Transmission of genetic
information
Dr. habil. Kőhidai László
- 2016.02.12. -
Regulation of cell cycle
?
Main phases
of cell cycle
G2
S
M
G1
G0
G = „gap”
S = synthesis
M = mitosis
Unicellular
Multicellular
 individual character
 short cycles
 blockers
 paracrine subst.
 intercellular communication
 activators
Nutrients
Growth factors
Cell cycle – Cell division
Molecular Biology of the Cell (© Garland Science 2008)
Molecular Biology of the Cell (© Garland Science 2008)
Main types of cell cycles
Embryonic:
- cell size decreased
- the only regulator is MPF
- no control point
Standard:
- cells dividing frequently
- regulators – SPF (G1-S)
- MPF (S-M)
Multicellular:
- cells in G0 (no cyclin)
- cyclins, CDK-s
- growth factors
Characteristics of cell cyle
phases
G1 = growth and preparation for replication
S = duplication of DNA and centrioles
G2 = preparation for mitosis
M = mitosis
4n
2n
2n
G2 phase
Molecular Biology of the Cell (© Garland Science 2008)
G2
DNS repair
S
Chromosomes
Mitosis
Mit. spindle
DNS replication
M
Checkpoints
Cytokinesis
G1
Cyclins/CDK-s
G0
Tumors
Ageing
Differentiation
Apoptosis
Asymmetric divisions
Growth factors (1)
 proteins
 specificity
 paracrine origine and action
 low acting concentration
 acting in G0 - G1
 influencing of differentiation
 adhesion of cells
 contact inhibition
Growth factors (2)
EGF – epidermis, mesenchyma, glia
IGF – chondroblast, fibroblast
FGF – fibroblast, endothel
TGF – several cells
NGF – axonos
PDGF – componenst of blood vessels
Some cytokines e.g. interferon, TNF
Growth factor
Surface
membrane
Tyrosine kinase cascade
Active Ras
Active Ser/Thr kinase cascade
Transcription factors
Transcription of „early-response” genes
(c-jun, c-myc, c-fos)
mRNA-s
Transcription of „delayed-response” genes
nucleus
Myc, Jun, Fos
Synthesis of
transcription factors
mRNA-s
Synthesis of CYCLIN
Tumor suppressor (e.g. p53)
Normal proliferation
Protooncogenes
Tumor suppressor
Enhanced mitotic activity (tumor)
Oncogenes (mutations)
Lack of tumor suppressor genes
Enhanced mitotic activity (tumor)
Protooncogenes
Expression of tumor suppressor genes
Degradation, apoptosis, aging
Protooncogenes
Regulatory elements of
cell cycle
 Cyclins
 Cyclin-dependent kinases (CDK)
 Cyclin-dependent kinase inhibitors (CDKI)
 Anaphase-promoting complex (APC)
Cyclins
 Regulatory proteins synthsized in different phases of cell cycle
 15 (or more) known mammal cyclin
 „cyclin-box” – 100 amino acid length, homologue CDK-binding domain
 G1-cyclins – rapid degradation – C-terminal „PEST” sequence
 S-cyclins
 M-cyclins – longer lifespan – degradation is ubiquitin-dependent
N-terminal „destruction-box”;
degradation before starting mitosis
 Degradation of cyclins results inactivation of CDK
Cyclin-dependent kinases
(CDK)
 Activated by forming a complex with cyclins
 Minimum 4 mammalian CDK-s are known
 Phosphorylation:
Activating:
CDK2 or CDK4 - Thr160  p40mol5  Cyclin H
Blocking (can be):
CDK2 – Thr14, Tyr15  wee1/mik1 protein kinase
 Their cytoplasmatic levels is constant – the regulation
takes place by the association to cyclin
 Types: G1-, S- and M-phase CDK-s
Cyclin-dependent kinase inhibitors
(CDKI)
 Minimum 7 different CDKI-s in mammals
 G1/S CDK binding – p21, p27, p57
 INK4 (inhibitor of CDK4) – p15, p16, p18, p19
these CDKI-s influence the cyclin D – CDK4 or
- CDK6 complexes
Anaphase-promoting complex (APC)
 proteolytic enzymes
 initiates degradation of cohesins which
keeps together the sister chromatids
 also responsible for degradation of M-phase cyclins
G0 phase
G0 - genes responsible for mitosis
are blocked
Transient or permanent phase;
in several organism majority of
cells are in G0
G0 !!!
