Download cell cycle control system

The cell Division
and Apoptosis
2013-12-12
藥理所 陳韻雯 老師
•Cell Division
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Overview of Cell Cycle
Phase of the cell cycle
The cell cycle consists of
Interphase – G1(gap1),
S (Synthesis),
G2 (Gap2) phase
The mitotic phase – cell division
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Figure 17-4 Molecular Biology of the Cell (© Garland Science 2008)
Interphase-G1
•G1 phase: START
• Begins immediately after
division
• Commitment point beyond S
phase
• cell growth
• New organelles formed
• Ensure the ability of cell to
replicate chromosome
• End of G1, cell has doubled in
size
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Interphase-S
•S phase:
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• The second part of interphase
• Cells make a perfect copy of
their DNA in its nucleus
• This process is called
REPLICATION
• DNA is found in the
chromatin in the nucleus
• Each daughter cell must have
a complete set of DNA to
survive
Interphase-G2
3-
•Commitment point beyond M
phase
•The cell produces structures
that it will use to divide into two
new cells
•Ensure the ability of cell to
divide
•Cell prepares to begin moiosis
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•G2 phase:
M PHASE
•M phase:
•The division of a cell into two
daughters
•Consists of nuclear division, or
mitosis, and cytoplasmic
division, or cytokinesis.
•Mitosis is itself divided into
five stages
•Together with cytokinesis are
described in this panel
Mitotic Division of an Animal Cell
PROPHASE
PROMETAPHASE
METAPHASE
Mitotic Division of an Animal Cell
ANAPHASE
TELOPHASE
CYTOKINESIS
Prophase-1st step in Mitosis
• The chromatin fibers become
more tightly coiled, condensing
into discrete chromosomes
observable with a light
microscope.
• The nucleoli disappear.
• Each duplicated chromosome
appears as two identical sister
chromatids joined together.
• The mitotic spindle begins to form.
It is composed of the centrosomes
and the microtubules that extend
from them. The radial arrays of
shorter microtubules that extend
from the centrosomes are called
asters (“stars”).
• The centrosomes move away from
each other, apparently propelled
by the lengthening microtubules
between them.
Prophase
Animal Cell
Plant Cell
Spindle fibers
Centrioles
Photographs from: http://www.bioweb.uncc.edu/biol1110/Stages.htm
Prometaphase
• Prometaphase starts abruptly with
breakdown of the nuclear envelope.
• Chromsomes can now attach to spindle
microtubules via their kinetochore and
undergo active movement
nd
Metaphase-2 step in Mitosis
• Metaphase is the longest stage of
mitosis, lasting about 20 minutes.
• The centrosomes are now at
opposite ends of the cell.
•The chromosomes convene on the
metaphase plate, an imaginary
plane that is equidistant between
the spindle’s two poles. The
chromosomes’ centromeres lie on
the metaphase plate.
• For each chromosome, the
kinetochores of the sister
chromatids are attached to
kinetochore microtubules coming
from opposite poles.
• The entire apparatus of
microtubules is called the spindle
because of its shape.
Metaphase
Animal Cell
Plant Cell
Photographs from: http://www.bioweb.uncc.edu/biol1110/Stages.htm
The Mitotic Spindle
• The spindle includes the centrosomes, the spindle
microtubules, and the asters
• The apparatus of microtubules controls chromosome
movement during mitosis
• The centrosome replicates, forming two centrosomes
that migrate to opposite ends of the cell
• Assembly of spindle microtubules begins in the
centrosome, the microtubule organizing center
• An aster (a radial array of short microtubules) extends
from each centrosome
The Mitotic Spindle
• Some spindle microtubules attach to the kinetochores of
