Cell Cycle

Budding
Cell cycle
 Cell cycle can be defined as the entire sequence of events

that leads to the duplication and division of a cell.

 Cells have the property of division and multiplication and

consist of three major phases namely

 Mitosis (M phase) or the nuclear division,

 Cytokinesis or the division of the cell and

 Interphase where replication of genetic material occurs.

Phases of the Cell Cycle
 Recognition of these phases began in 1882, when Walther

Flemming named the process of nuclear division mitosis

(from the Greek mito, or “thread”) after he first observed
the condensed chromosomes.

 Mitosis was a clear cell cycle landmark, and the rest of the

cell cycle between mitosis was called interphase.

Phases of the Cell Cycle
 Each cell is born at the completion of the M phase,
which includes mitosis, the partitioning of the
chromosomes and other cellular components, and
cytokinesis, the division of the cytoplasm.
 The chromosomal DNA is replicated during S phase
(synthetic phase).
 The remaining two phases are gaps between mitosis and
the S phase.
Phases of the Cell Cycle
 The G1 phase (first gap phase) is the interval between

mitosis and the start of DNA replication. The G2

phase (second gap phase) is the interval between the
completion of DNA replication and mitosis.

 All cycling cells have an M phase and an S phase.

 Different phases of mitotic cell cycle
Duration in hours
Vicia faba
Phases Mouse Human
(broad
liver cells Hela cells
bean)
G1 12 12 12
S 6 6-8 10
G2 12 3-6 3
M 1 1 1
Phases of the Cell Cycle
 The G1 and G2 phases vary in length and are very short in

some early embryos.

 The M phase lasts only for an hour in a period of 24 hour

required for a eukaryotic cell to divide.

 Cell cycles can range in length from as short as 30 minutes

in a cleaving frog embryo, whose cell cycles lack both G1

and G2 phases, to several months in slowly growing
tissues, such as the mammalian liver.
Interphase:
 The portion of the cell cycle when cells grow and replicate their
DNA.
 Interphase has three sections.

1. The G1 (first gap) phase is the interval between mitosis and

the onset of DNA replication.
2. The S (synthetic) phase is the time when DNA is replicated.
3. The G2 (second gap) phase is the interval between the
termination of DNA replication and the onset of mitosis.
Features of interphase
 The nuclear envelope remains intact.

 The chromosomes occur in the form of diffused, long, coiled and

indistinctly visible chromatin fibres.
 The DNA amount becomes double.

 Due to accumulation of ribosomal RNA (rRNA) and ribosomal proteins

in the nucleolus, the size of the latter is greatly increased.
 In animal cells, a daughter pair of centrioles originates near the already
existing centriole and, thus, an interphase cell has two pairs of
centrioles.
G1 Phase
 G1 is typically the longest and most variable cell-

cycle phase as it either occupies 30 to 50 per cent of

the total time of the cell cycle

 When cells are “born” at cytokinesis, they are roughly

half the size they were before mitosis, and during G1,
they grow back toward an optimal size.
G1 Phase
 It is also known as the first growth phase, since it

involves synthesis of RNA, proteins and membranes

which leads to the growth of nucleus and cytoplasm
of each daughter cell towards their enhancing size.

 During G1 phase, chromatin is fully extended and not

distinguishable as discrete chromosomes with the light

microscope.
G1 Phase
 Thus, it involves transcription of three types of RNAs,
namely rRNA, tRNA and mRNA; rRNA synthesis is
indicated by the appearance of nucleolus in the interphase
(G1 phase) nucleus.
 Proteins synthesized during G1 phase
 (a) regulatory proteins which control various events of mitosis

 (b) enzymes (DNA polymerase) necessary for DNA synthesis

of the next stage and
 (c) tubulin and other mitotic apparatus proteins.
G0 and Growth Control
 In multicellular organisms, many differentiated cells

no longer actively divide.

 These nondividing cells (such as neurons and striated

muscle cells) that no longer divide, are arrested

usually in the G1 stage, such a type of G1 phase is
called G0 phase, a branch of the G1 phase.
G0 phase
 Cells that are no longer capable of division, whether temporarily or
permanently, remain in G0 phase, where they remain metabolically
active but no longer proliferate unless called on to do so by
appropriate extracellular signals.
 A cell must receive a growth-promoting signal to proceed from the
quiescent stage or G0 into G1 phase and thus reenter the cell cycle.
 This process must be highly regulated, as the uncontrolled proliferation
of cells in a multicellular organism can lead to cancer.
S Phase
 During the S phase or synthetic phase of interphase,

replication of DNA and synthesis of histone proteins occur.

