Cell cycle, cell division and
its regulation
1. Cell division
2. Cell cycle
3. Mitosis, its individual phases
4. Meiosis, fundamental
differences from mitosis
5. Control of cell division and
cell growth
What is cell division?
 arranged sequence of macromolecular processes in
which the cell replicates its content and then splits
into two daughter cells, and each of them bears the
same chromosomes
 main goal: the reproduction of genetic material for
the next generation
Cell division in unicellular
Unicellular:
Harmonization of cell division with growth
Cell division in bacteria (E.coli)
It has a single circular chromosome, which is attached to the
plasma membrane and remains attached to it during chromosome
replication.
Both chromosomes are separated from each other by cell growth.
Cell wall and plasma membrane goes between between
chromosomes → two cells are formed.
= BINARY FISSION
1
Origin of
replication
E. coli cell
Two copies
of origin
Cell wall
Plasma membrane
Bacterial chromosome
Origin Origin
Chromosome
replication
begins.
Replication
continues.
Replication
finishes.
Two daughter
cells result.
2
3
4
Figure 12.12-4
Cell division -cell cycle in multicellular
• at the beginning of the development of a
multicellular organism is a single cell
• cell division necessary for
- growth of the organism
- replacement of damaged and old cells
- the spread of genetic information: mitosis,
meiosis
• before the division the cell must enlarge,
chromosomes duplicated and ensure their accurate
division into daughter cells
• Coordination of these processes takes place
within the cell cycle
• cell division is strictly controlled to maintain the
unity of multicellular organism
• the loss of dividing control leads to abnormal
development and may cause tumors
In adults the cells divide only in such places and
at the time when it is needed (e.g. replacement
of dying cells, restoring the injured tissue)
• Reproduction
• Development and
growth
• The renewal of
tissues
Eukaryotic cells
(a) Reproduction
(b) Growth and development
(c) Tissue renewal
20 m
100 m
200 m
Usually divide at all:
neural, muscle
Minimum is divided:
liver cells (1 per
year)
Intensively divided:
intestinal epithelial
cells, blood
precursor cells
(more than 1 day).
Each of us creates
million new cells
every second ,
division arrest leads
to death.
Cell cycle
cycle of cell life processes that begin with her birth and ends with the
formation of daughter cells
• precise continuity of cellular processes, two distinct phases:
Interphase
• cell growth, cell organelles duplication, increased metabolism
G1 first growth phase, the active synthesis of RNA and proteins
cell accumulates energy and prepares for DNA synthesis
(Replication proteins, histones)
S The synthesis of DNA, chromosomal duplication
G2 second growth phase continues synthesis of RNA and proteins
cell growth and preparation for M phase (mitotic spindle)
M phase
• mitosis (division of the nucleus)
and cytokinesis
(Cell division)
• chromatin condensation
• breakdown of the nuclear membrane
• Creation of spindle and kinetochore
• chromatid separation
• breakdown of spindles
• chromatin decondensation
• formation of membrane, cell division
Phase of the cell cycle of eukaryotic cells
G0 -PHASE
INTERPHASE (G1, S, G2) (normal cellular activity)
M-PHASE (cell division)
G1 and G2 phases
are phases when cell
growth, duplication of
cytoplasmic organelles are
in progress
G = gap
G0-phase (resting) maintained only basal metabolism
 It occurs only in certain cell types, especially
those that have been terminally
differentiated (neurons)
The synthesis of DNA during cell cycle
all structure and functions of cells are encoded by the DNA
• during cell division, each cell must obtain a complete genetic
information
• DNA is replicated before the cell division
• there is a duplication of chromosomes
• cell in G2 phase has 2x higher DNA content than in the G1 phase
• resulting copies of chromosomes are joined at the centromere
region, form sister chromatids which are separated during the
nucleus division
Synthesis of proteins and RNA during the cell
cycle
• still as fast during interphase
• in M phase the protein synthesis decreases and the
synthesis of RNA is stopped
• all proteins are formed during interphase continuously
• exception are the histones, which are formed only in G1
and S phase
• After completion of replication, histone mRNAs are
degraded
High accuracy requirements
correct sorting of phases
flawless replication
accurate chromosomes segregation
Unwanted risks:
replication before mitosis: loss of genetic
information at least in one cell
double replication of chromosomes before mitosis:
increase of genes copy number
The length of the cell cycle is different
Cell type Length of
teh cell
cycle
Yeast cells 1,5 – 3 hours
intestinal
epithelium cells
12 hours
mammalian
fibroblasts
20 hours
mammalian liver
cells
1 year
Time requirements of the cell cycle
• the speed of the cell cycle is determined by
internal and external stimuli
• cells with distinct specializations without the
ability to divide (neurons, muscle cells, red blood
cells)
• cells that divide only under certain conditions
(liver cells, lymphocytes)
• rapidly dividing cells (epithelial, blood stem cells)
yeasts 1.5 - 3 hours, intestinal epithelium 12 hours,
mammalian fibroblasts in culture for 20 hours,
mammalian liver cells about 1 year
interphase occupies most of the time, can last for
hours, days, weeks, months, years depending on the
type of cells
• proliferating human cell has about 22 hour cycle
• cell that stops to diferentiate enters the resting
phase G0
• variability is given by length of G1 phase
How is the accuracy of cycle achieved?
