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.