 New histones are required in massive amounts immediately

at the beginning of the S period of DNA synthesis to

provide the new DNA with nucleosomes.

 At the end of S phase, each chromosome has two DNA

molecules and a duplicate set of genes.

S Phase
 During replication, the duplicated DNA molecules,
called sister chromatids, become linked to each other
by a protein complex called cohesin.
 Cohesin is a member of the SMC, or structural
maintenance of chromosomes, family of proteins.
 SMC proteins are DNA-binding proteins that affect
chromosome architectures; indeed, cells that lack SMC
proteins show a variety of defects in chromosome stability
or chromosome behavior.
S Phase
 This pairing of sister chromatids is important for their

orderly segregation later in mitosis.

 S phase occupies roughly 35 to 45 per cent time of the

cell cycle
G2 phase
 This is a second gap or growth phase of interphase.

 During G2 phase, synthesis of RNA and proteins continues

which is required for cell growth. During G2, the cell synthesizes
a variety of proteins.
 Of particular significance to the cell cycle, most microtubules –
proteins that are required during mitosis – are produced during
G2.
 It may occupy 10 to 20 per cent time of cell cycle.

 As the G2 phase draws to a close, the cell enters the M phase.

M phase
 During M phase (mitosis and the subsequent
cytokinesis), chromosomes and cytoplasm are
partitioned into two daughter cells.
Mitosis
 The process of cell division whereby the
chromosomes are duplicated and distributed
equally to the daughter cells is called mitosis.

 The mitotic cycle is divided into: prophase,

metaphase, anaphase and telophase.

 The period between two mitotic cycles is called

interphase.
Cell cycle checkpoints
 A checkpoint is a stage in the eukaryotic cell cycle at which
the cell examines internal and external cues and "decides"
whether or not to move forward with division.
1. These checkpoints occur near the end of G1 (The
G1/Restriction point) ,
2. G2 Checkpoint at the G2/M transition , and
3. M (Metaphase) Checkpoint/ Spindle Assembly
checkpoint during metaphase to anaphase transition
Check for:
• DNA damage
• DNA replication
completeness

Check for:
• Chromosome
attachment to spindle
at metaphase plate

Check for:
• Cell size
• Nutrients
• Growth
Factors
• DNA Damage
The G1 Checkpoint
 The G1 checkpoint determines whether all conditions are
favorable for cell division to proceed.
 The G1 checkpoint, also called the restriction point (in yeast), is
a point at which the cell irreversibly commits to the cell division
process.
 External influences, such as growth factors, play a large role in
carrying the cell past the G1 checkpoint.
 The cell will only pass the checkpoint if it is an appropriate size
and has adequate energy reserves.
The G1 Checkpoint
 At this point, the cell also checks for DNA damage.

 A cell that does not meet all the requirements will not
progress to the S phase.
 The cell can halt the cycle and attempt to remedy the
problematic condition, or the cell can advance into G0
(resting) phase.
 Some cells stay permanently in G0 phase, while others
resume dividing if conditions improve.
The G2 Checkpoint
 The G2 checkpoint bars entry into the mitotic phase if

certain conditions are not met.

 As with the G1 checkpoint, cell size and protein

reserves are assessed.

 However, the most important role of the G2 checkpoint

is to ensure that all of the chromosomes have been

accurately replicated without mistakes or damage.
The G2 Checkpoint
 If the checkpoint mechanisms detect problems with the

DNA, the cell cycle is halted and the cell attempts to either
complete DNA replication or repair the damaged DNA.

 If the damage is irreparable, the cell may undergo

apoptosis, or programmed cell death. This self-destruction

mechanism ensures that damaged DNA is not passed on to
daughter cells and is important in preventing cancer.
The M/ Spindle Checkpoint
 The M checkpoint occurs near the end of the

metaphase stage of mitosis.

 The M checkpoint is also known as the spindle

checkpoint because it determines whether all the

sister chromatids are correctly attached to the
spindle microtubules.
The M/ Spindle Assembly Checkpoint
 Because the separation of the sister chromatids during

anaphase is an irreversible step, the cycle will not

proceed until the kinetochores of each pair of sister
chromatids are firmly anchored to at least two spindle
fibers arising from opposite poles of the cell.
Regulators Of The Cell Cycle
 The Nobel Prize in Physiology or Medicine for 2001 jointly to Leland
H. Hartwell, R. Timothy (Tim) Hunt and Paul M. Nurse for their
discoveries of "key regulators of the cell cycle"
 Among the above three Nobel Laureates, Hartwell was
responsible for the discovery of cell-division-cycle genes
(designated CDC genes) in budding yeast (Saccharomyces
cerevisiae), Nurse was responsible for the discovery of cell-division-
cycle genes (designated cdc genes) in fission yeast
(Schizosacharomyces pombe) and Hunt was responsible for the
discovery of cyclins in sea urchin, frog and mammals
Cdc genes – Cell-
division -cycle genes
Cell-Cycle Control Can Be Dissected
Genetically by Analysis of Yeast Mutants
Cdk and Cyclin
 Cyclin-dependent kinases (Cdks) belong

to a group of protein kinases and are the

central components that coordinate
activities throughout the cell cycle whose
activities in turn are regulated by Cyclin
(a Cdk regulatory protein) binding.
Cdk and Cyclin
 The Cyclin-Cdk complex causes the cyclical changes in
the phosphorylation of intracellular proteins initiate
regulate the major events in the cell cycle such as DNA
replication, mitosis or cytokinesis etc.
 CDKs phosphorylate proteins on Serine and Threonine
amino acid residues: they are Serine/Threonine kinases.
 Cyclins were originally named because they undergo a
cycle of synthesis and degradation in each cell cycle. The
levels of the cdk proteins, by contrast, are constant.
Figure.Cyclin-dependent
kinases (Cdks) are protein
kinases that, when fully
activated, can phosphorylate
and thus activate other proteins
that advance the cell cycle past a
checkpoint. To become fully
activated, a Cdk must bind to a
cyclin protein and then be
phosphorylated by another
kinase.
Cyclins can be divided into four
classes:
1. G1/S cyclins: They bind to Cdk at the end of G1 and commit
the cell to DNA replication. Their levels fall in the S phase.
2. S cyclins: They bind to Cdk during S-phase and require for
initiation of DNA replication and their level remain high until
mitosis.
3. M cyclins: Activate Cdks that stimulate entry into mitosis at
the G2/M checkpoint.
4. G1 cyclins: governs the activies of G1/S cyclins i.e. promote
cell cycle entry
 Table: Major Cyclins and Cdks of Verterbrates and
Budding yeast
Cyclin- Vertebrates Budding yeast
Cdk Cdk Cdk
Cyclin Cyclin
complex partner partner
Cdk 4, Cdk 1
G1-Cdk Cyclin D Cln 3
Cdk 6
G1/S-Cdk Cyclin E Cdk 2 Cln 1,2 Cdk 1
S-Cdk Cyclin A Cdk 2 Clb 5,6 Cdk 1
Ckb Cdk 1
M-Cdk Cyclin B Cdk 1
1,2,3,4
While yeasts use only 1 Cdk, vertebrates use 4 different Cdks
There are three D cyclins in mammals(cyclins D1,D2,andD3)
**The original name of Cdklwas Cdc2 in both vertebrates and flisionyeast, andCdc2B in budding yeast
Figure 2. The concentrations of cyclin proteins change throughout the cell cycle. There is a direct correlation between
cyclin accumulation and the three major cell cycle checkpoints. Also note the sharp decline of cyclin levels following
each checkpoint (the transition between phases of the cell cycle), as cyclin is degraded by cytoplasmic enzymes.
Fig.Cell cycle control by cyclin-CDK
Oscillations of Cdk activities (through cyclical expression/destruction of cyclins) leads to cyclical
changes in the phosphorylation of proteins that regulate DNA replication, mitosis and cytokinesis
 Figure. Cyclin-Cdk complexes of the cell-cycle control
system. The concentrations of the three major cyclin types
oscillate during the cell cycle, while the concentrations of Cdks
do not change and exceed the amounts of cyclins. In late
G1,rising G1/S-cyclin levels lead to the formation of G1/S-Cdk
complexes that trigger progression through the Start checkpoint.
S-Cdk complexes form at the start of S phase and trigger DNA
replication, as well as some early mitotic events. M-Cdk
complexes form during G2 but are held in an inactive state and
are activated at the end of G2 and trigger the early events of
mitosis. A separate regulatory protein, the APC/C, initiates the
metaphase-to anaphase transition.
 Cell cycle and Cyclin-Cdk complex
Cyclin E/Cdk2 Cyclin A/Cdk2
G1/S-Cdk S-Cdk
At the end of G1 and commit
the cell to DNA replication.
S Require for initiation of DNA
replication

G1 G2
Cyclin D/Cdk4,6
G1-Cdk
Governs the activities
of G1/S cyclins
;Promote cell cycle M Cyclin B/Cdk1
M-Cdk
entry Entry into mitosis at the G2/M
checkpoint
T-loop/activation T-loop move out of Phosphorylation
loop(red) blocks the the active site of Cdk2 by CAK
active site /ATP
binding site in the
when cyclein binds improves the
absence of cyclin active site
Inhibitory Phosphorylation and Cdk Inhibitory
Proteins (CKls) Can Suppress Cdk Activity

 The rise and fall of cyclin levels is the

primary determinant of Cdk activity during

the cell cycle.
 Several additional mechanisms, however,

fine-tune Cdk activity at specific stages of

the cycle.
Inhibitory Phosphorylation
 Phosphorylation at a pair of amino acids in the roof of
the kinase active site inhibits the activity of a cyclin-
Cdk complex.
 Phosphorylation of these sites by a protein kinase
known as Weel inhibits Cdk activity, while
 Dephosphorylation of these sites by a phosphatase
known as Cdc25 increases Cdk activity.
Inhibitory Phosphorylation
 During G1 and S phase, the CDK1 subunit of MPF is

inactive due to an inhibitory enzyme, Wee1. Wee1

phosphorylates the Thr-14 residues in yeast and Tyr-15
residues in humans of CDK1, rendering MPF inactive.

 During the transition of G2 to M phase, cdk1 is de-

phosphorylated by CDC25. The CDK1 subunit is now

free and can bind to cyclin B, activate MPF, and make the
cell enter mitosis.
Cdk Inhibitory Proteins (CKls)
 Binding of Cdk inhibitor proteins (CKIs) also regulates

cyclin-Cdk complexes.

 The three-dimensional structure of a cyclin-Cdk-CKl

complex reveals that CKI binding stimulates a large

rearrangement in the structure of the Cdk
active site, rendering it inactive.

 Cells use CKIs primarily to help govern the activities of

G1/S- and S-Cdks early in the cell cycle.

 Regulation of Cdk activity
 The activity of Cdk's during cell cycle progression is regulated by

four molecular mechanisms.

 First level of regulation involves the association of Cdk's with

their cyclin partners. Thus the formation of specific Cdk/cyclin

complexes is controlled by cyclin synthesis and degradation.

 Second, activation of Cdk/cyclin complexes requires

phosphorylation. This activating phosphorylation of the Cdk's is
catalyzed by an enzyme called CAK (for Cdk-activating kinase).
 Regulation of Cdk activity
 In contrast to the activating phosphorylation by CAK, the third
mechanism of Cdk regulation involves inhibitory
phosphorylation of tyrosine residues by the Weel protein
kinase. These Cdk's are then activated by
dephosphorylation of these residues by members of the
Cdc25 family of protein phosphatases.
 In addition to regulation of the Cdk's by phosphorylation, their
activities are also controlled by the binding of inhibitory
proteins (called Cdk inhibitors or CKIs)
The Cell-Cycle Control System
Depends on Cyclical Proteolysis
 Whereas activation of specific cyclin-Cdk complexes

drives progression through the start and G2/M

checkpoints; progression through metaphase to
anaphase transition is triggered not by protein
phosophorylation but by protein destruction,
leading to the final stages of cell division.
Anaphase- Promoting Complex
 The third major checkpoint is the metaphase-to-anaphase

transition, which leads to sister- chromatid segregation,

completion of mitosis and cytokinesis.

 Progression through this checkpoint occurs when M-phase

cyclin-Cdk complexes stimulate an enzyme called the

anaphase promoting complex, or cyclosome (APC/C),
which causes the proteolytic destruction of cyclins and of
proteins that hold the sister chromatids together.
Anaphase- Promoting Complex
 Anaphase promoting complex, or cyclosome (APC/C), a

member of the ubiquitin ligase family of enzymes.

