Lecture 10: Cell Cycle continues

Targets of phosphorylation and dephosphorylation to regulate mitosis:

- Chromosome condensation and decondensation- Nuclear breakdown and reassembly- Sister chromatid separation

Events Phase of cell cycle

Proteins required

CyclinB/cdk (MPF) activation (Phospho) or deactivation (dephospho)

Chromosome condensation

Prophase (M phase)

-Condensins-Topoisomeraser IICohesin-All 3 proteins are phosphorylated by MPF (cylcin B/CDK)

Condensins upon phosphorylation, form coils; thus coiling and condensing the DNA. Condensin is made of 4 subunits: Smc4,Smc2. Cohesin links two different DNA strands to condense it. Topo II prevents tangling of DNA strands, so that sister chromatids condense independently

Chromosome decondensation

Mitosis to G1

-Activity of phosphotases-Absence of kinases (sometime is sufficient to initiate dephosphorylation)-degradation of cyclin B to inactivate cyclin B/ CDK to bring to down MPF levels

Degradation of cyclin B by APC-cdh1 is important to decondense the DNA and allow for degradation of tubulin (mitotic spindle) experiment: added mRNA of non – degradable cyclin B with RNase treated extract, which showed after 80 minutes into the mitosis, there was tubulin found while in the wildtype, there was no tubulin found when the degradable cyclin was added with RNase treated extract.

Separation of sister chromatids

Anaphase -securin (anaphase inhibitor)-separase-APC cdh20

Securin is anaphase inhibitor as it is bound to separase; therefore prevents it’s activity which is to cut of cohesion. This secruin, is get rid of by APC cdh20 complex, which ubiquintinlates it to be degraded so that separase can do its thing experiment: cyclin B D – box was added with untreated extract. After a lot of it was added, it was observed that there was no decondensation of DNA, this was cyclin B D – box is another site of APC activity, and too much of it poses competition of APC; which is why when too much is added, decondensaiton is not observed because APC is busy chopping off cyclin B to bring

the MPF levels down and get the cell into another phase

Disassembly of nuclear envelope

Prophase Phosphorylation of serine residues in all three nuclear lamins

Nuclear lamina is composed of nuclear lamins (A,B,C polymer filaments); which underlies inner membrane and supports it. Nuclear lamina is composed of lamin A,B,C; during early mitosis, serine residues in all three are phosphorylated which leads to depolymerisation of lamins thus breaking down nuclear lamina,Only lamin B remains attached.experiment: only serine is phosphorylated, if serine is replaced by another amino acid, alanine, then it is not disintergrated

Reassembly of nuclear envelope

Telophase -degrdation of cyclin B- phosphataes (cdc14) reverses the effects of MPF

- dephosphorylation of nuclear membrane proteins important because nuclear membrane proteins associate with the chromosome and each chromosome is surrounded by karyomere which when combine together upon dephosphorylation form nuclear envelope

Mitotic spindle assembly

Prophase Myosin and microtubule associated proteins (MAPS

Activation of MPF

Trafficking of vesicles to ER

Phopho of ER and golgi proteins

Activation of MPF

Breakdown of mitochondrial network to smaller vesicle to promote transport

Phopho of mitochondrial proteins

Activation of MPF

Nuclear pore disassembly

Phopho of proteins composing nuclear envelope pores

Activation of MPF

Dissociation of chromatin from nuclear envelope

Phosphor of inner nuclear membrane proteins

Activation of MPF

DEPHOSPHORYLATION ENSURE THE REVERAL OF ALL EVENTS WHILE THE DEGRADATION OF PROTEINS ENSURES THAT THE PROCESS IS UNIDIRECTIONAL

Cohesion during cell cyclephase of cell cycle

Cohesin

G1 Cohesion rings loaded onto the chromosomeS DNA is replicated into the cohesion ringsG2 Cohesion rings is found throughout the sister chromatidsProphase Rings along the chromatid arms start to shedMetaphase Cohesion rings are only found near the centromere region of the

chromosomeAnaphase Cohesion rings along the centromere cut off by separase to allow for

the segregation of two sister chromatids

Checkpoint:-ensures that the progression of the cell cycle is correct: by either delaying it or leading to apoptosis (if error cannot be fixed)-4 types: DNA damage, DNA replication, spindle attachment and chromosome segregation

Checks for Proteins + pathway

Explanation

G1 arrest DNA damage: ionization radiation checks for breaks in DNA

ATM patwayP53, p21cpi

At two points: in the beginning of G1 and the S –phase entry

S arrest Unreplicated DNA At g2 entryG2 arrest DNA damage: UV

radiation checks for dimerization within DNA

ATR pathway: prevents dephospho of y15, which keeps kinase deactivated

At M phase entry

Metaphase arrest

Kinetochore attachment to spindle

Mad2 closed: inhibits APC –cdc20, as it binds to cdc20;

Prevents entry into anaphase, even if a single chromatid has not associated with the spindle

therefore, cdc20 cannot bind to APC keeping APC inactivated

Two conformations of Mad2: open and closed closed generated at the unattached kinetochore by binding to mad1, which then turns all the other open into closed conformation, which then bind to cdc20 and arrest the cell at metaphase. This change in conformation from close to open is done by p31. P31 binds to mad2, once it is released chromosome due to binding of chromosome to spindle, as it causes the degradation of mad1 this is important because mad1 + mad2 = mad2 close. P31 causes mad2 to release cdc20 which then activates APC and

Mutation: can cause aneuploidy due to missegregatio of chromosomes.Experiment that identified spindle assembly checkpoint: genetic screen of mutants that failed to arrest during cell division even if the chromosomes had not attached. Mutants created by treating yeast with benomyl (destabilizes microtubules)

deactivates the checkpoint and causes the cell to proceed to anaphase.

Anaphase arrest: Tem1 not activated, cdc14 not activated, spindle invert

Chromosome segreagation

-APC-Cdh1 activated by cdc14-Cdc14 activated by segregation of sister chromatids, (inactivated cdc14 remains in the nucleolous) as segregation of sister chromatids activate Tem1 (GTPase) which activates and releases cdc14-MEN (mitotic exit network) set of proteins that monitor checkpoint

For telophase and cytokinesis to occur MPF must be inactivated done by degradation of cyclin B which is done by APC-cdh1 for cdh1 to bind to APC, it must be dephosphorylated which is done by cdc14

After cyclin B degradation MPF inactivated all the phosphrylation events are reversed (by cdc14) and cell exits mitosis.

Cdc14 also prevents the phosphorylation of sic1 by g1 cyclin – cdk; thereby keeping s-cyclin –cdk inactivated in G1

P53 is highly unstable but is stabilized by phosphorylation. When phosphorylated it generates two types of genes, P21( cdk inhibitor) and pre-apoptosis genes