 the use of checkpoints, which stop the beginning of
each subsequent step before completing the previous
step
 damage of checkpoints by mutation: chromosomal
rearrangements, abnormal numbers of chromosomes,
changes in gene expression, etc. (cancer risk)
 system of regulation of DNA replication and
segregation of chromosomes is in all eukaryotic cells
identical
 similar to the transcription and translation: the
management system of the cell cycle is conservative
and evolutionary stable
Restriction point
• in late G1 phase
• for transition from G1 to S an
extracellular signal is needed
(growth factor, mitogen)
• Without this signal cell leaves the
cycle and enters the G0
S-phase (synthetic)
 replication of nuclear DNA
 synthesis of histones
G1-phase (presynthetic)
probíhají syntetické procesy = ribozomy, ER, mitochondrie,
enzymy, nukleotidy
M-phase
(mitotic)
G2-phase
(postsynthetic)
 protein synthesis, RNA
Mitosis (karyokinesis) = division of the
nucleus
cytokinesis = division of the cytoplasm
G0-phase (resting)
 maintained only basal
metabolism
 It occurs only in certain
cell types, esp. those
which are already
terminally differentiated
(neurons)
The cell cycle of eukaryotic cells
INTERPHASE
S –phase (synthetic)
The cell replicates nuclear DNA
- duplication of chromosomes
M–phase (mitotic)
Cell growth stops and is followed by
1) Nuclear division
- condensation of chromosomes
- formation of the mitotic
spindle
2) Cytoplasmic division
- formation of contractile ring
- division of organelles
MT and ChL are divided,
GA and ER disintegrates into small
fragments, which increases the
probability of uniform distribution.
G1 phase
First growth phase after cell division, follows the cytokinesis
• cell needs to reach its original parent cell size
• in early embryos G1 and G2 phase is missing, the cell divides and diminishes
creation of cytoplasm and new organelles, synthesis of RNA, structural and regulatory
proteins
• preparation of cells for S phase - synthesis of replication proteins and histones
• normal metabolic activity
• length depends on the cell type, availability of nutrients and growth factors, the presence of
inhibitors of proliferation
• In optimal conditions, 9-11 hrs
• the genetic material in the form of decondensed chromatin, 2n DNA molecules (46 in
humans)
• restriction point is sensitive to external factors, the first cell cycle checkpoint
• At the end of the phase, the cell decides how it will proceed
i) entry into the G0 phase: resting phase, cell does not divide
the absence of growth factors, senescence, differentiated cells
ii) entry into S phase: after overcoming the restriction point, the cell continues by next cell
division
S phase
semi-conservative DNA replication
• within the replicon based in places ori bidirectional replication fork
• replication proteins - initiating proteins, helicases, polymerases, RNases,
ligases, telomerase
• DNA synthesis requires a template DNA strand and the RNA primer
• leading strand is formed continuously lagging strand is formed through Okazaki
fragments
• replication of the ends of linear chromosomes is ensured by telomerase
correctness of replication is ensured by accuracy and proofreading DNA
polymerase activity, error base pairing repair
• duplication of genetic material, chromosomes composed of sister chromatids
G2 phase
second growth phase, premitotic phase
• protein synthesis and formation of organelles, systematic preparation for M phase
• synthesis of proteins of mitotic spindle, centriole duplication
• replicated genetic material, 2 x 2n DNA molecules, two sister chromatids in
chromosome
• 2nd cell cycle checkpoint, checking the integrity and accuracy of DNA
replication
Centriole
• paired organelle of eukaryotic cell, not in plant and fungi cells
• capable of independent division
• nine microtubule triplets around the central cavity
• two connected perpendicularly oriented in M and G1 phase
• duplication in S and G2 phase, in the resulting pair there is always one original
centriole and one new
• together with its surroundings form the centrosome, which organizes the mitotic
spindle
M phase It includes nukleus division (mitosis, karyokinesis)
and the cytoplasm (cytokinesis)
• based on changes in morphology and behavior of chromosomes
mitosis is divided into 5 phases
Interphase Mitotic
prophase
Mitotic
promethaphase
Mitotic
metaphase
Mitotic
anaphase
Mitotic
telophase
MITOSIS
Early mitotic prophase
Late mitotic prophase (prometaphase)
Prophase a prometaphase
chromosome
condensation
occurs
Outside
the core
begins to
rise the
mitotic
spindle
M-phase
1) Prophase
• condensation of chromosomes to form visible under light microscope
• sister chromatids connected in centromeric region
• centrosome shift to opposite sides of the cell