 Ubiquitin is a small (8.5 kDa) regulatory protein found in

most tissues of eukaryotic Ubiquitination affects proteins in

many ways: it can mark them for degradation via the
proteasome, alter their cellular location, affect their
activity, and promote or prevent protein
interactions.organisms, i.e. it occurs ubiquitously.
Anaphase- Promoting Complex
 They transfer multiple copies of the small protein

ubiquitin in a process known as ubiquitination, to

specific target proteins (such as cyclins, Cdk inhibitor
proteins and other cell-cycle regulators) , resulting in
the ubiquitinated proteins to recognized and destroyed
by giant protease complexes called proteasomes.
The APC/C catalyzes the ubiquitylation and
destruction of two major proteins.
 The first is securin, which normally protects the

protein linkages that hold sister chromatid pairs

together in early mitosis.

 Destruction of securin at the metaphase-to-anaphase

transition activates a protease that separates the sisters

and unleashes anaphase.
The APC/C catalyzes the ubiquitylation
and destruction of two major proteins.
 The S- and M-cyclins are the second major targets of the APC.

 Destroying these cyclins inactivates most Cdks in the cell.

 As a result, the many proteins phosphorylated by Cdks from S

phase to early mitosis are dephosphorylated by various
phosphatases that are present in the anaphase cell.
 This dephosphorylation of Cdk targets is required for spindle
disassembly and the completion of mitosis, and for
cytokinesis.
SCF
 The cell-cycle control system also uses another ubiquitin ligase
called SCF (after the names of its three subunits SCF, whose
name is derived from three of its central components—Skpl,
cullin and the F-box.).
 It ubiquitylates certain CKI proteins in late G1and thereby helps
control the activation of S-cdks and DNA replication.
 SCF is controlled instead by changes in the phosphorylation state
of its target proteins, as F-box subunits recognize only
specifically phosphorylated proteins.
Figure 3-1 A simplified view of the cell-cycle control system
Maturation-promoting factor (MPF)
 A famous example of how cyclins and Cdks work together
to control cell cycle transitions is that of maturation-
promoting factor (MPF).
 The name dates back to the 1970s, when researchers found
that cells in M phase contained an unknown factor that
could force frog egg cells (stuck in G2 phase) to enter M
phase.
 This mystery molecule, called MPF, was discovered in the
1980s to be a Cdk bound to its M cyclin partner
Function of MPF
 It stimulates the mitotic and meiotic phases of the cell cycle.

 MPF promotes the entrance into mitosis (the M phase) from

the G2 phase by phosphorylating multiple proteins needed during
mitosis.
 MPF is activated at the end of G2 by a phosphatase, which
removes an inhibitory phosphate group added earlier.
 The MPF is also called the M phase kinase because of its ability
to phosphorylate target proteins at a specific point in the cell
cycle and thus control their ability to function
Overview of functions
 Triggers the formation of mitotic spindle through microtubule
instability.
 Promotes mitosis i.e. chromatin condensation through phosphorylation
of condensins.
 The three lamins present in the nuclear lamina, lamin A, B & C, are
phosphorylated by MPF at serine amino residues. This leads to
depolymerisation of the nuclear lamina & breakdown of nuclear
envelope into small vesicles.
 Causes phosphorylation of GM130, which leads to the fragmentation of
the Golgi and the ER
 The following are affected by MPF.
 condensins, which enable chromatin condensation
(see prophase)
 various microtubule-associated proteins involved in mitotic
spindle formation
 lamins, interaction contributing to degradation of the nuclear
envelope
 Histones, H1 and H3

 Golgi matrix, to cause fragmentation

 MPF is composed of two subunits:

 Cyclin-dependent kinase 1 (CDK1), the cyclin-dependent kinase

subunit. It uses ATP to phosphorylate specific serine and threonine
residues of target proteins.
 Cyclin, a regulatory subunit. The cyclins are necessary for the kinase
subunit to function with the appropriate substrate. The mitotic cyclins
can be grouped as cyclins A & B.
 These cyclins have a nine residue sequence in the N-terminal region
called the “destruction box”, which can be recognized by the ubiquitin
ligase enzyme which destroys the cyclins when appropriate.
Activation
 MPF must be activated in order for the cell to transition from G2 to M
phase. There are three amino acid residues responsible for this G2 to M
phase transition. The Threonine-161 (Thr-161) on CDK1 must be
phosphorylated by a Cyclin Activating Kinase (CAK). CAK only
phosphorylates Thr-161 when cyclin B is attached to CDK1.
 In addition, two other residues on the CDK1 subunit must be activated
by dephosphorylation. CDC25 removes a phosphate from residues
Threonine-14 (Thr-14) and Tyrosine-15 (Tyr-15) and adds a hydroxyl
group. Cyclin B/CDK1 activates CDC25 resulting in a positive
feedback loop.