1. Chapt. 16 Eukaryotic Cell cycle; Chapt. 17 Stem cells Chapt. 16 Student learning outcomes: Explain basic phases of eukaryotic cell cycle Regulators, checkpoints Describe events of mitosis Regulation by MPF, phosphorylation, proteolysis Explain regulators of progression: cyclins, cyclin-dependent kinases, kinase inhibitors (Explain meiosis and fertilization) Explain features of stem cells Adult, embryonic, Induced pluripotent

2. Introduction Self-reproduction is a fundamental cell characteristic All cells reproduce by dividing in two: parental cell gives rise to two daughter cells after 1 cycle Cell division is carefully regulated and coordinated. Progression through cell cycle is controlled by protein kinases (conserved from yeasts to mammals) Defects in cell cycle regulation are common cause of abnormal proliferation of cancer cells.

3. Fig 16.1 Phases of the cell cycle Four phases of cell cycle: M phase: Mitosis (nuclear division) usually ends with cell division (Cytokinesis). Interphase — period between mitoses: G 1 phase (gap 1): Metabolically active, growing. S phase (synthesis) DNA replication. G 2 phase (gap 2) Cell growth continues, Proteins synthesized in preparation for mitosis Yeast cycle 90 min; human cell 24 hrs Fig. 16.3

4. Fig 16.3 Determination of cellular DNA content Identify phases of interphase by DNA content. Animal cells in G 1 are diploid (2 copies of each chromosome); DNA content is 2n. During S phase, replication increases DNA content of cell to 4n. Analyze fluorescence intensity of individual cells stained with DNA dye flow cytometer or fluorescence-activated cell sorter Fig. 16.3 DNA content of asynchronous population of cells

5. The Eukaryotic Cell Cycle - yeast Fig. 16.4 Budding yeast Saccharomyces is model eukaryote: Size of bud shows cell cycle phase Cell cycle is Regulated: Extracellular and internal signals: Major control point START (G 1 to S ) Once cells pass START committed to S phase, one division cycle. Can enter resting stage if nutrients lacking

6. The Eukaryotic Cell Cycle – animal cells Animal cells have restriction point in late G 1 Regulated by extracellular growth factors Once past restriction point, cell committed to proceed through S phase, rest of cell cycle. Lack of growth factors → stop at restriction point, cells enter resting stage G 0. Fig. 16.5

7. The Eukaryotic Cell Cycle Coordination between different phases of cell cycle depends on series of cell cycle checkpoints: DNA damage checkpoints ensure damaged DNA is not replicated and passed on Spindle assembly checkpoint arrests mitosis at metaphase if chromosomes not properly aligned on mitotic spindle. Fig. 16.7

8. Fig 16.8 Restriction of DNA replication Genome must replicate only once per cell cycle: Control mechanisms prevent re-initiation of DNA replication until cell cycle completed. MCM helicase proteins bind origins of replication with ORC (origin recognition complex) proteins ( required for initiation of replication). Displacement of MCM proteins from origin prevents rereplication Fig. 16.8

9. Regulators of Cell Cycle Progression 2. Regulators of Cell Cycle progression: Conserved set of protein kinases trigger major cell cycle transitions – (expts led to Nobel prizes): Frog oocytes arrest in G 2 until hormonal stimulation triggers entry into M phase (MPF factor) Yeast ts mutants defective cell cycle at high temp (cdc mutants) Protein synthesis in sea urchin embryos (cyclins)

10. Fig 16.9 Identification of MPF 1. Oocytes enter M phase after microinjection of cytoplasm from hormonally-stimulated oocytes. Cytoplasmic factor is maturation promoting factor (MPF) MPF also in somatic cells, induces entry into M phase. MPF general regulator of transition from G 2 to M. Fig. 16.9 key experiment Masui & Markert, 1971

11. Regulators of Cell Cycle Progression 2. Genetic analyses of yeasts: found ts (temperature- sensitive) mutants defective in cell cycle progression (called cdc for cell division cycle mutants) cdc genes are required: for passage through START, entry into mitosis Some cdc encode Protein kinases : Cdk1 protein kinase Fig. 16.10 Hartwell’s cdc28 mutant

12. Regulators of Cell Cycle Progression 3. Protein synthesis in early sea urchin embryos:  2 proteins (cyclins A, B) accumulate in interphase,  rapidly degraded toward end of mitosis → suggests role in inducing mitosis. Fig. 16.11 Hunt’s Microinjection of cyclin A into frog oocytes triggers G 2 to M transition.

13. Regulators of Cell Cycle Progression In 1988 MPF was purified: two subunits: Cdk1 and Cyclin B. Cyclin B is regulatory subunit required for catalytic activity of Cdk1 protein kinase. MPF is regulated by phosphorylation and dephosphorylation of Cdk1. Fig. 16.12 MPF = cyclin + Cdk

14. Fig 16.13 MPF regulation MPF is regulated by phosphorylation and dephosphorylation of Cdk1. Cyclin B forms complexes with Cdk1 during G 2. Cdk1 gets 3 PO 4, → accumulate inactive Cdk1/cyclin B in G 2 Removal of inhibitory PO 4 activates Cdk1 MPF phosphorylates proteins to initiate M phase. Cyclin B degraded by ubiquitin-mediated proteolysis Fig. 16.13

15. Regulators of Cell Cycle Progression Cdk1 and cyclin B belong to protein families Different members control progression through phases Cdk = cyclin-dependent kinases Yeast only Cdk1; animal cells have multiple Cdks Fig. 16.14

16. Regulators of Cell Cycle Progression 4 mechanisms regulate activity of Cdks : 1. Association of Cdk’s and cyclin partners cyclin synthesis and degradation. 2. Activation requires phosphorylation Thr161 catalyzed by CAK (Cdk-activating kinase), composed of Cdk7/cyclin H 3. Inhibitory phosphorylation Thr14,Tyr15 by Wee1 protein kinase. Cdks activated by dephosphorylation by Cdc25 protein phosphatase Fig. 16.15

17. Regulators of Cell Cycle Progression 4. Binding of inhibitory proteins Cdk inhibitors (CKIs). Mammalian cells have two families of Cdk inhibitors:  Ink4 and Cip/Kip Fig. 16.15

18. Regulators of Cell Cycle Progression *Growth factors stimulate animal cell growth: D-type cyclins - one link between growth factor signaling and cell cycle progression. Growth factors stimulate cyclin D1 synthesis through Ras/Raf/MEK/ERK pathway, Cyclin D1 synthesized if growth factors present Cyclin D1 is rapidly degraded Cdk4,6/cyclin D1 drives cells through restriction point. Defects in cyclin D1 regulation contribute to loss of control characteristic of cancer cells. Fig. 16.16*

19. Regulators of Cell Cycle Progression * Tumor suppressor Rb is key substrate of Cdk4, 6/cyclin D complexes; Rb often mutated in tumors ( retinoblastoma, rare inherited childhood eye tumor ) Rb is prototype tumor suppressor gene — gene whose inactivation leads to tumor development. Rb couples cell cycle machinery to expression of genes required for cell cycle progression. Rb binds to E2F Transcription factor: Represses synthesis of Cyclin E & others Rb inactivated by PO 4 by Cdk4,6/cyclin D Fig. 16.17

20. Regulators of Cell Cycle Progression Progression through Restriction point, entry into S: Requires inactivation of Rb to permit E2F stimulation of transcription of Cyclin E & other genes Requires activation of Cdk2/cyclin E complexes In G 1, Cdk2/cyclin E inhibited by p27 (Cip/Kip family). Fig. 16.14

21. Regulators of Cell Cycle Progression Cdk2/cyclin E passes Restriction Point : In G 0 and early G 1, Cdk2/cyclin E is inhibited by p27 (Cip/Kip family); Transcription of p27 inhibited by growth factors Cdk2/cycD binds p27, sequesters Activation of Cdk2/cyclin E: Degrades p27 Activates MCM helicase Initiates DNA replication at ORC sequences Fig. 16.18 Cdk2/cycE role

22. Regulators of Cell Cycle Progression DNA damage checkpoints arrest cell cycle Mediated by protein kinases, ATM and ATR, Activated by DNA damage. Activate signaling path: cell cycle arrest, DNA repair, (programmed cell death)  ATM, ATR activate Chk1 and Chk2 kinases  Chk1 and Chk2 phosphorylate and inhibit Cdc25 Phosphatase (necessary to activate MPF) Fig. 16.19

23. Regulators of Cell Cycle Progression *p53 tumor suppressor: Arrests cells at G 1 checkpoint p53 is phosphorylated by ATM and Chk2 kinases. p53 is transcription factor; its increased expression leads to induction of Cdk inhibitor p21. p21 inhibits Cdk2/cyclin E complexes, -> cell cycle arrest G 1 p53 frequently mutated in cancer Fig. 16.20 p53

