Cell cycle I

what is the role of the cell cycle and what is the goal of mitotic division

• the goal of cell cycle is to ensure accurate chromosome transmission (DNA, organelles and regulation factors)

• the goal of mitotic division is to generate 2 identical daughter cells with same genetic make up as the parent cell (heterozygosity → parental and maternal copy)

cell cycle consist of

G1, S, G2, M DUS G1/S transition and S/G2 transition and S/M

M consist of

pro, meta, ana and telophase met belangrijke transition: meta to ana phase and ana to telophase

how did we discover cell cycle phase specific biochemical molecules

exp fusing cells in diff cell cycles (G1 and S → both replicate, G2 & S → niet both, want G2 niet licensed)

how did they discover MPF

by injecting cytoplasm form a mature frog oocyte into an immature one, causing it to enter M phase without progesteron. this showed that a soluble factor, MPF can trigger cell cycle progression leading to identification of cyclin CDK complexes as key cell cycel regulators

MPF

maturating prmoting factor, consist of cyclin and CDK, phoshphorylates at recognition site: [S/T](-)prolineX[arginine/lysine](+)

what did they discover about cyclin in relation to MPF (en over DNA/RNA)

cyclin levels fluctuate synchronously with MPF levels. in enucleated cells the cyclin cycle could still be initiated so no need for DNA, wel voor mRNA

what is the destruction box (& quick exp to test its function)

cyclin B contains a destruction box = sequence of 8 aa that signal for its own destruction, its needed so that late mitotic events can take place

(exp: fuse it to a reporter p (B-galactosidase) en determine wether its still present or no vs control)

how does the destruction box trigger destruction of cyclin B

through ubiquitination, poly ubiquitin is added to cyclin B, marking it for degradation by the proteasome (4 rings of 7 proteases), recognized by cap of proteasome the Ub is recycled en the p is unfolded (ook door cap), it enters the proteasome and is degraded by the 28 proteases (active sites) inside

what happens if you remove the destruction box

cell cycle arrest during mitosis in the anaphase

cyclin/CDK substrates

• S phase promoting factors

• positive regulators for mitosis

• negative regulators for mitosis

• structural proteins

Functions of Cdk/MPF

• P lamins → demantle nuclear membrane

• P condensin → chromosomes condense

• P contractile ring → cannot contract

• P golgi → remodel & seperate between 2 cells

• P microtubule associated proteins → Microtubule can grow and shorten

• P E3ligase → activate its own inhibitor

S pombe budding or elongate

elongate

what is the S pombe equivalent of Cyclin B and CDK

• cyclin B = cdc13

• CDK = cdc2

what are the regulates cdc2 (CDK) and cdc13 (cyclinB) (s.pombe)

• cdc25 (phosphatase), stimulates cell division (de P)

• wee1 (kinase) inhibits cell division, P CDK

how does wee1 inhibit cell division

it P the tyrosine residue close to the ATP binding site, so ATP doesnt bind due to compition for P

what type of transition is blocked if cells grow but dont divide

G2/M

what type of transition is blocked if cells are round (budding yeast S. cerivisae)

G1/S (cell doesnt start replicating, dus ook geen division)

werking cdc13 and cdc2 in yeast (s.pombe, elongating)

1. cdc13 (cyclin B) and cdc2 (CDK) associate together

2. if wee1 P Y15, cdc13/cdc2 is unable to bind ATP or any target proteins

3. when Cak kinase P Y161, cdc13/cdc2 can bind its target proteins but is still inactive

4. cdc25 de P the y15, allowing ATP to bind, activating cdc13/cdc2

DUS to be active a phosphorylated Y15 and Y161

how do active replication forks inhibit the start of mitotic prophase

1. ATR (=kinase associate with active replication forks) P Chk1 (=kinase)

2. Chk1 amplifies ATR signal and P other proteins, including cdc25

3. cdc25 is then inactive, cannot P (activate) cdc2

4. cell remain in G2/S phase

when are all mitotic p produced

considering mitosis is only 1 hour, the proteins are already available just inactive