chromosomes and move the chromosomes to the metaphase plate
• In anaphase, sister chromatids separate and move along the
kinetochore microtubules toward opposite ends of the cell
Aster
Microtubules
Sister
chromatids
Chromosomes
Centrosome
Metaphase
plate
Kinetochores
Centrosome
1 µm
Overlapping
nonkinetochore
microtubules
Kinetochore
microtubules
0.5 µm
Anaphase- 3rd step in Mitosis
• Anaphase is the shortest stage of
mitosis, lasting only a few minutes.
• Anaphase begins when the two sister
chromatids of each pair suddenly part.
Each chromatid thus becomes a fullfledged chromosome.
• The two liberated chromosomes begin
moving toward opposite ends of the cell,
as their kinetochore microtubules
shorten. Because these microtubules are
attached at the centromere region, the
chromosomes move centromere first (at
about 1 µm/min).
• The cell elongates as the
nonkinetochore microtubules lengthen.
• By the end of anaphase, the two ends of
the cell have equivalent—and
complete—collections of chromosomes.
Anaphase
Animal Cell
Plant Cell
Photographs from: http://www.bioweb.uncc.edu/biol1110/Stages.htm
Telophase- 4th step in Mitosis
• Two daughter nuclei begin to
form in the cell.
• Nuclear envelopes arise from
the fragments of the parent
cell’s nuclear envelope and
other portions of the
endomembrane system.
• The chromosomes become
less condensed.
• Mitosis, the division of one
nucleus into two genetically
identical nuclei, is now
complete.
Telophase
Animal Cell
Plant Cell
Photographs from: http://www.bioweb.uncc.edu/biol1110/Stages.htm
Cytokinesis
•
Cleavage of cell into two
halves
– Animal cells
 Constriction belt of
actin filaments
– Plant cells
 Cell plate
– Fungi and protists
 Mitosis occurs
within the nucleus
Cytokinesis In Animal And Plant Cells
Cleavage furrow
Contractile ring of
microfilaments
100 µm
Vesicles
forming
cell plate
1 µm
Wall of
patent cell Cell plate
New cell wall
Daughter cells
(a) Cleavage of an animal cell (SEM)
Daughter cells
(b) Cell plate formation in a plant cell (SEM)
Animal Mitosis -- Review
Interphase
Prophase
Metaphase
Anaphase
Telophase
Cytokinesis
Plant Mitosis -- Review
Interphase
Prophase
Metaphase
Anaphase
Telophase
Cytokinesis
Cell Cycle
28
Cell Cycle Control System
The Cell Cycle Control System
• The sequential events of the cell cycle are directed by a distinct
cell cycle control system, which is similar to a clock
• The cell cycle control system is regulated by both internal and
external controls
• The clock has specific checkpoints where the cell cycle stops
until a go-ahead signal is received
The Major Cell-Cycle Regulatory Protein
Overview of the Cell -Cycle control system
Figure 17-21 Molecular Biology of the Cell (© Garland Science 2008)
Checkpoint control system
• Checkpoints
• cell cycle controlled by STOP & GO chemical signals at
critical points
• signals indicate if key cellular
processes have been
completed correctly
G0
G1 checkpoint
G1
(a) Cell receives a go-ahead
signal.
G1
(b) Cell does not receive a
go-ahead signal.
Checkpoint control system
• 3 major checkpoints:
• G1/S
• can DNA synthesis begin?
• G2/M
• has DNA synthesis been completed
correctly?
• commitment to mitosis
• spindle checkpoint
• are all chromosomes attached to
spindle?
• can sister chromatids separate
correctly?
Spindle checkpoint
G2 / M checkpoint
Chromosomes attached at
metaphase plate
• Replication completed
• DNA integrity
Inactive
Active
Inactive
Cdk / G2
cyclin (MPF)
M
Active
APC
C
cytokinesis
mitosis
G2
G1
S
MPF = Mitosis
Promoting Factor
APC = Anaphase
Promoting Complex
Cdk / G1
cyclin
Active
G1 / S checkpoint
Inactive
• Growth factors
• Nutritional state of cell
• Size of cell
“Go-ahead” signals
• Protein signals that promote cell growth & division
• internal signals
• “promoting factors”
• external signals
