Surender Rawat

M. Sc. Microbial Biotech

Roll no. 1784

CELL CYCLE• Includes 4 coordinated processes-

– Cell growth

– DNA replication

– Disrtribution of chromosomes

– Cytokinesis

In bacteria, cell growth and DNA replication takes place throughout the cell cycle.

In eukaryotes, it consists of four phasesG1 phase – Gap 1Synthesis phase- S phaseG2 phase- Gap 2Mitosis phase – M phase

CELL CYCLE

Different systems-- yeast: cell cycle mutations-- frog: big dividing embryos-- sea urchin & clam: many

embryos

Asynchronously dividing cells DNA/nucleus staining Flow cytometry

Synchronously dividing cells

DAPI stained cells

REGULATION OF CELL CYCLE BY EXTERNAL FACTORS

• Major regulatory point in G1 in Saccharomyces cerevisiae

• Called START

• Once START is passed cells are commited to enter the S phase.

• It is highly regulated and controlled by extracelluar signals.

– Nutrients

– Mating factors

– Cell size

• If signals are absent the cells are arrested at G1.

• This quiescent stage is called G0 in which they are metabolically active but cease growth.

• Eg. Skin fibroblasts, nerve cells

CELL CYCLE CHECKPOINTS

• Prevent entry into next phase of the cell cycle.

• Also called DNA damage checkpoint

MOLECULES OF CELL CYCLE REGULATION

• Three experimental approaches contributed to identification of key molecules responsible for cell cycle regulation

1. Identification of MPF in frog oocytes

2. Identification of cdc molecules in Saccharomyces cerevisiae mutants

3. Identification of cyclins in sea urchin embryos

DISCOVERY OF MPF

YOSHIO MASHUI

IDENTIFICATION OF CDK• Studied cdc mutants of Saccharomyces cerevisiae

• These required Cdc28 to pass START

•Studied cdc mutant of Schizosaccharomyces pombe•Discovred cdc2 which arrest cell cycle at G1 and G2 to M transition

LEE HARTWELL

Cdc28 and cdc2 were homologous and coded for a kinase known as Cdk1

PAUL NURSE

IDENTIFICATION OF CYCLINS• Stuied in sea urchin and calm embryo in

1983

• Accumulation in interphase and degradation in the end of mitosis

• Hunt called these cyclin A and cyclin B

• In 1986, Joan Ruderman showed cyclin A triggers G2 to M transition in frog oocyte

STRUCTURE OF MPF

• Purified in 1988 by James Maller from frog eggs

• MPF is composed of two subunits

– Cdk1 – catalytic subunit

– Cyclin B – regulatory subunit

• Cyclin B is synthesized and form complexes with cdk1 during G2.• Phosphorylation of cdk2 at threonine 161 is required for its activity• Phosphorylation of tyrosine 15 by wee1, inhibits cdk1 activity and

leads to the accumulation of cdk and cyclin B complex.• Activation is by deposphorylation of threonine 14 and tyrosine 15 by

the phosphotase cdc25 for G2 to M transition

MPF Regulation

Mechanism of cdk regulation

Association with cyclins

Phosphorylation at threonine 160 by Cak composed of cdk7 and cyclin H

Inhbitory Phosphorylation at threonine 14 and tyrosine 15

2 families of cdk inhibitors Ink family cip/kip family

•Ink family (p15, p16, p18, p19) cdk4/cdk6 G1

•Cip/kip family (p21, p27, p57) cdk2/cyclin E G2cdk2/cyclin A S

Families of cyclins and cyclindependent kinases

• In eukaryotes

– G1 to S - cdk2, cdk4,cdk6 + cyclin D & E

– G0 – cdk4, cdk6 + cyclin D1,D2 & D3

– Late G1 - cdk2 + cyclin E1 &E2

– Through S – cdk2 + cyclin A1 & A2

– S to G2 – cdk1 + cyclin A1 & A2

– G2 to M – cdk1 + cyclin B1, B2, & B3

GROWTH FCTORS AND REGULATION OF G1 CDKs

• Cyclin D synthesis is induced in response to growth factor stimulation

• If growth factors are removed cyclin D level decreses

• Cdk4, 6/cyclin D complex drive cells through START

• Mutations in continual unregulated expression of cyclin D is associated with many lymphoma and breast cancer.

• Mutations that inactivate cdk4 and cdk6 are found in cancer cells• A substrate of cdk4/6 is Rb protein which is a tumor suppresor• Inactivation of Rb gene leads to cancer• This complex is inhibited by p16

Role of Rb

• When Rb is phosphorylated by cdk4,6/cyclin B then it binds to E2F which transcribes cyclin E

• Prgression through restriction point and entry into S phase is mediated by cdk2/cyclin E complex

• MCM helicase is activated by cdk2/cyclin E complex

• It is degraded by p27

DNA DAMAGE CHECKPOINTS

• These checkpoints are operative in G1, S & G2

• Cell cycle arrest is mediated by 2 proteins

– ATM – ds breaks

– ATR- ss breaks

• These are activated in response to DNA damage and then activate the signalling pathway that leads to cell cycle arrest, activation of DNA repair and apoptosis

ROLE OF p53

• Progression to anaphase is mediated by activation of Anaphase promoting complex/cyclosome (APC/C)

• Unattached kinetochores lead to the assembly and activation of a complex of Mad/Bub proteins that inhibit APC/C binding to cdc20.

