M-cyclin concentration mitosis interphase M-Cdk (MPF) activity triggers progression to M-phase 21 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) Discovery of MPF -1971: Masui & Markert, Smith & Ecker - Microinjection of M-phase vs. interphase cytoplasm into oocytes of frog (Rana pipiens, northern leopard frog) M-phase cytoplasm contains a factor inducing meiosis (M-phase) -> MPF INJECT CYTOPLASM FROM MPHASE CELL nucleus spindle easily detected OOCYTE IS DRIVEN INTO M PHASE INJECT CYTOPLASM FROM INTERPHASE CELL V IB) OOCYTE DOES NOT ENTER M PHASE 22 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) Function of MPF (M-Cdk) MPF-s — Phosphorylates target proteins -> initiates processes essential for M-phase - Nuclear envelope breakdown: phosphorylates nuclear pore complex subunits & proteins of nuclear lamina - Initiates and promotes spindle assembly: phosphorylation of motor proteins associated with microtubules - Increases microtubule dynamics - Chromosome condensation: phosphorylation of condensin proteins -Progression through G2/M checkpoint 23 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) Regulation of M-Cdk activity Cyclin B levels & protein kinases - Wee1: Inhibitory phosphorylation - Activating phosphorylation by Cdk activating kinase (CAK) - Cdc25: Activating dephosphorylation ^Active M-Cdk: M-phase Tagged for proteasomal degradation by APC/C G,/S-cyclin S-cydin M-cydin -' —* start >- g2/m 1^ meta phase-a nap hase I (31 i " i © : :m [g]| ,cyclin G,/S-Cdk S-Cdk Cyclin G ° f>'>^> flegr.itl.il i on (f a Dep h usp h a ry 1 .1 f i u n 24 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) APC/C (anaphase promoting complex/cyclosome) - Ubiquitin ligase: adds multiple ubiquitin molecules to target proteins - Polyubiquitylated proteins tagged for degradation by proteasome -APC/C target specificity regulated by activating subunits - Degradation targets - Securin (holds sister chromatid pairs) -> promotes transition to anaphase - S- and M-cyclins -> dephosphorylation and inactivation of Cdks (APC/C active to early G1) - Progression through spindle checkpoint - Resets the cell-cycle control system (Cdks inactivation) 25 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) fl Proteolysis by proteasome - Ubiquitination (ubiquitylation) - tag for proteasomal degradation - Important regulatory role: Rapid degradation of cyclins of preceding phases control of proteolysis by APC /C ^ E2 (peptides) ubiquitylation enzymes 26 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) Ubiquitin-dependent proteolysis Multiple cycles of ubiquitination Ubiquitin transferred from ubiquitin conjugating enzyme, E2, to target protein by E3 ubiquitin ligase Ubiquitin covalently attached ATP AMP © © ut —^=—ei vi?)——^ >C^! Polyubiquitinated proteins 'fyp* transferred to proteasome -> Cleavage ///s ADP ATP Peptides 27 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) Signals regulating cell cycle entry Unicellular - Determined by nutrients availability (similar to prokaryot Multicellular - Mitogenic signals (growth factors) - Produced by surrounding or distant cells within the organism - Homeostasis - only when necessary to proliferate: growth, renewal - Lack of mitogenic signaling - Cell cycle halted/blocked (GO) - intrinsic braking mechanisms prevail 28 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) Cell cycle arrest in GO - Lack of mitogenic signaling May be restored by mitogens -Terminally differentiated cells - Expression of Cdks and cyclins permanently turned off - cell cycle not responsive to mitogens - e.g., neurons, skeletal muscle cells - Reprogramming: dedifferentiation restores cell cycle - Replicative senescent cells - Shortened telomeres (DNA damage) prevent progression through G1/S checkpoint - Restricts number of cell divisions 29 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) Mitogenic signaling leads to cyclin D expression Mitogen 1 Signal transduction pathway Cyclin D Helps to initiate / cell cycle progression CDK4 Epidermal growth factor (EGF) induced expression of cyclin D EGF Guanine nucleotide exchange factor p p Receptor tyrosine kinases Cyclin D Transcription <- p p MAP kinase I Phosphorylation I Myc 30 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) How cyclin D helps to overcome restriction point? Cyclin D/CDK4 phosphorylates protein Rb - release of E2F transcription factors - expression of cyclin E and other S-phase proteins i á ''E2F r*_ Enhancer Repressor Cyclin E Cyclin E ■ E2F1 ■ / I 1_ Enhancer Repressor Cyclin E 31 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) Protein Rb (retinoblastoma) - Tumor suppressor - Intrinsic negative cell cycle regulator - Nuclear protein binding E2F - Prevents transcription of E2F targets: genes encoding proteins involved mainly in DNA replication