• the formation of the mitotic spindle begins in centrosomes
2) Prometaphase (late prophase)
• formation of the kinetochores on chromosomes
- A large protein complex in centromeric region
- Allows to bind chromosomes to spindle microtubules
- Inner layer recognizes and binds to centromere DNA
- An intermediate layer connects the outer and inner layer
- The outer layer is connected to the (+) ends of microtubules, comprising
contains motor proteins, based on the fibrous corona
M-phase prometaphase
disintegration of the nuclear membrane and nucleolus
• stabilization of centrosomes position on opposite sides of the cell
• movement of chromosomes into the center of the cell
• formation of the mitotic spindle
- Tubulin polarity: (-) end in the centrosome, (+) end grows at the opposite end
- Dynamic structure, shortening and lengthening (de) polymerization of tubulin
- After the disintegration of the nuclear membrane (+) the ends of the spindle
contact the kinetochore
- Kinetochore microtubules: connected to kinetochores
- Polar microtubules: connected to tubules from the opposite pole of the spindle
- Astral microtubules: attached to the plasma membrane proteins
Chromosomes are grouped in the
equatorial plate and form a
metaphase plate.
M-phase Metaphase
Chromosome condensation is completed
• chromosomes are pulled / pushed by kinetochore microtubules
• sorting of chromosomes in the equatorial plate between the
poles of the mitotic spindle (metaphase plate)
• sister chromatids of each chromosome are connected to
opposite poles of the spindle
• establishing the karyotype from the cells which were stopped
in metaphase (colchicine)
Metaphase 1. Metaphase beginning is defined
by formation of metaphase
plate. Chromosomes are aligned
in the equatorial plane midway
between the poles. Kinetochores
of all chromosomes are also
aligned in a plane.
Kinetochore
microtubule
Centrosome on the
spindle pole
2. Chromosomes in metaphase plate
are held with considerable force.
Microtubular molecular motors (motor proteins) and a gradual buildup
and breakdown of microtubules are involved in the establishment
and maintenance of this state. Tubulin units are either added or
deleted, which leads to movement.
Colchicine = mitotic spindle poison, blocks addition of microtubule
subunits
Sister chromatids split and
migrate towards opposite
ends of the cell
M-phase Anaphase
• chromosomes separate at
centromeres, sister chromatids split
• chromatids move towards spindle
- kinetochore microtubules shorten
- spindle poles become more distant
- participation of motor proteins
• the same set of genetic information
is assembled at each spindle pole,
establishing a base for daughter cells
Chromosomes
move towards
spindle
apparatus
Spindle poles
become more
distant
Anaphase
1. The connection between sister
chromatids is broken by proteolytic
enzymes.
Shortening
kinetochore
microtubule
Spindle pole
travels to the
cell outer
boundary
Sister chromatids
Each chromatid (daughter
chromosome) is pulled toward
the spindle pole, to which it is
connected.
2. This segregation of
chromosomes leads to a
chromosome distribution into
two identical sets at opposite
ends of the cell.
The chromosome movement speed
is 1μm per minute. The movement
speed is the result of two
independent processes (anaphase A
- anaphase B)
New nuclear envelope is created around each set of
chromosomes to produce two daughter nuclei
M-phase Telophase
Polar microtubules continue to lengthen, preparing for cytokinesis
• chromosomes, which arrived to the poles of the spindle begin to
decondense
• kinetochores and mitotic spindle deteriorate, new nuclei forms
• nuclear membrane restore around each set of sister chromatids
Telophase A new nuclear envelope begins
to assemble around each set of
chromosomes, producing two
daughter nuclei.
Nuclear membrane vesicles
gather around individual
chromosomes and later fuse
together, establishing nuclear
envelope. Intermediate filaments,
which were phosphorylated in
prometaphase, are now
dephosphorylated and associate
back into nuclear lamina, which
is under the nuclear envelope
(inner x outer nuclear membrane)
Nuclear proteins penetrate through the freshly established nuclear
membrane and the nucleus continues to grow.
Chromosomes decondense into so called interphase state, reenabling
transcription. Mitosis ends.
Cytokinesis
Cytokinesis is a
division of cytoplasm
and all of its
components. Begins
already in anaphase
– cleavage furrow is
created
perpendicular to the
mitotic spindle
division axis.
Contractile ring
appears in anaphase
as well.
Contractile ring is formed by bundles of actin and myosin filaments. It is
attached to proteins associated with the inner side of the membrane and
is able to develop a considerable force.
