1. Cell cycle regulation

2. Cell cycle phases Mitosis – nuclear division
Cytokinesis – cytoplasmic division

3. G1-phase checkpoint (restriction point; start) – Is environment favorable? Have required signal been obtained? Whether cell cyle is ok?
G2-phase checkpoint – is all DNA replicated? Is all DNA repaired?
M-phase checkpoint – are all chromosomes attached to the mitotic spindle?
Checkpoints

4. Cell cycle control
Two types of cell machinery: reproducing and allocating
Cell cycle stages are regulated by Cdk/cyclins
Cdks persist in the cell during all the cycle being in an inactive or active state in dependence on cyclins and other regulatory elements
Concentrations of cyclins are changeable

5. The major mammalian cyclins are designated A, B, D, E
The most important Cdks are Cdk1 (previously known as Cdc2), Cdk2, Cdk4, Cdk6
MPF (mitosis promoting factor or Maturation- promoting factor) = cyclin B/Cdk1 complex = M- cyclin/M-Cdk complex
Cdk4/Cdk6/D cyclin= = G1-Cdk/G1-cyclin complex control G1
Cdk2/E cyclin controls G2/S transition
Cdk2/cyclin A controls S phase
Major Cdks/cyclins

6. Regulation of cyclin concentration
Cyclin concentration are regulated by ratio expression/degradation
Cyclins B and A are ubiquitylated by anaphase -promoting complex (APC) and are degraded by proteasome

7. Regulation of the MPF
Cdk1/CyclinB complex is inactive when Cdk1 is phosphorylated on Tyr15 by Wee1
Cdk1/CyclinB complex is activated when phosphatase Cdc25 dephgosphorylates Tyr 15
Thus, PKs and Pjosphateases regulate activity of specific cyclin-Cdk complxes to control cell cycle

8. Cdk activity can be blocked by Cdk Inhibitor proteins
Cdk2/Cyclin A is inhibited by p21 and p27
Cdk1/CyclinA – by p21
Cdk2/Cyclin E – by p21
Cdk4,6/Cyclin D – by p15/p16/p18/p19
Zafonte et al., 2000

9. General regulatory mechanisms
Cyclins A abd B: APC inhibitors
MPF: Regulators of Cdc25
Cdks: Cdk inhibitor proteins

10. Cdks are stably inactivated at G1
Stable inactivation is due to elimination of all cyclins while the synthesis of novel cyclins is blocked
All existing Cdk/cyclin complxes are blocked

11. pRb (retinoblastoma protein) is an inhibitor of the transcriptional factor E2F
The pRB is an important target for CycD/Cdk4/6 and CycE/Cdk2 that affects the G1/S transition
pRB represses S-phase genes including cyclin A and B genes
Figure 2 Cumulative phosphorylation of the pRb tumour suppressor by cyclin D-CDK4/6 and cyclin E-CDK2 alleviates its inhibitory activity towards the E2F transcription factor
Bioscience Reports (2010) 30, Randy Suryadinata, Martin Sadowski and Boris Sarcevic

12. G1/S transition The G1/S transition depends on mitogen signaling
Mitogens activate TKRs
TKRs activate the MAPK and PI3K/Akt//NF-kB cascades
MAPK and NF-kB activate the Cyclin D expression – the Cdk4/6 Cyclin D complex
The Cdk4/6/Cyclin D complex HYPOPHOSPHORYLATES pRB
The pRB/E2F dissociate from HDAC = Activity of E2B transcription factor is low but enough to start expression of Cyclin E= R point!
The Cdk2/CyclinE complex hyperphosphorylates pRB to release E2F and to activate G1/S genes
G1/S transition
Symonds et al., 2009

14. DNA damage can arrest the cell in G1 phase
DNA damage causes increase in concentration and activity of p53.
p53 is a transcriptional regulator
p53 positively regulates expresion of p21 and p16 which are Cdk2/Cyclin E and Cdk4/6/Cyclin D inhibitors, respectively

15. G1/S transition
ORC is bound with the replication origin throughut the cycle
Cdc6 binds with ORC at G1
Cdt1 binds with MCMs
Cdc6 and Cdt1 promote ORC/MCM complex which is known as a prereplicative complex
Cdk2/Cyclin E phosphorylates MCMs and Cdc6/ Cdt1 that allows the assembly of other proteins (DNA-pol, Cdc45, GinS)= replicative complex=replisome  and results in Cdc6 and Cdt1 releasing and
Mcms and GINS and Cdc45 unwind DNA to expose template DNA.

16. G2 checkpoint Incomplete replication arrests cells in the G2 phase
Perry and Kornbluth Cell Division 2007
Incomplete replication arrests cells in the G2 phase
Cdc25 driven activation of Cdk1/CyclinB is the critical step
During interphase, Cdc25 is held inactive via by inhibitory phosphorylation at Ser287 and binding.
At the G2/M transition, Cdc25 is activated in a stepwise fashion.
First, Cdk2/cyclinA phosphorylates CDC25 at Thr138 to release
Exposed Ser287 is then readily dephosphorylated by PP1, inducing the activation and nuclear translocation of Cdc25 and dephosphorylation of Cdk1 =Cdc2. Once activated, Cdk1/Cyclin B phosphorylates multiple sites on Cdc25, enhancing its activity and preventing inactivation.

