1. The document discusses various aspects of the cell cycle, including its key phases and regulating molecules. It notes that the cell cycle includes growth, DNA replication, chromosome separation, and cytokinesis. 2. Major regulatory molecules discussed include cyclins, CDKs, Rb protein, and checkpoints like START that ensure DNA damage is repaired before progression. 3. External factors like nutrients and growth signals regulate the cell cycle at transition points like the G1/S boundary through pathways involving cyclins, CDKs, and Rb.

1. Surender Rawat
M. Sc. Microbial Biotech
Roll no. 1784

2. 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 phases
G1 phase – Gap 1
Synthesis phase- S phase
G2 phase- Gap 2
Mitosis phase – M phase

3. CELL CYCLE

6. 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

7. 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

8. CELL CYCLE CHECKPOINTS
• Prevent entry into next phase of the cell cycle.
• Also called DNA damage checkpoint

9. 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

10. DISCOVERY OF MPF
YOSHIO MASHUI

11. IDENTIFICATION OF CDK
• Studied cdc mutants of Saccharomyces cerevisiae
• These required Cdc28 to pass START
LEE HARTWELL
PAUL NURSE
•Studied cdc mutant of Schizosaccharomyces pombe
•Discovred cdc2 which arrest cell cycle at G1 and G2
to M transition
Cdc28 and cdc2 were homologous and coded for a kinase known as
Cdk1

14. 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

16. 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

18. MPF Regulation

19. 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 G2
cdk2/cyclin A S

20. Families of cyclins and cyclin
dependent 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

22. 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

23. 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

24. 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

27. ROLE OF p53

30. SPINDLE ASSEMBLY CHECKPOINT
• 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.
• 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.

31. SPINDLE ASSEMBLY CHECKPOINT

32. 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.

33. Cdk1/cyclin B
Chromatin condensation
Nuclear envelope
breakdown
Spindle formation
Fragmentation of
Golgi apparatus

35. • 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.

36. 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

37. webbed fingers

38. 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.

39. Programmed cell death eliminates unwanted cells
Two distinct forms of cell death – apoptosis and necrosis

40. Classical View of Cell Death: Apoptosis vs Necrosis
Murder? Suicide?
www.imm.ki.se/ sft/bilder/Image1.jpg

41. Comparison between two forms of cell death, apoptosis and necrosis

42. Apoptotic cells are biochemically recognizable
Characteristic biochemical changes in cells undergoing apoptosis
1. Cleavage of DNA into fragments at internucleosome site
2. Chromatin condensation
3. Change in the plasma membrane – phosphatidylserine in the
outer leaflet
4. Cytoplasm shrinkage
5. Membrane blebbing
6. Loss of electrical potential across the inner membrane of the
mitochondria
7. Relocation of cytochrome c
8. Corpse clearance via phagocytosis

43. Fragmentation of Golgi bodies
Apoptotic
cell
Nuclear
fragments
Phagocytosis of apoptotic bodies

44. 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

45. PHOTOGRAPH OF A NORMAL WORM AND A Ced 3 MUTANT

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

47. Caspases : The executioner of Apoptosis
Cysteine-dependent aspartate specific proteases
•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

48. APAPTOSOME

49. Caspase
Nuclear lamins
Inhibitors of DNase
Golgi matrix proteins
Cytoskeletal proteins

50. CENTRAL REGULATORS OF APOPTOSIS
Bcl2 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

51. INTRINSIC PATHWAY OF APOPTOSIS

52. The intrinsic pathway of apoptosis depends on mitochondria

53. 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

54. ROLE OF p53 IN APOPTOSIS

55. EXTRINSIC PATHWAY

56. Three ways that extracellular survival factors can inhibit apoptosis

57. AUTOPHAGY – ALTERNTIVE WAY OF
CELL DEATH
• 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