Cell cycle regulation

Cell cycle regulationCell cycle phasesMitosis nuclear divisionCytokinesis cytoplasmic division

CheckpointsG1-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?

Cell cycle controlTwo types of cell machinery: reproducing and allocatingCell cycle stages are regulated by Cdk/cyclinsCdks persist in the cell during all the cycle being in an inactive or active state in dependence on cyclins and other regulatory elementsConcentrations of cyclins are changeable

Major Cdks/cyclinsThe major mammalian cyclins are designated A, B, D, EThe most important Cdks are Cdk1 (previously known as Cdc2), Cdk2, Cdk4, Cdk6MPF (mitosis promoting factor or Maturation-promoting factor) = cyclin B/Cdk1 complex = M- cyclin/M-Cdk complexCdk4/Cdk6/D cyclin= = G1-Cdk/G1-cyclin complex control G1Cdk2/E cyclin controls G2/S transitionCdk2/cyclin A controls S phase

Regulation of cyclin concentrationCyclin concentration are regulated by ratio expression/degradationCyclins B and A are ubiquitylated by anaphase -promoting complex (APC) and are degraded by proteasome

Regulation of the MPFCdk1/CyclinB complex is inactive when Cdk1 is phosphorylated on Tyr15 by Wee1Cdk1/CyclinB complex is activated when phosphatase Cdc25 dephgosphorylates Tyr 15Thus, PKs and Pjosphateases regulate activity of specific cyclin-Cdk complxes to control cell cycle

Cdk activity can be blocked by Cdk Inhibitor proteins

Cdk2/Cyclin A is inhibited by p21 and p27Cdk1/CyclinA by p21Cdk2/Cyclin E by p21Cdk4,6/Cyclin D by p15/p16/p18/p19Zafonte et al., 20008General regulatory mechanisms

Cyclins A abd B: APC inhibitorsMPF: Regulators of Cdc25Cdks: Cdk inhibitor proteins

Cdks are stably inactivated at G1Stable inactivation is due to elimination of all cyclins while the synthesis of novel cyclins is blockedAll existing Cdk/cyclin complxes are blocked

pRb (retinoblastoma protein) is an inhibitor of the transcriptional factor E2F

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, 243-255 - Randy Suryadinata, Martin Sadowski and Boris Sarcevic The pRB is an important target for CycD/Cdk4/6 and CycE/Cdk2 that affects the G1/S transitionpRB represses S-phase genes including cyclin A and B genes

G1/S transition

Symonds et al., 2009The G1/S transition depends on mitogen signalingMitogens activate TKRsTKRs activate the MAPK and PI3K/Akt//NF-kB cascadesMAPK and NF-kB activate the Cyclin D expression the Cdk4/6 Cyclin D complexThe Cdk4/6/Cyclin D complex HYPOPHOSPHORYLATES pRBThe 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

DNA damage can arrest the cell in G1 phase

DNA damage causes increase in concentration and activity of p53. p53 is a transcriptional regulatorp53 positively regulates expresion of p21 and p16 which are Cdk2/Cyclin E and Cdk4/6/Cyclin D inhibitors, respectivelyG1/S transition

ORC is bound with the replication origin throughut the cycleCdc6 binds with ORC at G1Cdt1 binds with MCMs Cdc6 and Cdt1 promote ORC/MCM complex which is known as a prereplicative complexCdk2/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 andMcms and GINS andCdc45 unwind DNA to expose template DNA. G2 checkpointIncomplete replication arrests cells in the G2 phaseCdc25 driven activation of Cdk1/CyclinB is the critical stepDuring interphase, Cdc25 is held inactive via by inhibitory phosphorylation at Ser287 and 14-3-3 binding. At the G2/M transition, Cdc25 is activated in a stepwise fashion. First, Cdk2/cyclinA phosphorylates CDC25 at Thr138 to release 14-3-3. 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.

Perry and Kornbluth Cell Division 2007M phaseCdk1/CyclinB=MPF =M-Cdk is the only regulator of mitosisCdk1/CyclinB accumulates during G2 phase in the inactive stateEntrance in mitosis is switched by Cdc25Cdk1 activates condensins to condense chromatin during mitosis

condensinsCohesins vs condensinsIn contrast to condensins, cohesins are required to held sister chromatids togetherCohesins bind DNA in S phase, G2 and into early mitosisCondensin bind DNA in M phase

MitosisCentrosome duplication starts together with DNA replicationCdk2/CyclinE (Cdk-S) regulates centrosome duplication

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

Mitotic spindlePrometaphase starts with nuclear membrane breakdownThe nuclear membrane breakdown is driven by nuclear lamina and results in membrane vesicle formationContact 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 microtubulesIn plant cells, there are no centrosomes. Still, bi-direction exists. Chromosomes are involved in spindle formationChromosome line up at the equator at the methaphase

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.Mitotic checkpoint= spindle assembly checkpoint (SAC)Pablo Lara-Gonzalez et al. The Spindle Assembly CheckpointCurrent Biology, Volume 22, Issue 22, 2012, R966 - R980During 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. Chromosome segregation in the anaphaseAnaphase begins with cohesin breakageAnaphase A: kinetochore microtubules shortenAnaphase B: spindle poles move apart

kinesinsdyneinsTelophaseThe nuclear envelope re-forms in the telophase

Nuclear envelope breakdown and re-assembly

NE is invaginated around centrosomes due to microtubules that are attached to the nuclear envelope (NE) in conjunction with dyneinAt 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. Gttinger et al., 2009NE re-assembly 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.

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

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;122:1071-1079

Pier Paolo D'Avino J Cell Sci 2009;122:1071-1079Peripheral (or aquatorial) microtubules interact with cell cortex microfilamentsInteraction 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.