S. cerevisiae (budding) phenotypes

• large buds → no division: G2/M blockade

• round cells (no growing buds) → no growth or replication: G1/S blockade

what is the role of cohesin

to keep the sister chromatid together until the metaphase to anaphase transition. they need to be dissolved for transition to occur

what does the meta-anaphase transition depend on

the spindle checkpoint

cohesins need to be dissolved, for this centromeres need to be attached to centrosomes on opposite sides and under tension

1. if centromeres are not all attached the cdc20-MAD2 complex is formed, keeping cdc20 inactive (dan gebeurt wat hier onder staat dus allemaal NIET dus cdc20 = substrate adaptor for meta-anaphase transition)

2. if all centromeres are attached and under tension cdc 20 is released from MAD2 and APC (anaphase promoting complex) can bind to it (cdc20 is dus substrate adaptor for APC)

3. APC ubiquitilates PDS1 (anaphase inhibitor) that normally keeps Esp1 inactive

4. Esp1 is now active and activates Scc1

5. Scc1 cuts a cohesin subunit, dissolving the cohesins

6. meta to anaphase transition occurs

what is another function of APC (apart from ubiquitylating PDS1) and what regulates this

it destroys cyclin at telophase, this is regulated by cdh1 (=substrate adaptor for the ana-telophase transition)

SO E3 ubiquitin ligase activity of APC is regulated by cdc20 and cdh1

how does the cell transition from anaphase into telophase

everything that was activated/phosphorylated by cyclinB need to be inactivated (deP), this is mainly done through deP by cdc14 which recognizes the same site.

pathway:

1. TEM1 hydrolyzes GTP in presence of GAP, keeping it inactive (on centrosome)

2. once the centrosomes touch the cortex, TEM1 is brought in contact with GEF, GEF shift TEM1 into a GTP bound state, activating it

3. Tem1 then activates cdc14

4. cdc14 dephosphorylates cdh1

5. cdh1 then bring APC E3 ligase to the destruction box of cyclin, this leads to cyclin degradation, and cell exits mitosis

what happens during telophase

cell divide. chromosomes decondense (condins deP), lamins deP → nuclear enevlope forms, origin of replications are licensed

what is licensing of the ORC

loading of MCM helical, this can only occur during telophase or early G1, which is when cdt1 and cdc6 are NOT phosphorylated, so when CDK levels are low (hierdoor nooit refiring of ORC during M)

how are licensed origins fired and prevented from refiring (tip: same molecule)

during S phase, cyclin A (no destruction box)

• activates cdc45, which P MCM helicase, leading to firing of ORC

• P cdc 6 so it becomes inactive (inhibits licensing)

what is the S cerevisiae equivalent of CDK1 (cdc2 in S. Pombe) and what happens if its weakly or strongly mutated

• cdc28 = CDK

• when weakly mutated → G1/S block

• when strongly mutated → G2/M block

This revealed that cdc28 functions as bot MPF and SPF

yeast have different cycins for the G1, S, M, G2 phase, which ones are functional in G1, and which during S,G2,M

• G1; cyclin A → no destruction box and promotes entry into S phase (early cell cycle)

• S/G2/M: cyclin B → has a destruction box

• you need both types, but cyclin 3 is an expection and sufficient on its own (geldt voor yeast dit allemaal)

LET OP: cyclin A van humans = B type cyclin (rot benoemd)

what drives yeast cell cycle progression (G1/S transitition) in s. cerevisiae (budding yeast)

1. to initiate S phase, Clb5, Clb6-cdc28 kinases (= s phase cyclin-cdk complexes) need to be activated, this is a post translational event bc they are already there, just inactive (inhibitor, Sic1 needs to be removed)

2. G1 cyclin is activated based on metabolic status of the cell (are there enough nutrients)

3. Sic1= CDK inhibitor (CKI) of clb5/6 is phosphorylated by G1 cyclin (type A), is then ubiquitylated and destroyed, Clb5, Clb6-cdc28 are activated and DNA replication (s phase starts)

what determines the cell cycle phase

the combination of cyclin-CDK

Cdk-Cln1/2/3 phosphorylate two transcription factors (MBF en SBF)which are together responsible for ….

transcription of M/G1, G1 and S-phase genes

what for decision is deciding to start the cell phase

a transcriptional decision