Lyphocytes – return to G1 phase by antigen stimuli
Tumor cells CAN NOT enter G0 – this is one reason of
their continuous division
Cyclin – CDK relation
kinase
phosphatase
substrate-P
CKI
CDK
CDK
+
CKI
kinase
transcription
proteolysis
translation
substrate
CDK – Cyclin:
Molecular level of activation
Molecular Biology of the Cell (© Garland Science 2008)
CDK – Cyclin:
Effect of p27 (CDKI)
Molecular Biology of the Cell (© Garland Science 2008)
Cell cycle-specific regulatory effects
of cyclins (1)
Molecular Biology of the Cell (© Garland Science 2008)
Cell cycle-specific regulatory effects
of cyclins (2)
Tumor-suppressor function
E7 = human papilloma virus product
Rb = retinoblastoma protein
E2F = transcription factor
E7
Rb
E2F
Rb
E2F
Promoter (c-myc)
Turned off – G0 phase
Turned on – Mitosis
M-phase
G1-phase
Restriction point
S-phase
pRb-PO4
(p107 -PO4,p130 -PO4)
Cdk2-Ec
Dc
CKI 
(p15, 16 ,18, 19)
Growth
factors
pRb-E2F
Cdk4/6-Dc
(p107, p130)
CKI 
(p21, p27, p57)
p53 
DNA
damage
E2F
Ac 
Ec 
Cdk2-Ac
Rb
p53
E2F
Turned off – G0 phase
– molecular signal of DNA damage (G1 and G2 phases)
- E2F binding to transcription factors
- blocks binding to E2F promoter (c-myc or c-fos)
- in the case of „small” errors results stop of cell
cycle
- in the case of major damages of DNA results
apoptosis
p53
- p53 gene inherited recessively
- the controlling function is lost only when both copies
are mutant  tumor-suppressor gene
- when only the mutant is present in the cell – it may
result tumors (in 50% of human tumors there is no
functioning p53 protein !)
- very high p53 levels – early aging, death
Multilevel functions of p53
p53 - DNA
„Checkpoint”-s
 Is S-phase completed?
Until Okazaki fragments are detectable the cell
can NOT exit S-phase
 DNA quality?
G1 –checkpoint
S-phase
G2 –checkpoint
 Quality of mitotic spindle?
S
G2
M
G1
M-checkpoint – relation of spindle-kinetochore
orientation of microtubuli in the spindle
irreparable error  induction of APOPTOSIS
Regulation by the
checkpoints
Ciklin B – CDK1
Ciklin A – CDK1
G2
Ciklin A – CDK2
S
Ciklin E – CDK2
Ciklin E – CDK2
M
G1
Ciklin D – CDK4 & CDK6
MPF
-G1 and S cyclin-CDK-s
(cyclin E)
-M cyclins
-building of mitotic spindle
-breaking down nucl env.
G2
S
S-phase;
Metaphase
M
SPF
-preparation of cell
for entry to the
-chromosome condensation
MPF
G1
-for DNA duplication
G1 cyclins- CDK
APC
-isolation of chromatíides
-breakdown ofM cyclins
-synth. G1 cyclins
-geminin degradation
-preparation of DNA for replication
Phosphorylation can block, too…
Molecular Biology of the Cell (© Garland Science 2008)
MPF = Mitosis promoting
factor
CDK1 and Cyclin B complex
Cyclin
Cyclin
CDK1
CDK1
Tyr Thr
Tyr Thr
P
inactive
Inhibitor kinase Activator kinase
!!! CDK1 = cdc2 !!!