Actin filaments movement against myosin fibers is similar to muscle
contraction. The contractile ring structure is however only temporary !! It
disappears.
cytokinesis does
sometimes not follow
immediately after mitosis -
SYNCYTIUM
(polynuclear cell), is thus
created
In cytokinesis, cell
membranes are being
formed at both daughter
cells simultaneously =
CELLULARISATION
Cells in animal tissues usually adhere firmly to their neighbors,
they tend to be rather flattened and adhered to some sort of
base.
Once the cell enters M phase, integrin's
(responsible for holding the cells together in tissues) start to
phosphorylate weakening their interactions and
bonds, leading to cell rounding.
After cytokinesis finishes the cells reintroduce their adhesion
forces and flatten once again. Reorganizing the contacts with
neighboring cells allows for proper cell incorporation into tissue.
Meiosis
Asexual reproduction
• cell divides to produce two new
identical daughter cells
• e.g. mitosis, binary fission
Sexual reproduction
• two cells fuse to produce a new cell
(zygote), that is different from original
cells
Meiosis
• reduction division (chromosomes)
• occurs in germ cells, produces
gametes (eggs, sperm)
• chromosomes replicate in
interphase, there are two meiotic
divisions
• the original cell is diploid (2n) and
produces four daughter haploid (n)
cells
• chromosome number is reduced by
half, fertilization restores the
diploid status
Diploid – 2n
46 dsDNA
Diploid – 2n
92 dsDNA
haploid – n
46 dsDNA
Homologous
chromosome
pair
Sister
chromatids
Four gametes (haploid, n, 23 dsDNA)
DNA replication
Meiosis I
Meiosis II
Meiosis
• occurs in germ cells, produces
gametes (eggs, sperm)
Spermatogenesis
oogenesis
Spermatogonium
(diploid)
Oogonium
(diploid)
Spermatids
(haploid)
Sperms
(haploid)
Polar bodies
(haploid)
Oocyte
(haploid)
Meiosis I – reduction division
• homologous chromosomes with
different alleles for the same genes
separate
• unusual cell division type, that
creates haploid cells with
chromosomes originating from sister
chromatids
Prophase I
• homologous chromosomes pair up,
bivalent assembly (2 chromosomes),
tetrads (4 chromatids)
• crossing-over (homologous
recombination) takes place between
homologous chromosomes connected
by a synapse
- genetic recombination in offspring
is ensured by replacing parts of
chromosomes
• chromosomes condense, spindle
apparatus forms, nuclear membrane
disintegrates
• consists of five phases
Sister chromatids
(same genes, same
alleles)
Homologous
chromosomes (same
genes, different alleles)
Gene X
Synapse
begins
Recombined
chromosomes
Synapse
disintegrates,
visible chiasma
Synapse
finished,
crossing over
Duplicated
chromosomes
condense
Leptotene Zygotene Pachytene Diplotene Diakinesis
Meiosis I – reduction division
Metaphase I
• chromosome homologous pairs assemble in equatorial plane
Anaphase I
• homologous chromosomes separate and move towards spindle apparatus
• sister chromatids remain connected by centromeres
Telophase I
• spindle apparatus disintegrates, nuclear membrane restores
• cytokinesis splits the cell into two
Interphase Prophase I Metaphase I Anaphase I Telophase I
Chromosomes replicate Homologous chromosomes pair
up, crossing over
Tetrads assemble in
equatorial plane
Homologous
chromosomes split up
Two haploid cells,
chromosomes still doubled
Nuclear
membrane
chromatin Sister
chromatids
tetrads
Centromere with
kinetochore Homologous chromosomes
separate
spindlecentrosomes
Kinetochore
microtubules
Sister chromatids
untouchedMetaphase
plate
chiasma
Cleavage furrow
Meiosis II – separation division
• just one chromosome homologue is present in cell after meiosis I
• during meiosis II sister chromatids, that contain matching
information separate into newly forming gametes
Prophase II nuclear membrane disintegrates, spindle apparatus
forms
Metaphase II chromosomes align in equatorial plane
Anaphase II sister chromatid separation and their movement
towards opposite poles of spindle apparatus
Telophase II spindle apparatus disintegrates, nuclear membrane
restores, cytokinesis
Sister chromatids (same
genes, same alleles)
Gene X
Two haploid cells,
chromosomes still doubled
Sister chromatids
Sister chromatids split during meiosis II. Four haploid cells containing a
copy of each chromosome arise.