17. M phase Cdk1/CyclinB=MPF =M-Cdk is the only regulator of mitosis
condensins
Cdk1/CyclinB=MPF =M-Cdk is the only regulator of mitosis
Cdk1/CyclinB accumulates during G2 phase in the inactive state
Entrance in mitosis is switched by Cdc25
Cdk1 activates condensins to condense chromatin during mitosis

18. Cohesins vs condensins
In contrast to condensins, cohesins are required to held sister chromatids together
Cohesins bind DNA in S phase, G2 and into early mitosis
Condensin bind DNA in M phase

19. Mitosis Centrosome duplication starts together with DNA replication
Cdk2/CyclinE (Cdk-S) regulates centrosome duplication

20. Mitotic spindle formation: prophase
`Dynamic instability is the foundation of mitotic spindle formation
MPF decrease microtubule stability
The plus ends of microtubules nucleated at centrosomes grow, shrink and re-grow, randomly exploring space.
Interpolar microtubule interactions stabilize them

21. Mitotic spindle Prometaphase starts with nuclear membrane breakdown
The nuclear membrane breakdown is driven by nuclear lamina and results in membrane vesicle formation
Contact with a kinetochore inhibits microtubule's dynamics.
As a result, the kinetochore establishes a relatively stable connection to the pole.
Kinetochores of sister chromatids look at opposite directions. So, chromatids bind to opposite poles = bi-direction
Three classes of microtubules
In plant cells, there are no centrosomes.
Still, bi-direction exists. Chromosomes are involved in spindle formation
Chromosome line up at the equator at the methaphase

22. Mitotic checkpoint= spindle assembly checkpoint (SAC)
During prometaphase unattached kinetochores catalyse the formation of the mitotic checkpoint complex (MCC)
MCC inhibits APC.
Once all the chromosomes are aligned with their kinetochores attached to the spindle (metaphase), generation of the MCC ceases, allowing activation of the APC, leading to the ubiquitylation and degradation of securin and cyclin B1.
Pablo Lara-Gonzalez et al.
The Spindle Assembly Checkpoint
Current Biology, Volume 22, Issue 22, 2012, R966 - R980
Degradation of securin liberates separase which cleaves the cohesin ring; this opens the ring, allowing sister chromatids to separate (anaphase).
Degradation of cyclin B1 inactivates Cdk1, leading to mitotic exit.

23. Chromosome segregation in the anaphase
dyneins
kinesins
Anaphase begins with cohesin breakage
Anaphase A: kinetochore microtubules shorten
Anaphase B: spindle poles move apart

24. Telophase
The nuclear envelope re-forms in the telophase

25. Nuclear envelope breakdown and re-assembly
Güttinger et al., 2009
NE is invaginated around centrosomes due to microtubules that are attached to the nuclear envelope (NE) in conjunction with dynein
At prometaphase, phosphorylation of nuclear lamins and INM proteins by CDK1, protein kinase C (PKC) and probably other kinases results in lamina disassembly
Then NE dissassembly results in (1) formation of vesicles OR (2) the retraction of NE membranes into the endoplasmic reticulum (ER).
In metaphase, most soluble components of the NE are dispersed throughout the cytoplasm, whereas INM proteins reside in the tubular mitotic ER.
Nuclear envelope breakdown and re-assembly

26. During late anaphase, membrane tubules start binding to the chromatin surface.
During telophase, the retraction of membrane-bending proteins (reticulons) into the peripheral vesicles allows the remodelling of tubules into flattened membrane sheets on the chromatin surface. Binding of INM proteins to DNA/chromatin supports the attachment of membrane sheets to chromatin.
The first traces of lamins can be detected on chromatin at this stage. NPC are formed.
Finally, transport-competent NPCs allow for the nuclear import of lamins to complete the assembly of the nuclear lamina.
NE re-assembly

27. Cytokinesis Cytokinesis is a process of cytoplasm division.
Cytokinesis is dependent on a contractile ring which is formed underneath the membrane by actin and myosin microfilaments
The furrowing occurs perpendicularly to the long axis of the mitotic spindle

28. Contraction is governed by Rho GTPase
Actomyosin filaments in the contractile ring are connected with the spindle microtubules via the centralspindlin complex.
Contraction is governed by Rho GTPase
Pier Paolo D'Avino J Cell Sci 2009;122:

29. (i) profilin- and formin- mediated actin polymerization;
Peripheral (or aquatorial) microtubules interact with cell cortex microfilaments
Interaction between the centralspindlin complex and the RhoGEF Pbl/ECT2 induces the formation of GTP-bound RhoA, which in turn promotes
(i) profilin- and formin- mediated actin polymerization;
(ii) phosphorylation of the myosin regulatory light chain (MRLC) by Rho kinase (ROK)
Together, these events promote the sliding of myosin heads along actin filaments, and therefore the formation and ingression of the cleavage furrow.
Pier Paolo D'Avino J Cell Sci 2009;122: