Chapt. 16 Eukaryotic Cell cycle; Chapt. 17 Stem cells

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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

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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.

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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
• G1 phase (gap 1)
• Metabolically active, growing.
• S phase (synthesis)
• DNA replication.
• G2 phase (gap 2)
• Cell growth continues,
• Proteins synthesized
• in preparation for mitosis
• Yeast cycle 90 min human cell 24 hrs

Fig. 16.3
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Fig 16.3 Determination of cellular DNA content

• Identify phases of interphase by DNA content.
• Animal cells in G1 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
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The Eukaryotic Cell Cycle - yeast

• 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 (G1 to S)
• Once cells pass START
• committed to S phase,
• one division cycle.
• Can enter resting stage
• if nutrients lacking

Fig. 16.4
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The Eukaryotic Cell Cycle animal cells

• Animal cells have restriction point in late G1
• 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 G0.

Fig. 16.5
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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
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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
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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 G2 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)

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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 G2 to M.

Fig. 16.9 key experiment Masui Markert, 1971
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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 Hartwells cdc28 mutant
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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 Hunts Microinjection of cyclin A into
frog oocytes triggers G2 to M transition.
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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
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Fig 16.13 MPF regulation

• MPF is regulated by phosphorylation and
  dephosphorylation of Cdk1.
• Cyclin B forms complexes with Cdk1 during G2.
• Cdk1 gets 3 PO4, ? accumulate inactive
  Cdk1/cyclin B in G2
• Removal of inhibitory PO4 activates Cdk1
• MPF phosphorylates proteins
• to initiate M phase.
• Cyclin B degraded by
• ubiquitin-mediated proteolysis

Fig. 16.13
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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
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Regulators of Cell Cycle Progression

• 4 mechanisms regulate activity of Cdks
• 1. Association of Cdks 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
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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
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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
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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 PO4
• by Cdk4,6/cyclin D

Fig. 16.17
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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 G1, Cdk2/cyclin E inhibited by p27 (Cip/Kip
  family).

Fig. 16.14
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Regulators of Cell Cycle Progression

• Cdk2/cyclin E passes Restriction Point
• In G0 and early G1, 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
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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
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Regulators of Cell Cycle Progression

• p53 tumor suppressor
• Arrests cells at G1 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, -gt cell
  cycle arrest G1
• p53 frequently mutated in cancer

Fig. 16.20 p53
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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
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Fig 16.21 Stages of mitosis in an animal cell
Mitosis
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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
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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
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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
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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
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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
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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 -gt
• 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)

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Fig 16.29 The spindle assembly checkpoint

• Activation of APC/C starts anaphase
• by Mad/Bub release inhibition Cdc20

Fig. 16.29 anaphase
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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
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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
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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

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Fig 16.33 Comparison of meiosis and mitosis
Fig. 16.33
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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 Ca2 levels in
  egg cytoplasm, probably hydrolysis of (PIP2).
• Surface alterations prevent additional sperm
  entering egg ensures normal diploid embryo
• Complex developmental pathways

Fig. 16.3
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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?