Telophase I Prophase II Metaphase II Anaphase II Telophase II
Cleavage furrow
• sexual reproduction is a source of genetic diversity
stemming from:
i) unbiased chromosome choice: chromosomes split
randomly in meiosis I
ii) random fertilization: random fertilization of an
egg by a sperm resulting in zygote
iii) crossing-over: new allele combinations coming
from parent chromosomes
Meiosis
outcome in four haploid cells, each with a copy of every chromosome
• crossing-over generates altered allele combinations within chromosomes
• meiosis is the basis for sexual reproduction, depends on sex chromosome presence
• offspring produced by sexual reproduction is genetically different from its parents
and siblings
Mitosis Meiosis
DNA replication During interphase, before
mitosis
During interphase, before
meiosis, only once
Number of divisions 1 2
Homologous chromosome
linked by a synapse
Does not occur Occurs together with
crossing-over between nonsister
chromatids in prophase
I
Daughter cell number and
their genetic content
Two diploid (2n) daughter
cells, genes identical with
parent cell
Four haploid (n) daughter
cells, genes different from
parent cell
Role in human body Growth, repair Genetic diversity in sexual
reproduction
Regulating cell cycle
decision whether to split is a cellular reaction to its size and
extracellular signals (nutrients, growth factors, stress factors,
DNA damage)
• yeasts decide mainly according to cell size and nutrient availability
• mammals adjust according to growth hormones and mitogens
Principles in cell cycle regulation
• mitosis wont happen before DNA replication finishes,
DNA replication wont happen without splitting the cell
• damaged DNA must not replicate and be passed onto
daughter cells
• erroneously paired chromosomes must not finish mitosis
• cell cycle deregulation can lead to carcinogenesis
Healthy tissue: organized cell growth cancer: uncontrolled cell growth
Cell cycle regulation elements
Foundation of this regulation lies in specifically
activating proteins that regulate cell cycle
i) Posttranslational protein modifications are a part of
cell cycle regulations
- phosphorylation: major change to protein structure
and function, reversible
proteinkinases are among the main cell cycle
regulators
- proteolysis: unneeded proteins are labelled by
ubiquitin and decomposed in proteasome, irreversible
degradation system
ii) Cell cycle checkpoints
kinases of the CDK group, cyclines,
cyclin inhibitors
CDK – cyclin dependent kinases
heterodimer proteinkinases
with a regulatory (cyclin)
and catalytic (kinase)
component (cyclindependent
kinase, CDK)
They phosphorylate key
regulator proteins and are
thus involved in cell cycle
management
Systems for protein degradation:
• targeted degradation of proteins regulating cell
cycle, results in unidirectional irreversible cell phase
shift
• Ubiquitin ligase:
• SCF
• „anaphase promoting complex“ = cyclosome (APC/C)
• Ensures labelling targeted protein by polyubiquitin
mark, that predetermines proteins for degradation
in proteasome
is a multi-protein E3 ubiquitin ligase complex catalyzing the
ubiquitination of proteins destined for proteasomal
degradation. It has important roles in the ubiquitination of
proteins involved in the cell cycle and also marks various
other cellular proteins for destruction
Ubiquitin-proteasome system
Ubiquitin activation
• Catalyzed by ubiquitin-activating enzyme E1
• ATP dependent reaction
• Ubiquitin is attached by C-terminus to E1 protein
• Ubiquitin is consequently carried over from E1 onto
ubiquitin-conjugating enzyme E2
• Enzyme E2 works in conjunction with E3 enzyme, this
complex is termed ubiquitin-ligase
Proteasome degradation: major targets
• Cell cycle and growth regulators:
• Components of cell signal cascades
• transcription factors
• metabolism enzymes
• protein defective variants resulting from mutations or
faulty proteosynthesis
• major histocompatibility complex I antigens
• Proteasome inhibition is lethal to the cell
• Mammals posses hundreds of ubiquitin-ligases, that
recognize degrons (signals for degradation) on target
proteins by recognition section E3
• Ubiquitin-ligases then manage the ubiquitination
itself, producing polyubiquitin chains attached in
several steps to target protein or previous ubiquitin
at lysine residue
• Binding of poly-Ub labeled substrate to
Ub receptor subunit of the proteasome
• Cleavage of Ub through izopeptidase
bound to hatch
• substrate translocation to the
proteasome column
• degradation of the substrate into small
peptides
Degradation in the
proteasome
Cyclin-dependent protein kinases (CDK)
• key enzymes which regulate the transition between cell cycle phases, transfer
cells from the G0 to G1
• various phases ofCC BC triggered by phosphorylation of specific target proteins
• their activity is strictly regulated
i) Catalytic activity only in complex with cyclin
ii) Tyr phosphorylation sites – activating, inhibitory
- phosphorylation by specific kinases, dephosphorylation by
phosphatases
iii) inhibitors
• in yeast one enzyme, in humans five enzymes
G1 CDK4, CDK2, CDK6
S CDK2
G2 CDK2, CDK1
M CDK1
• negative regulators of CC passage, two families (INK, CIP)
• They bind to CDK and inhibits their activity, responsible for the arrest in G1
• e.g. P21 is actively synthesized during DNA damage, binds to CDK and interrupts
the progress of CC
Cyclins
• proteins that form complexes with CDK, activate Cdk and determine their
substrate specificity
• regulate the passage of the phases of CC
• its concentration fluctuates during the CC phases - specific synthesis /
ubiquitination and proteolysis
Cyclins of G1-phase (D): Participation in the regulation of cyclins G1 / S
Cyclins of G1/S-phase (E): CDK activation in late G1, contributing to overcome
the restriction point
Cyclins of S-phase (A): CDK activation point after the passage of restriction
poinz, on duplication of chromosomes, the S phase cyclin remains elevated until
mitosis, because they contribute to the regulation of early mitosis.