SPINDLE ASSEMBLY CHECKPOINT

• Once all chromosomes are aligned on the spindle, the Mad/Bub complex dissociates, relieving inhibition of Cdc20 and leading to APC/C activation.

• APC/C ubiquitinates cyclin B leading to inactivation of cdk1.• In addition, APC/C ubiquitinates securin, leading to activation

of separase.• Separase degrades a subunit of cohesin, breaking the link

between two sister chromatids and initiating anaphase.

SPINDLE ASSEMBLY CHECKPOINT

SPINDLE ASSEMBLY CHECKPOINT

• APC/C degrades cyclin B which is necessary for exiting mitosis and return to interphase

• Inactivation of cdk1 is triggers cytokinesis

• Abnormalities in chromosome segregation is resulting from failure of spindle assembly checkpoint are common in cancer cells and are thought to play an important role in many tumors.

Cdk1/cyclin B

Chromatin condensation

Nuclear envelopebreakdown

Fragmentation of Golgi apparatus

Spindle formation

• Programmed cell death is a active process which usually proceeds with a distinct series of cellular changes known as apoptosis.

– Chromosomal DNA is fragmented

– Chromatin condenses

– Nucleus breaks up

– Cell shrinks into apoptotic bodies

• Apoptosis, or programmed cell death, is a normal component of the development and health of multicellular organisms.

• Cells die in response to a variety of stimuli and during apoptosis they do so in a controlled, regulated fashion.

SIGNIFICANCE

• Balances cell proliferation

• Maintains constant cell numbers in tissues

– 5 × 1011 blood cells eliminated daily in bone marrow

• Provides a defense mechanism

– Eliminates Virus infected cells

– Eliminates DNA damaged cells

• Plays a key role in development

– Elimination of tissues between the digits

– 50% neurons are eliminated by apoptosis

webbed fingers

Apoptosis during the metamorphosis of a tadpole into a frog.

The cells in the tadpole tail are induced to undergo apoptosis stimulated by

the increases in thyroid hormone that occurs during metamorphosis.

The nematode

Caenorhabditis elegans has

also been a very important

model system for studying

apoptosis in development.

Two distinct forms of cell death – apoptosis and necrosis

Programmed cell death eliminates unwanted cells

www.imm.ki.se/ sft/bilder/Image1.jpg

Murder? Suicide?

Classical View of Cell Death: Apoptosis vs Necrosis

Comparison between two forms of cell death, apoptosis and necrosis

Apoptotic cells are biochemically recognizable

Characteristic biochemical changes in cells undergoing apoptosis

1. Cleavage of DNA into fragments at internucleosome site2. Chromatin condensation3. Change in the plasma membrane – phosphatidylserine in the

outer leaflet4. Cytoplasm shrinkage5. Membrane blebbing6. Loss of electrical potential across the inner membrane of the

mitochondria7. Relocation of cytochrome c8. Corpse clearance via phagocytosis

Fragmentation of Golgi bodies

Phagocytosis of apoptotic bodies

Apoptotic

cellNuclear

fragments

HISTORY

•Studied Development of C. elegans in 1986

• Specific 130 out of 1090 cells are eliminated by PCD

• Identified 3 genes by mutagenesis

•Ced 3 - PCD •Ced 4 - PCD•Ced 9 - Regulator

ROBERT HORVITZ

PHOTOGRAPH OF A NORMAL WORM AND A Ced 3 MUTANT

Lodish et al. 6th Figure 21.37 Evolutionary conservation of apoptotic pathways

Caspases : The executioner of Apoptosis

•Caspases involved in inflammation caspases 1 , 4, 5

•Caspases involved in apoptosis Initiator caspases caspases 2, 8, 9, 10 Executioner caspases caspases 3, 6, 7

Cysteine-dependent aspartate specific proteases

APAPTOSOME

Caspase

Inhibitors of DNase

Nuclear lamins

Cytoskeletal proteins

Golgi matrix proteins

CENTRAL REGULATORS OF APOPTOSISBcl2 proteins regulate the intrinsic pathway of apoptosis

The three classes of Bcl2 proteins

inhibit apoptosis

promotes apoptosis

bind and regulate the anti-apoptotic BCL-2 proteins to promote apoptosis

INTRINSIC PATHWAY OF APOPTOSIS

The intrinsic pathway of apoptosis depends on mitochondria

IAPs inhibit caspases

• IAP – inhibitors of apoptosis directly intract with caspases and supress apoptosis by

• inhibiting caspase activity• ubiquitination and degradation of caspases

• Present in Dorsophila and mammals

ROLE OF p53 IN APOPTOSIS

EXTRINSIC PATHWAY

Three ways that extracellular survival factors can inhibit apoptosis

• Uptake of proteins or orgenelles into vesicles (autophagosomes) that fuse with lysozomes.

• Promotes cell survival under starvation

• Does not require caspases

• Characterized by accumulation of lysozomes

• Alternative pathway of cell death when apoptosis is blocked

• Bak/Bax cells die by autophagy

AUTOPHAGY – ALTERNTIVE WAY OF CELL DEATH