and cell cycle progression - Rb phosphorylation - binding inhibited, E2F released Mitogenic signals Cyclin E CDK2 Cyclin D Cyclin-dependent CDK4/6^ kinase inhibitors p * # (CDKN1A, 2Aetc.) P P M G2 f Cell cycle G1 pointy S v ^^^^ ^ Histone acetylase i-► E2F targets (cyclin A, cyclin E etc.; No Mitogenic signals VYA\ 32 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) Protein Rb phosphorylation & dephosphorylation Hyperphosphorylation - overcomes restriction point Dephosphorylated at late M-phase (protein phosphatase 1) extent of pRb phosphorylation dephosphorylation hypophosphorylation hyperphosphorylation Hypo-phosphorylation is catalyzed by cycD-CDK4/6 Hyper-phosphorylation is catalyzed by cycE-CDK2 CDK4/6 hypophosphorylated -I unphosphorylated hyperphosphorylated E-CDK2 3S hypophosphorylated 33 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) Defects in the cell cycle -Cell cycle (13 Apr2022) Cell cycle arrest: Cdk inhibitor proteins -Cell intrinsic Cdk inhibitors -Transcription induced upon stress stimuli, e.g., DNA damage DNA / x-rays DNAdamage Mdm2 ATM/ATR kinase activation I Chkl/Chk2 kinase activation PHOSPHORYLATION OF pS3 Ink family p16lnk4a p15lnk4b p1glnk4c p19lnk4d _L Cip/Kip family p21 p27 p57 ± CDK4/6 D Cyclin CDK1/2/3 Cyclin P53 p53 UBIQUITYLATION AND DEGRADATION IN PROTEASOMES DNA damage: - p53 induces p21 transcription - P21 inhibits CDK2 and CDK1 1 i'fe. stable, ' active p53 ACTIVE p53 BINDS TO REGULATORY REGION OFp27 GENE li'- Rc p21 gene TRANSCRIPTION TRANSLATION | i p21 mRNA 9 p21 (Cdk inhibitor protein) ACTIVE Gn/S-Cdk and S-Cdk INACTIVE G,/S-Cdkand S-Cdk complexed with p21 35 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) p53 - guardian of the genome Tumor suppressor Integrates various stress signals Regulates expression of downstream targets Cell context-dependent response - Repair mechanisms, normal function restored, cell-cycle arrest, or apoptosis Nutrient deprivation Hypoxia □ NA Oncogene damage expression Ribosomal Oxidative stress dysfunction Telomere attrition Senescence lXI'i>Uitt! DNA repair Autophagy Migration Tumor suppression - p53 mutated in ~50% of human cancers 36 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) fl Cancer: A cell cycle defect -Cell cycle positive regulators - typical proto-oncogenes - Cyclin DIE, growth factors, mitogenic signaling kinases and transcription factors (all promote cyclin D/E expression) - Amplification, overexpression, constitutive activity of kinases -> oncogenes promote (uncontrolled) cell cycle progression - Cell cycle negative regulators - typical tumor suppressors - Intrinsic Cdk inhibitors, protein Rb, protein p53 and DNA damage recognizing kinases Inactivating mutations, deletions -> cell cycle checkpoint mechanisms disabled 37 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) Models to study the cell cycle 38 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) Schizosaccharomyces pombe - Fission yeast; Cell cycle: 4 phases (A) FISSION YEAST (Schizosaccharomycespombe) CO) C^KCTÖ) (TO Gi S t START -Small genome: 4,824 genes, ~70% human orthologs - Non-pathogenic, fast cell cycle (90 min) -Can exist as haploid: easy loss-of-function studies (only one allele) 39 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) fl Schizosaccharomyces pombe - Optimal to study G2/M transition - G2 is the longest - size is a parameter of progression - Cdc2 (Cdk1), Wee1, Cdc25 identified in S. pombe -! Differences from multicellular organisms — Closed mitosis dosedM^sis Nuclear envelope does not break down Open Mitosis 40 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) Saccharomyces cerevisiae - Budding yeast (B) BUDDING YEAST (Saccharomyces cerevisiae) START - Bud appears in S-phase, grows through cell cycle -Optimal to study G1/S transition 41 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) How to study loss-of-function effects - Loss of cell cycle - no cell division, non-viable culture - Conditional mutants - temperature sensitive - Low temperature - protein functional vs. High temperature - protein not functional (A) PERMISSIVE (LOW) TEMPERATURE (B) RESTRICTIVE (HIGH) TEMPERATURE 42 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) Mammalian cell cultures - Primary (non-cancerous) cells - Replicative senescence studies - Normally 25-50 (max. 80) divisions - telomere shortening -Immortalized/cancercell lines Not restricted by replicative senescence - Approximate mimic of in vivo regulation in normal healthy cells -Genetic manipulation by RNA silencing, CRISPR-Cas9 editing 43 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022) CELL CHECKPOINT All violators must fix their mistakes ...or face apoptosis. The cell checkpoints were always a site of intense scrutiny. 44 Bi1700en Cell Biology / 06 - Cell cycle (13 Apr 2022)