Cyclins of M-phase (B): Cdk activation stimulating entry into mitosis after
passing through the checkpoint G2 / M, degradation in the middle of phase M.
The activity of cyclin-cdk complexes
- concentration of cyclins varies during the cell cycle
- concentration of CDK is more stable, but their activity is zero
without cyclin
- cyclins co-decide on the choice of substrate phosphorylation by
complex CDK / cyclin
Three functional states of Cdk
A) inactive - no cyclin - Cdk active site is blocked by the portion of the
protein, called T-loop
(B) partially active - after binding of cyclin T-loop leaves the active site
of Cdk and partially activates it
(C) fully active - threonine phosphorylation of the T-loop Cdk kinase by
CAK (cdk-activating kinase): conformational changes, increased affinity
for the respective substrates
The regulation of CDK activity:
a) phosphorylation
binding of cyclins primarily determines the activity of Cdk
assistance mechanisms contribute to the fine regulation of CDK activity:
- phosphorylation of pair of amino acids near the active site of the kinase
Wee1 inhibits the activity of cyclin / CDK complexes
- dephosphorylation of amino acids by phosphatase Cdc25 enhancing activity
of cyclin / CDK complexes
- other activating phosphate adds CAK to T-loop
• bind to cyclin / CDK complexes and inhibit in
• due to changes in conformation of the active site
in Cdk
The regulation of CDK activity: b) inhibitory
proteins CKIs
a) family Cip/Kip
b) family INK4
CDK inhibitors: Cip/Kip family
three members in mammals: p21 Cip1 , p27 Kip1 , p57 Kip2
distinct roles in cell cycle regulation
p21 is induced by DNA damage using p53 - ensures arrest of
cell cycle in G1 and G2 phases
p27 applies when leaving the cell cycle when they accumulate;
upon re-entry into the cell cycle from dormancy is rapidly
degraded
p57 is applied during embryogenesis, is expressed tissuespecifically
during development
CDK inhibitors: INK4 family (“inhibitors of
CDK4“)
p16 INK4A
p15 INK4B
p18 INK4C
p19 INK4D
Common signs:
inhibiting the formation of a
complex of cyclin D and CDK4 /
CDK6 and inactivation of already
formed complexes
cell cycle arrest in the G1 phase
(prior to the restriction point)
• expression of p16 stimulated by
growth inhibitors (e.g. TGFβ)
and E2F (negative feedback)
Pharmacological inhibitors of CDKs
• CDKs contol cell cycle, control gene expression, differentiation,
apoptosis, processes in the nervous system
• deregulation of CDKs in diseases, pharmacological inhibitors blocking
the ATP binding site of CDK
• investigation of the selective action of these inhibitors and their
possible use for medical purposes
• - tumors, neurodegenerative diseases (Alzheimer's disease, ALS,
stroke)
• - cardiovascular diseases (atherosclerosis, heart hypertrophy)
• - viral infections (HCMV, HIV, HSV, HPV)
• - parasitic protozoa (malaria, sleeping sickness)
Palbociclib (PD-0332991) (inhibitor of
CDK4 and CDK6) gave encouraging
results in a phase II clinical trial on
patients with estrogen-positive, HER2negative
advanced breast cancer
CC checkpoints-molecular brakes
They control :
 initiate S phase
 initiation of mitosis
 distribution daughter chromosomes in anaphase
 beginning of telophase and cytokinesis
 DNA integrity
ensure that major events of cell cycle tate place in a fixed
order
sensors monitor key going cycle and provide a feedback control
system
if the error is logged, the control system will extend the stage
that the error could be corrected
provide extremely accurate cell division (receiving the correct
number of chromosomes correctly replicated by daughter cells)
They perceive external and internal stimuli and
ensure the proper operation of CC
• checking the completion of the previous phase,
and fulfillment of conditions for the next phase
G1 checkpoint - restriction point
• external factors - the presence of nutrients,
mitogens, antiproliferative factors
• after crossing restriction ponit, CC can be stopped
only by internal factors
• DNA integrity
• size of cells (yeast)
G2 checkpoint
• completion of DNA replication
• DNA integrity
• size of cells (yeast)
M checkpoint
• metaphase-anaphase transition
• proper connection of mitotic chromosomes to the
spindle
CC checkpoints-molecular brakes
External stimuli - chemical
• animal cells require addition of essential nutrients, extracellular signals for
growth, division and survival
• signaling molecules produced by other cells of the same organism:
i) mitogens - stimulate cell division, overcome natural braking mechanisms