• “growth factors”
• Primary mechanism of control
• phosphorylation
• kinase enzymes
• either activates or inactivates cell signals
Cell cycle signals
inactivated Cdk
• Cell cycle controls
• cyclins
• regulatory proteins
• levels cycle in the cell
• Cdk’s
activated Cdk
• cyclin-dependent kinases
• phosphorylates cellular proteins
• activates or inactivates proteins
• Cdk-cyclin complex
• triggers passage through different stages of cell
cycle
The Major Cyclins and Cdks
The Cyclin-Cdk complexes: the major
components of the cell cycle control
system
Regulation of cyclin-CDK activity
• Three mechanisms for controlling cyclin-CDK activity:
• (1). Regulation of the cyclin concentration of (mitotic) cyclins by ubiquitin-protein
ligases (SCF and APC/C).
• (2). Regulation of the kinase activity of CDKs by phosphorylation.
• (3). Regulation of cyclin-CDK activity by CDK inhibitors (CKIs).
(1)
(2)
(3)
APC/C ubiquitin-protein ligases
• Anaphase-promoting complex (APC/C) is a ubiquitin-protein ligase which is involved in
regulating two transitions in cell cycle:
• (A) from metaphase to anaphase- the APC/C catalyzes the destruction of securin, leading to the
separation of sister chromatins and move to opposite of the spindle during anaphase.
• (B) from anaphase to cytokinesis- the APC/C targets the S- and M-cyclins for destruction, leading
to the loss of most CDK activity in anaphase.CDK inactivation allows phosphatases to
dephosphorylate the many CDK substrates in the cell, as required for the completion of mitosis
and cytokinesis.
DNA damage and
checkpoint
Nature 2000;408:433-9
DNA damage arrests the cell
cycle in G1
Figure 17-63 (part 1 of 2) Molecular Biology of the Cell (© Garland Science 2008)
Human DNA damage response pathway
DNA damage
Replication block
Signals
Common
intermediate
ATM
or ATR
Rad17/RFC
Transducers
Hus1/Rad1/Rad9
Chk1 BRCA1 Others
Cell cycle Apoptosis
arrest
Sensors
p53
DNA repair
Effectors
Checkpoints in Cell-cycle
Regulation
Checkpoint and
Cancer
Cancer & Cell Growth
• Cancer is essentially a failure
of cell division control
• unrestrained, uncontrolled cell growth
• What control is lost?
• lose checkpoint stops
• gene p53 plays a key role in G1/S restriction point
• p53 protein halts cell division if it detects damaged DNA
p53 is the
Cell Cycle
Enforcer
• options:
• stimulates repair enzymes to fix DNA
• forces cell into G0 resting stage
• keeps cell in G1 arrest
• causes apoptosis of damaged cell
• ALL cancers have to shut down p53 activity
p53 discovered at Stony Brook by Dr. Arnold Levine
p53 — master regulator gene
NORMAL p53
p53 allows cells
with repaired
DNA to divide.
p53
protein
DNA repair enzyme
p53
protein
Step 1
Step 2
Step 3
DNA damage is caused
by heat, radiation, or
chemicals.
Cell division stops, and
p53 triggers enzymes to
repair damaged region.
p53 triggers the destruction
of cells damaged beyond repair.
ABNORMAL p53
abnormal
p53 protein
Step 1
Step 2
DNA damage is
caused by heat,
radiation, or
chemicals.
The p53 protein fails to stop
cell division and repair DNA.
Cell divides without repair to
damaged DNA.
cancer
cell
Step 3
Damaged cells continue to divide.
If other damage accumulates, the
cell can turn cancerous.
Development of Cancer
• Cancer develops only after a cell experiences ~6 key
mutations (“hits”)
• unlimited growth
• turn on growth promoter genes
• ignore checkpoints
• turn off tumor suppressor genes (p53)
• escape apoptosis
• turn off suicide genes
• immortality = unlimited divisions
• turn on chromosome maintenance genes
• promotes blood vessel growth
• turn on blood vessel growth genes
• overcome anchor & density dependence
• turn off touch-sensor gene
It’s like an
out of control
car!
What causes these “hits”?
• Mutations in cells can be triggered by




