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
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.
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
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
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
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
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
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
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)
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
Regulators of Cell Cycle Progression
2. Genetic analyses of yeasts: found ts (temperaturesensitive) 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
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 G2 to M transition.
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
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
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
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
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
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*
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
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
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
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
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, -> cell cycle arrest G1
• p53 frequently mutated in cancer
Fig. 16.20 p53
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
Fig 16.21 Stages of mitosis in an animal cell
Mitosis
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
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
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
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
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
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)
Fig 16.29 The spindle assembly checkpoint
Activation of APC/C starts anaphase
by Mad/Bub release inhibition Cdc20
Fig. 16.29 anaphase
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
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
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
sister chromatids)
Meiosis II:
• Resembles mitosis - sister chromatids separate and
segregate to different daughter cells
Yeast can have mitosis as either haploid or diploid
(2
Fig 16.33 Comparison of meiosis and mitosis
Fig. 16.33
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
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?