G1 / S (e.g. Rb)
ii) growth factors - stimulate increase in mass and cell growth, induce
the synthesis of macromolecules - e.g. PDGF: formation in platelets,
promotion of wound healing
iii) factors for survival - suppress programmed cell death
External stimuli – physical
for growth and proliferation cells require space and surface for adherence
Internal stimuli • DNA integrity, connecting chromosomes to the spindle
DNA damage
due to physical and chemical mutagens
• repairable damage - stop of CC to the time of repairing DNA
• extensive damage - programmed cell death
• CC arrest in G1 (inactivation of CDK2) or G2 (Cdk1 inactivation)
• the key role of tumor suppressor genes
ATM, ATR - recognition of DNA damage (UV photoproducts, dsDNA breaks),
activation of CHK1 CHK2
CHK1, CHK2 – activation of p53, inhibition of Cdc25
p53 - increased levels of CKIs
Cell cycle checkpoints
p53 deficiency
• through the cell cycle with damaged DNA - the accumulation of
mutations – risk of tumors (mutations in the p53 gene in the
majority of cancer)
• Li-Fraumeniho syndrome
- hereditary syndrome caused by p53 inactivating mutations
- increased risk of cancer (sarcomas, brain tumors, breast
cancer, adrenal gland, leukemia)
- 50 % risk of cancer up to 40 years of age, 90% to 60 years
of age
p53
• important cell cycle regulator, acts as a tumor suppressor
• Under normal conditions, a low level, continuous degradation
- Mdm2 provides transport p53 from the core to the
proteasome
- Feedback: p53 triggers the expression of Mdm2
• stabilized in stressful situations, especially when DNA damage
• acts as a transcription factor, induces
- cell cycle arrest: expression of p21/CIP
- stop DNA replication: inactivation of DNA polymerase δ
- mitosis arrest: inactivates cdc25
- apoptosis: expression of Bax, Puma
Cell cycle checkpoints
Spindle checkpoint
• for entry into anaphase cells must receive confirmation of the connection of
all chromosomes to spindle microtubules
• damage to this control mechanism leads to damage and nondisjunction of
chromosomes
i) checkpoint ON
• unattached kinetochore activate Mad
and Bub proteins
• complex APC-Cdc20 in inactive state
ii) checkpoint OFF
• Sister chromatid chromosomes binding on
microtubules from opposite ends of
spindle causes mechanical stress in the chromosomes
• complex of proteins Mad and Bub not formed
• APC-Cdc20 complex is active and provides
securine degradation
Mitogenes
pro-growth signaling molecules,
that activate genes of
immediate response by
intracellular signaling cascades
products of immediate response
genes activate genes of delayed
reaction (eg. cyclin D)
cyclin D/CDK phosphorylates Rb
positive feedback: E2F
enhances the transcription of its
own gene; G1/S-Cdk and S-Cdk
increased phosphorylation Rb
E2F proteins
E2F are transcription factors that activate gene expression encoding
proteins necessary for DNA replication, cyclins of late G1 and S
phase and CDK of S phase
The absence of mitogenes:
gene expression controlled by E2F is unabled by interaction between E2F
and Rb protein
The presence of mitogens:
G1-CDK phosphorylates Rb,
Rb-E2F bound is released
and E2F subsequently ensure expression of target genes
Crossing point restrictions: participation of Rb
Rb protein is a key substrate of cyclin-CDK complex of G1 phase
phosphorylation Rb in several places prevents its association with E2F
phosphorylation Rb is further increased during a cell cycle
by phosphatase PP1 Rb passes to the hypophosphorylated state of G2
phase
Factors influencing the passage
of the cell cycle
presence of mitogenes – expression of cyclin D
• presence of antimitogene, differentiation factors – expression of CKI
• cell aging – expression of CKI
• damage of DNA – arrest of cell cycle through p53
• viral infections (eg. HPV, human papillomavirus) – inhibition of p53 and
pRb, cell proliferation
Cell cycle control – early phase of G1
• cyclin D - CDK4/6
• Three forms of cyclin D (D1, D2, D3) with tissue-specific expression form complex with
CDK4 or CDK6
• cyclin D acts as a sensor of mitogenic signals and prepares the cell for the S-phase
• activation of transcription factors, which provide expression of replication proteins and
cyclin E
• dephosphorylation of components pre-initiation complexes, building of new complexes in
places ori
• response to mitogenic signals: eg. EGF - EGFR - GEF factor - Ras protein - MAP kinase
pathway - transcription factors – expression of cyclin D
• inhibition of antimitogenes signals
eg. TGFβ - TGFβR - transcription factor Smad3, Smad4 – expression of CDK inhibitor