Cyclin
P
P
CDK1
Tyr Thr
active
Cdc25
P
Substrates of MPF





H1 histone – chrs. condensation
Lamins – decomposition of nuclear envelope
MAP-s – mitotic spindle
Phosphatase – increases number of MPF
Myosin L chain – morphological changes
cdc2 gene
p34
CDK
S-cyclin
S-cyclin
p34
CDK
M-cyclin
p34
CDK
M-cyclin
ATP
ATP
ADP
S-phase
ADP
Mitosis-specific
reactions
Control of DNA replication
Positive control („licensing”):
ORC (origin recognition complex) genes (ORC1-6) and proteins
Accessory proteins (concentration increases in G1-phase)
Cdc6, Cdt1 – with ORC they cover DNA
coverage of MCM proteins is REQUIRED
for DNA replication (MCM=mini chromosome maintenance)
After entry into S-phase: Cdc6 and Cdt1 are removed
MCM remains on the replication
fork
Negative control:
-Geminin - inhibits MCM to associate with the new DNA in G2
Molecular Biology of the Cell (© Garland Science 2008)
Members of
holmologue
chrs. pairs
isolated
Isolation of
homologue chrs.
n
2n
Metaphase
chromosoma
Chromosome
Condensed
chromatin
nucleosomes
Anchoring
to proteins
Chromatin
Solenoid
DNA
Nucleosome
DNA helix
Chrs 1 (human)
DNA length 50 mm
Chrs. length 3-4 mm
10.000 x
condensation
Two chromatide chromosoma
Centromer
region
Chromatides
Schematic and TEM
image of chromosome
Interphase
Prophase
Prometaphase
Interphase
Metaphase
Prophase
Anaphase
Prometaphase
Cytokinesis
Metaphase
Cell division
Anaphase
Metaphase
Telophase
Cytokinesis
Prophase
Telophase
MITOSIS
Metaphase
Late anaphase
Early anaphase
Reorganisation of the nuclear envelope in mitosis
Anaphase
Telophase
Cytokinesis/G1
Nucleoporins
Nuclear envelope
Lamins
Formation of nuclear pores after dividion
Assembly
Fusion
Development of spindle
Centrioles
anya-centriolum
distalis „függelékek”
subdistalis
„függelékek”
microtubulus
PCM
leány-centriolum
összekötő rostok
Division of
centrioles
Division of centrioles – cell cycle
Semi-conservative process
Molecular Biology of the Cell (© Garland Science 2008)
Pathological forms of
centrioles
(light microscopy)
Endogenuous processes:
- centrosome – duplication
- microtubuli - nucleation
- microtubuli - removal
- microtubuli - anchoring
Exogenuous processes:
- cytokinesis
- checkpoint control
- cell cycle
- actin release
- mit.-spindle orientation
- movements of nucleus
- cilia and flagellum form.
- regulatory proc.
