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

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Title: The Cell, 5e Author: Betz, Joan Last modified by: l-admin Created Date: 10/16/2000 7:08:56 PM Document presentation format: On-screen Show (4:3) – PowerPoint PPT presentation

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


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
  • 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
4
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
5
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
6
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
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 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)

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 G2 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 Hartwells 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 Hunts Microinjection of cyclin A into
frog oocytes triggers G2 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 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
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 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
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 PO4
  • 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 G1, 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 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
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 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
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 -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)

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 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
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?
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