Cell cycle control – transition of G1/S
cyclin D - CDK4/6, cyclin E - CDK2
• further prepare the cell to the S phase
• transcription of cyclin E controlled by a set of transcription factors E2F (activators 1-3,
inhibitors 4-5)
• Rb protein (pRb) active in unphosphorylated form, inhibited by phosphorylation
• in G0 and G1 pRB inhibits by its binding activating E2F and activates E2F inhibition,
suppresses the expression of cyclin E
• after phosphorylation by complex cyclin D - CDK4/6 pRb is released and E2F triggers
expression of cyclin E, cyclin A and replication proteins
• Positive feedback – cyclin E formes complex with CDK2 and complets pRb phosphorylation,
at this point it is through the cell cycle independent of the presence of a mitogen
• after initiation of S phase, cyclin E is degraded and CDK2 forms a complex with cyclin A
Cell cycle control – S phase
cyclin A - CDK2
• phosphorylation of components of
the pre-initiation complex
- initiation of DNA replication
(activation complex MCM)
- block of repeat replication within
a cell cycle (cdc6 releasing)
• The result of S phase is the
emergence of two sister chromatids
of each chromosome associated by
cohesin molecules
Cell cycle control – transition of G2/M
MPF (mitosis-promoting factor) = cyclin B -
CDK1
• cyclin B concentration increases continuously throughout
interphase
• CDK1 - inhibitory kinase phosphorylation by Wee1
removed by phosphatase Cdc25
- activating phosphorylation by kinase CAK
• increase in MPF activity is jumping, at maximum level of
cyclin B and after removal of inhibitory phosphorylation
• Positive feedback - activated MPF activates Cdc25
and inhibits Wee1
MPF phosphorylates target proteins involved in early
stage of mitosis
• chromatin condensation - activation of condensins
• disintegration of the nuclear membrane – depolymerization of
laminates, phosphorylation of nuclear pore and proteins of inner
membrane
• formation of the mitotic spindle - activation of centrosomes and
proteins associating with microtubules
Control of cell size in yeast cells
• low activity of Cdc25 and Wee1 high activity - delayed
mitosis, prolonged G2, cell enlargement
• high activity of Cdc25 and low activity of Wee1 - Fast Track
mitosis, shorter G2, reduce cell
Cell cycle control- M phase
APC, anaphase-promoting complex
• ubiquitin ligase connecting ubiquitin to proteins that control mitosis, and thus
predetermines degradation
• activity during the cell cycle fluctuates, activated in late mitosis by treatment MPF
• only works when connected Cdc20 protein, the protein is blocked by Mad and Bub, who
released him up after connecting the kinetochore of chromosomes to spindle microtubules
• substrate are e.g.:
i) securin
• its decomposition relaxes separase which degrades proteins linking sister chromatids
ii) cyclin B
• a sudden drop in his level inactivates MPF
• absence of MPF allows constitutively active phosphatases to remove phosphate groups from
proteins that were phosphorylated MPF, mitotic endings, entry into a new G1
Disorders of cell cycle control
Tumour diseases
• tumor cells do not need to divide the growth factors
- themselves can generate growth factors
- May signal in the absence of growth factor
- May have impaired cell cycle checkpoints
• increased proliferation of cells to support its
transformation into tumor cells
• tumor is basically failure to control cell division,
uncontrolled cell growth
• protooncogenes
- normally activate cell proliferation
- an abnormal activation or losing of inhibition - support
tumorigenesis
- eg. growth factors, cyclines, CDK, E2F, Mdm2, Ras, Myc
• tumor suppressors
- normally suppress cell division
- inactivation or increased inhibition promotes tumor
formation
- eg. pRb, p53, p21, p14, p16, TGF-β, ATM
Disorders of cell cycle control
Numerical chromosomal abnormalities (aneuploidy)
• normal karyotype 46, XX(Y) monosomy - eg. 45, X trisomy - eg. 47, XY +21
• Errors in the mitotic spindle checkpoint, the result of delays and non-disjunction of
chromosomes
i) Errors in mitosis
• chromosome delays in anaphase and non-inclusion in the core subsidiary
• formation of mosaicism, in one organism occurring cell lines with different karyotype
ii) Errors in meiosis
• non-disjunction - creation of nulisomic/trisomic gametes monosomy/trisomy after
fertilization
- in MI: error in the distribution of homologous chromosomes
- in MII: error in the chromatids division
• chromosome delays in anaphase : the formation of nulisomic gamete