- aster formation
- organisation of cytoplasme
- membranetransport
- mit. spindle degradation
Pathology of centrosomal regulation
Centrosome duplication
Ectopic MTOC assembly
Centrosome errors
(number, structure, size)
Spindle errors
Chromosome division
Genetic instability
Tumor progression
Microtubule disassembly
Deformities and polarity errors
Tumor progression
Tumor metastases
Division – Centriolum - Evolution
Mammalian cell
S. cerevisiae
Kinetochore
Molecular Biology of the Cell (© Garland Science 2008)
Molecular Biology of the Cell (© Garland Science 2008)
Kinetochore structure
Cohesin
SCF
Cohesin
CEN
Inner
Kinetochore
Midle
Kinetochore
Spindle
components
Motors
Cohesin
Spindle
checkpoint
Cohesin
APC
Anaphase
progression
Effects of APC
Inhibitionof
anaphase
APC
Progression of
anaphase
Cohesins
Centromer
Defect of
spindle,
kinetochore
Checkpoint
of spindle
assembly
Smc - Structural Maintenance of Chromosomes
Smc1+Smc3 = Cohesin
Smc2+Smc4 = Condensin central part
Smc5, Smc6 = DNA repair
Molecular Biology of the Cell (© Garland Science 2008)
Effects of SCF
(SCF = Skp, Cullin, F-box containing complex)
Molecular Biology of the Cell (© Garland Science 2008)
Mitotic spindle
M-cyclin – APC regulation
Molecular Biology of the Cell (© Garland Science 2008)
Molecular
background of
chromatide
separation
Prophase
Metaphase
Anaphase
Cohesin
Adhered kinetochore
Free kinetochore
Microtubuli
Ubiquitin
Phosphorylation
Spindle – chromosome relations
Molecular Biology of the Cell (© Garland Science 2008)
inner lamine outer lamine (BubR1)
microtubuli
Development of
kinetochore – spindle
connection
fibrosus „corona”
(CENP-E, dynein)
centromeric
heterochromatine
bipolar
adhesion
tightness
Mad2 (Rod/Zw10/CENP-E)
pole
Dynein dependent movement
of checkpoint-proteins
Development of spindle errors
Cell biological
error
Sensors
Signaltransduction
Effectors
Cell cycle
error
Metaphase
Anaphase
Motor proteins of the spindle
Molecular Biology of the Cell (© Garland Science 2008)
Spindle – Movement of chromosomes (1)
Prophase
Prometaphase
Spindle – Movement of chromosomes (2)
Metaphase
Anaphase
Telophase
kinetochore
Does
kinetochore
microtubule
pull
chromosome?
kinetochore
microtubule
depolymerisation
Polarization of chromosome in spindle
Molecular Biology of the Cell (© Garland Science 2008)
Spindle errors
Molecular Biology of the Cell (© Garland Science 2008)
Molecular Biology of the Cell (© Garland Science 2008)
Molecular Biology of the Cell (© Garland Science 2008)
„Back and forth” in the spindle
Molecular Biology of the Cell (© Garland Science 2008)
Molecular Biology of the Cell (© Garland Science 2008)
Molecular Biology of the Cell (© Garland Science 2008)
Molecular Biology of the Cell (© Garland Science 2008)
Formation of midbody
Molecular Biology of the Cell (© Garland Science 2008)
Molecular Biology of the Cell (© Garland Science 2008)
Molecular basis of
midbody
formation
Molecular Biology of the Cell (© Garland Science 2008)
Origin of photos and references used:
Alberts, Johnson, Lewis, Raff, Roberts, Walter
Molecular Biology of the Cell 5th Ed. Garland Sci. (2008)
Bruke, B., Ellenberg, J. Remodelling the walls of the nucleus.
Nature Reviews-Molecular Cell Biology, 3:487 (2002)
Craig, et al., Exp. Cell Res. 246:249 (1999)
Doxsey, S. Re-evaluating centrosome function
Nature Reviews-Molecular Cell Biology, 2:688 (2001)
Kitagawa, K, Hieter, Ph. Evolutionary conservation between
budding yeast and human kinetochores
Nature Reviews-Molecular Cell Biology, 2:678 (2001)
Musacchio,A., Hardwick, K.G. The spindle checkpoint: Structural
insights into dynamic signalling
Nature Reviews-Molecular Cell Biology, 3:731 (2002)
Walczak, C.E., Mitchison, T.J. Kinesin-related proteins at mitotic
spindle poles: Function and regulation
Cell, 85:943 (1996)
Walczak, C.E., Mitchison, T.J., Desai, A.
Cell, 84:37 (1996)
Web.uvic.ca/~bioweb/people/choy/Parslow/tsld035.htm