UV radiation
chemical exposure
radiation exposure
heat




cigarette smoke
pollution
age
genetics
•Apoptosis
are born,
Cells Cells
are born,
livelive
for for
a given
a given
period of
time period
and then die
of time and then dieBowen, 1998
Bowen, 1998
APOPTOSIS
--- Physiological cell death
--- Cell suicide
--- Cell deletion
--- Programmed cell death
Cell Death
• The body is very good at maintaining a constant number
of cells. So there has to exist mechanisms for ensuring
other cells in the body are removed, when appropriate.
• Two forms
• Apoptosis - suicide - programmed cell death
• Necrosis - killing - decay and destruction
Apoptosis – Programmed Cell Death
Apoptosis – Programmed Cell Death
Necrosis vs. Apoptosis
• Apoptosis – programmed cell death
• Necrosis – un-programmed cell death
Apoptotic cells are biochemically recognizable
Characteristic biochemical changes in cells undergoing apoptosis
1. Chromosomal DNA cleaved into fragments
2. Change in the plasma membrane – phosphatidylserine (PS)
in the outer leaflet
3. Loss of electrical potential across the inner membrane of the
mitochondria
4. Relocation of cytochrome c from the intermembrane space of
the mitochondria to the cytosol
Cleavage of chromosomal DNA into a characteristic ladder of fragments
Apoptosis Signaling
What is a Caspase?
• Single chain of pro-enzymes
• Contains an N-terminal domain, a small subunit and
a large subunit (similar to a ribosome)
• Apoptotic stimulus Activation Substrate
Cleavage Enzyme
• Proteins which degrade other proteins are employed
by apoptosis - caspases
• Made as inactive precursors - procaspases
• These are activated by other proteins when the right
signal is received
• One caspase cleaves the lamin proteins resulting in
the irreversible breakdown of the nuclear membrane.
3 Types of Caspases
• Inflammatory Caspases: -1, -4, and -5
• Initiator Caspases: -2, -8, -9, and -10
• Long N-terminal domain
• Interact with effector caspases
• Effector Caspases: -3, -6, and -7
• Little to no N-terminal domain
• Initiate cell death
Two Caspase Pathways
Extrinsic Pathway

Death Ligand

Death Receptors

Caspases

Cell Death
Cell-surface death receptors activate the extrinsic pathway
of apoptosis
The extrinsic pathway of apoptosis activated through Fas death receptors
Two Caspase Pathways
Intrinsic Pathway

Mitochondria

Cytochrome C

Apoptosome
Complex

Caspases

Cell Death
The intrinsic pathway of apoptosis depends on mitochondria
Apoptosis depends on an intracellular proteolytic cascade that is
mediated by caspases
Procaspase activation during apoptosis
Detection of cell cycle by
Flow Cytometry
• Cell Cycle Analysis by Propidium Iodide (PI) Staining
2n
4n
Detection of Apoptosis by
Flow Cytometry
• Early stage
• Mid stage
• Late stage
Annexin V/7-AAD(PI)
TUNEL assay
< Go/G1 DNA content
Annexin V: An Early Marker of Apoptosis
One of the earliest indications of apoptosis is the
translocation of the membrane phospholipid
phosphatidylserine (PS) from the inner to the outer leaflet
of the plasma membrane.
Once exposed to the extracellular environment, binding
sites on PS become available for Annexin V, a 35-36 kDa,
2+
Ca -dependent, phospholipid binding protein with a high
affinity for PS.
• TUNEL（Terminal deoxynucleotidyl
transferase-mediated dUTP nick-endlabeling）
TUNEL assay
Sub-G0/G1
• Apoptosis by Propidium Iodide (PI) Staining
2n
4n
Any Questions??