The Cell Cycle
Every cell is the product of a cell cycle.
The cell cycle comprises two alternating events:
Cell division & cell growth
S phase: Doubling of DNAM phase: halving of DNA during mitosis
The Cell Cycle
So, the cell cycle consists of: •G1 = growth and preparation of the chromosomes for replication •S = synthesis of DNA•G2 = preparation for mitosis •M = mitosis
When a cell is in any phase of the cell cycle other than mitosis, it is said to be in interphase.
S phase: Doubling of DNAM phase: halving of DNA during mitosis
Cell division in animals
• chromosomes move toward a discrete spindle pole
The nuclear envelope remains intact during mitosis, and the spindle forms withinthe nucleus in contrast with higher eucaryotic cells.
The behavior of a temperature-sensitive cdc mutant.
In a cdc15 mutant grown at the restrictive temperature, cells completeanaphase but cannot complete the exitfrom mitosis and cytokinesis.
>1 mm in diameterCarries 100,000 times more cytoplasm than an average cell in the human body.212 cells (4096 cells) within 7 hrs without detectable G1 or G2 phases.
1 division cycle every 30 min.
fibroblasts
3H-thymidine incorporation Fluorescent anti-BrdU Abs
DNA Replication
DNA replication is strictly controlled during the cell cycle.
Many molecules are involved in controlling S-phase progression.
Stained cells with DNA-binding dye (flow cytometer)
Checkpoints: Quality control of the cell cycle
The cell has several systems for interrupting the cell cycleif something goes wrong.
•Cyclins •G1-cyclins •G1/S- and S-phase cyclins •M-phase cyclins
The passage of a cell through the cell cycle is controlled by proteins in the cytoplasm:
•Their levels in the cell rise and fall with the stages of the cell cycle.
Cyclin-dependent kinases (CDKs) •G1 CDKs •A CDK shared by both G1/S- and S-cyclins •M-phase CDK
•Their levels in the cell remain fairly stable, but each must bind the appropriate cyclin (whose levels fluctuate) in order to be activated.
•They add phosphate groups to a variety of protein substrates that control processes in the cell cycle.
Phosphorylation of a CDK inhibits the protein kinase activity of the enzyme
CKI (Cdk inhibitor prot.)
Mechanisms of cell cycle control
Specific kinase complexes advance the cell through the cell cycle.
SCF serves as a ubiquitin ligase with the help of E1 and E2
APC (anaphase-promoting complex) serves as ubiquitin ligase for M-cyclin.
Only G1 cells are competent to initiate DNA replication.Cells that have completed S phase (G2 phase) are not able to rereplicate their DNA.
The initiation of DNAreplication once percell cycle.
Cdc25 is stimulated in part by Polo kinase and is further stimulated by active M-Cdk
The DNA replication checkpoint.Hydroxyurea blocks DNA synthesis activates checkpoint mechanismAddition of caffeine makes the checkpoint mechanism fail.
Mitosis
Structural and regulatory moleculesare involved in controlling the initiation
of mitosis
APC (anaphase-promoting complex)
The triggering of sister chromatid separation by the APC.
Mechanism whereby proteolysis regulates cell cycle progression
The APC • triggers the events leading to destruction of
the cohesins and thus allowingthe sister chromatids to separate.
• degrades the mitotic (M-phase) cyclins.
The anaphase-promoting complex (APC) and other proteolytic enzymes.
Mad2 protein on unattached kinetochores
Any kinetochore that is not properly attached to the spindle sends out a negative signal to the cell-cycle control system, blocking Cdc20-APCactivation and sister chromatid separation.
The creation of a G1 phaseby stable Cdk inhibitionafter mitosis.
The control of G1 progression by Cdk activity in budding yeast.
The Rb protein acts as a brake in mammalian G1 cells.
Cell size control throughcontrol of the cell cycle in yeast.
A hypothetical model of how budding yeast cells might coordinate cell growth.As the cell grows, the total # of Cln3 molecules increases in parallel withtotal cell protein.
Cell-cycle progression isblocked by DNA damage and p53: DNA damage checkpoints
Mdm2: acts as a ubiquitin ligase
An overview of the cell-cycle control system. The activity of each cyclin-Cdk complex is influenced by various inhibitory checkpointmechanisms, which provide information about the extracellularenvironment, cell damage, and incomplete cell-cycle events.
Mouse embryo
Apoptosis during the metamorphosis of a tadpole into a frog.
Neuronal cell death may occur through diverse mechanisms.
In classical study of cell death during development, Schweichel a
nd merker(1973,Teratology) divided cell death into three types ba
sed on the differences in the ultrastructural molphological featur
e
Schweichel, J. and Merker, H. (1973) Teratology 7, 243-266
Diversity in the Mechanism of neuronal Cell death
Type I cell death
Type II cell death
Type III cell death
Apoptosis
Autophagy
Necrosis
Type I cell death : Apoptosis
: Previously termed apoptosis by kerr, wyllie, and currie
(Kerr etal, 1972 Br.J.Cancer)
Cytoplasmic condensation
Nuclear pyknosis
Chromatin condensation
DNA fragmentation
Formation of membrane bound apopototic bodies
Cell rounding
Membrane blebbing
Cytoskeleton collapse
necrosis apoptosis
Intrinsic apoptotic pathway
Caspase 3,7
ICAD(Inhibitor of caspase activated Dnase)
CAD Caspase activated Dnase
DNA cleavage
50-200kb
(Apoptosis inducing factor)->Block IAP (Inhibitor of Apoptosis)
Proposed caspase functions and structureProposed caspase functions and structure
(Initiator caspases)
Extrinsic Death receptor pathways
DISC :Death-Inducing Signaling complex
“Induced proximity” model
Type II cell death : Autophagic cell death
Autophagic vacuoles of the lysosomal origin
Mitochondrial dilation
Enlargement of the ER and the Golgi apparatus
Other hallmarks of apoptosis such as nuclear pyknosis and
membrane blebbing may also may occur later in autophagic
death but are less prevalent
Autophagic cell death has been described in neurons during
development and in association with neurodegenerative disea
ses.
Ubiquitin-proteasome pathwayMost intracelullar short-lived proteins
Most long-lived proteins Lysomes
The mechanism to deliver cytoplasmic components to the lysosomes is called au
tophagy in general
Autophagy - autophagosome
At the ultrastructural level, the main criterion for recognizing autophagy is
the appearance of intracellular double membrane vacuoles containing cytopl
asmic Components such as fragments of ER or MT and lysosomal hydrolase
s.
The size of autophagosomes varies among mammalian cell (usually 0.5-1.5m)
Intracellular lysosome-mediated catabolic mechanism
Three types of autophagy have been proposed
Macroautophagy
Microautophagy
Chaperone-mediated autophagy
ex) Starvation-induced proteolysis
De novo formation of a sequestering vesicle in the cytosol
and is the main mechanism involved in the degradation
and recycling constituent of intracellualr organelle
Operates by protruding or invaginating a portion of pre-existing
vacuolar membrane to engulf cytosol or organelles
Result from the delivery of the proteins with the signature sequence
of KFERQ to lysosomes through designated lysosomal transporters
Mechanism of vacuole formation & delivery of materials
The role of autophagy in neuronal cell death : Prod
eath ?
Metamorphosis of Drosophila
The destruction of obsolete larval tissue such as midgets and salivary glandsinduced by pulses of the steroid hormone ecdysone is accompaniedby massive accumulation of lysosomes as well as hall marks of apoptosis
The neuron of ION (isthmo-optic neuron) that make inappropriate projections are eliminated through a cell death mechanism involving formation of autophagic vacuoles
Chick isthmo-optic nucleus(ION)
NGF-deprivation-induced sympathetic neuronal cell death
Serum-deprivation-induced death of PC12 cells
Steroid regulation of autophagic programmed cell death during development
Lee et al , Development 128, 1444-1455 (2001)
Nerve cells that receive enough survival factor live, while theothers die by apoptosis.
Xue et al, Molecualar and cellular neuronscience 14,180 (1999)
Autophay is activated by apoptotic signaling in sympathetic neurons : alternative mechanism of death execution
NGF deprivation –induced autophgy
Autophagy-related protein
Although the mechanism of autophagy in mammalian cells remains skechy
genentic studies in yeast have revealed a complex pathway and execution of
autophagy.
Yeast Mammalian
Apg 6/VPS 30 Beclin 1
Can promote autophagy when overexpressed
in cultured MCF7.
Can inhibit tumorigenesis.
Is monoallelically deleted in 40-75% of spo
radic Human breast cancers and ovarian c
ancers.
Activation of autophagy allows yeast to
survive nutrient starvation, and yeasts
lacking Apg6/vpg30 are defective in bo
th their ability to undergo nitrogen dep
rivation-induced autophagy.
Liang et al, Nature 402,627 (1999)
Bcl-2 interacting protein
The role of autophagy - tumorigenesis
Liang et al, Nature 402,627 (1999)
Autophagy-promoting activity of beclin-1 in MCF cell is associated with inhibition of MCF7 cellular proliferation, in vitro clonigenicity and tumorigenesis in nude mice
Beclin-induced autophagy acts as a defense mechanism
that results in lysosomal capture and removal of sindbis virus
Beclin 1-induced autophgy – defense mechanism
Liang et al , J virol Vol 72, 8586 (1997)
One of the most obvious differences between canonical apoptosis and a
poptosis with autophagic features may be the mechanism of dead cell d
egradation
metamorphosis or large-scale tissue histolysis
Canonical apoptosis
Apoptosis with autophagy
Lysosome of professional or nonprofessional phagocytes
Autophagy(Internal process)
Hypothesis
Autophagy during neuronal cell death serves as clearance mechanism
and does not participate critically or directly in the execution process
of cell death
Summary of Autophagy
Thus, despite of autophagy during neuronal cell death and cell death in other systems, the conclusive evidence for a prodeath role of autophagy during neuronal cell death is still missing
The functional consequence of increased autophagic activity for neurodegeneration is also not clear.
Autophagy is activated as a compensatory mechanism for a defect or an insufficiency in the proteasome pathway, which may be impaired in chronic neuronal degenerative disease
It is possible that autophagy might actually facilitate the removal of mutant or otherwise misfolded proteins.
Type III cell death : Necrosis
Clarke, (1990. Anat. Embryol.181, 195-213 ) further subdivided the type III cell death into types 3A and 3B.
They differ in the apparent manner of cell destruction
Nucleus of type 3B shows Karyolysis or edema and cytoplasmic membarne round up
Both of the nucleus and the cytoplamic membrane are destroyed by fragmentation
Type 3A Type 3B
Distinguished from the type II cell death by its lack of lysosomal involvement Swelling of intracellular organelles followed by empty spaces in the cytoplasm
Although necrosis has been frequently associated with pathological neuronal death, certain developmental neuronal cell death has also been found to exhibit features of necrosis
Receptor-mediated necrosis
Fas, TNF-alpha family When caspases are inhibited in certain cell type
Necrosis
Swelling of mitochondria and ER
Intracellular vacuolization
Dilation of the nuclear membrane
FADD
Caspase 8
Caspase 3
Canonical apoptosis
Matsumura et al J Cell Biol. 2000 Dec 11;151(6):1247-56
Vercammen et al J Exp Med. 1998 Sep 7;188(5):919-30
Mitochondrial damage
Loss of the mitochondrial transmembrane potential
Necrosis
Swelling of Mitochondria
Early loss of ATP synthesis
The release of cytochrome C
Apoptosis
Cyt-c/Apaf-1/caspase-9
Energy-dependent suicide mechanism
A certain level of ATP synthesis
is maintained until late in the process
What may be the mechanism of the mammalian necrotic cell death?
Amiloride sensitive Na+ channel
Type I cell death
Type II cell death
Type III cell death
Apoptosis
Necrosis
Autophagy
Conclusion
AIF (Nucleus condensation, DNA cleavage)
Caspases independent
Caspases
Autophagosome
Independent of lysosomal activity
Cell deathLysosome
Mechanisms of caspase activation
Pathways to cell death in C.elegans and mammals
Two major apoptotic pathways in mammalian cells
How caspases disassemble a cell
When blood clots, platelets incorporatedin the clot are triggered to release the contents of their secretory vesicles.PDGF
Mitogen stimulates cell division.
Cell-cycle arrest or apoptosis induced by excessive stimulationof mitogenic pathways.
Overcoming replicative cell senescence by the forced expression of telomerase
One way in which growth factorsPromote cell growth.
Cell growth and division can be controlled by separate extracellularSignal proteins in some cell types.The size of sympathetic neurondepends on the amount of NGFsecreted by the target cells it innervates.
The wider Bcl-2 family
Possible mechanisms of action of Bcl-2 family members
Density-dependent inhibition of cell divisionseems to reflect the ability of a cell to depletethe medium locally of extracellular mitogens.
Anchorage dependence of cell division
Actin filaments : greenP-Tyr : red Overlap : orange (Focal adhesion kinase activation)
The effect of a myostatin mutation on muscle size.Myostatin is a TGF-β family member that normally inhibits the proliferation of myoblasts that fuse to form skeletal muscle cells.
Sections of kidney tubules from salamander larvae of different ploidies.The size of organs and organisms depends on mechanisms that can somehowmeasure total cell mass. maintain body form
haploid
tetraploid
The hindbrain in a haploid and in a tetraploid salamander.