24. The Events of M Phase Mitosis is activated by MPF (Cdk1/cycB) Major reorganization of cell components: Chromosomes condense, nuclear envelope breaks down, cytoskeleton reorganizes to form spindle, chromosomes move to opposite poles. Cell division (cytokinesis) usually follows. Mitosis divided into 4 stages: 1. Prophase 2. Metaphase 3. Anaphase 4. Telophase *Fig. 16.14

25. Fig 16.21 Stages of mitosis in an animal cell Mitosis

26. Fig 16.22 Fluorescence micrographs of chromatin, keratin, and microtubules during mitosis of newt lung cells DNA blue, keratin red, microtubules green Fig. 16.22 mitosis

27. Fig 16.24 Targets of Cdk1/cyclin B Cdk1/cyclin B protein kinase (MPF) is master regulator of M phase transition: –Activates other mitotic protein kinases –Phosphorylates structural proteins involved in reorganization. Fig. 16.24

28. The Events of M Phase Condensation of chromatin: Driven by condensins, members of “structural maintenance of chromatin” (SMC) proteins. Condensins and cohesins (another family of SMC proteins) contribute to chromosome segregation: Condensins are activated by Cdk1/cyclin B phosphorylation, replace most cohesins, sister chromatids linked only at centromere. Fig. 16.24

29. Fig 16.26 Breakdown of the nuclear envelope Breakdown of nuclear envelope Nuclear membranes fragment Nuclear pore complexes dissociate Nuclear lamina depolymerizes: after phosphorylation of lamins by Cdk1cycB Golgi apparatus fragments into small vesicles Fig. 16.26

30. Fig 16.28 The metaphase spindle Chromosomes on spindle Balance of forces on chromosomes leads to alignment on metaphase plate in center of spindle. Spindle: kinetochore and chromosomal microtubules attached to chromosomes, and polar microtubules, which overlap in center of cell Fig. 16.28

31. The Events of M Phase Spindle assembly checkpoint: progression to anaphase mediated by activation of the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase. Checkpoint is mediated by Mad/Bub proteins that inhibit Cdc20, required component of APC/C. Activation of APC/C -> Degradation of securin, regulatory subunit of separase. Separase degrades cohesin, breaks link between sister chromatids; they segregate and move to poles Cyclin B gets degraded, inactivates Cdk1 (Fig. 8.43 44)

32. Fig 16.29 The spindle assembly checkpoint Activation of APC/C starts anaphase by Mad/Bub release inhibition Cdc20 Fig. 16.29 anaphase

33. Fig 16.31 Cytokinesis of animal cells Cytokinesis usually starts after anaphase Triggered by inactivation of Cdk1. Yeast and animal cells contractile ring of actin and myosin II filaments forms beneath plasma membrane (Figs. 12.30,31) Fig. 16.31

34. Fig 16.32 Cytokinesis in higher plants Plant cell cytokinesis has new cell walls and plasma membranes. vesicles carrying cell wall precursors from Golgi accumulate at former site of metaphase plate. vesicles fuse polysaccharides form matrix of new wall Fig. 16.32

35. Meiosis and Fertilization 4. Meiosis specialized cell cycle reduces chromosome number → 4 haploid daughter cells: 2 sequential rounds of nuclear and cell division (meiosis I and meiosis II), after 1 DNA replication Meiosis I: Homologous chromosomes pair with one another, Recombination occurs between homologs Homologs segregate to different daughter cells. Daughter cells contain 1 of each chromosome pair (2 sister chromatids) Meiosis II: Resembles mitosis - sister chromatids separate and segregate to different daughter cells Yeast can have mitosis as either haploid or diploid

36. Fig 16.33 Comparison of meiosis and mitosis Fig. 16.33

37. Fig 16.42 Fertilization Fertilization: sperm binds receptor on surface of egg, fuses with egg plasma membrane. Mixes paternal and maternal chromosomes, induces changes in egg cytoplasm for further development: Binding sperm signals increase in Ca 2+ levels in egg cytoplasm, probably hydrolysis of (PIP 2 ). Surface alterations prevent additional sperm entering egg; ensures normal diploid embryo Complex developmental pathways Fig. 16.3

38. Review questions 4. What are the medchanisms that regulate the activity of cyclin-dependent kinases? 6. What cellular processes would be affected by expression of siRNA targeted against Cdk7 (CAK)? 3. Radiation damages DNA and arrests cell cycle progression at checkpoints in G1, S and G2. Why is this advantageous for the cell? 9. What substrates are phosphorylated by Cdk1/cycB (MPF) to initiate mitosis?