3rd checkpoint regulation

Question 1

What is the role of the metaphase-anaphase checkpoint

Answer 1

1. Detects chromatid attachment problems
2. Normal timing of anaphase
3. Metaphase-anaphase checkpoint (can block cell cycle)
4. Anaphase trigger (progresses cell cycle)

Question 2

What does APC/C do

Answer 2

1. APC/C targets cyclins for degradation
2. Levels of s-CYLCIN are reduced at start of transition
3. M-cyclin levels are reduced just after transition start
4. APC/C = anaphase promoting complex or the cyclosome.
5. APC/C is an E3 ubiquitin ligase
6. Proteins ‘tagged’ with K48-linked polyubiquitin are degraded via the 26S proteasome. The addition of ubiquitin to a protein is a post-translational modification.

Question 3

What activates APC/C

Answer 3

1. M-Cdk activates APC/C
2. Cdc20 had to bind to APC/C and be phosphorylated
3. Phosphorylation by m-cdk

Question 4

What does Plk1 do

Answer 4

1. Plk1 can repress inhibitors of the cyclosome

2. Inhibitors bound to cyclosome and when high enough concentrations plk1 can remove inhibitors

Question 5

What happens if chromatids aren’t all attached

Answer 5

1. Mad2 blocks APC/C activation
2. Mad2 can bind to cdc20 sequester it away from being able to bind and activate cyclosome
3. When removed cdc is free to bind again

Question 6

What does Mad2 act as

Answer 6

1. Mad2 is a checkpoint break
2. recruited to unattached kinetochore
3. sequesters and inhibits Cdc20
4. blocks anaphase entry until every chromatid is attached to the spindle

Question 7

What does APC/C trigger

Answer 7

1. APC/C triggers entry into Anaphase
2. Sister chromatids are held together by cohesin.
3. When cohesin is broken down the sister chromatids can then be pulled apart by the mitotic spindle. APC/C initiates this process.
4. Once M-Cdk phosphorylates APC/C, Cdc20 is able to bind to and activate APC/C.
5. Activated APC/C causes the ubiquitylation (covalent attachment of a K48-linked [Lysine 48-linked] poly-ubiquitin chain) of securin targeting it for destruction and causing the release of separase.
6. Separase is a protease which cleaves one of the subunits of cohesin.
7. When cohesin is cleaved the sister chromatids can then be pulled apart to the opposite sides of the cell by the forces exerted by the mitotic spindle fibres.

Question 8

What does mitotic exit require

Answer 8

1. M-Cyclins must be degraded in anaphase and telophase
2. Mitotic exit- requires reversal of early mitotic events that are induced by M-Cdk
3. S-CYCLINS also need to be degraded

Question 9

What targets M- and S- cyclins for destruction

Answer 9

1. APC/C
2. APC/C is a ubiquitin ligase that can transfer one or more ubiquitin molecules to proteins that need to be degraded via the proteasome.

Question 10

What activates APC/C

Answer 10

1. In mitosis it is activated by its association with the Cdc20 subunit.
2. The Cdc20 subunit recognizes and binds to a site within APC/C.

Question 11

How does APC/C target M and S- cyclins for destruction

Answer 11

1. In conjunction with E1 and E2, APC/C then orchestrates the addition of ubiquitin molecules onto the cyclin of the M-Cdk complex (and the cyclin of the S-Cdk complex), targeting them for degradation.
2. This results in a drastic decrease in levels of active M-Cdk.
3. A negative feedback loop is established whereby active APC/C causes its own downregulation; this is because active APC/C levels are decreased due to the decrease in active M-Cdk levels (active M-Cdk phosphorylates inactive APC/C).
4. Although APC/C becomes activated at the start of mitosis, it can only cause degradation of S-cyclin at this stage.
5. Later, at the metaphase-to-anaphase transition the M-cyclins are targeted for degradation.

Question 12

What happens in telophase and cytokinesis

Answer 12

1. An actin-myosin ring drives cytokinesis
   2 Begins in anaphase, completes in telophase
2. As the sister chromatids separate in anaphase a contractile ring forms below the plasma membrane
3. This ring contracts slowly and a new membrane is inserted in the gap between the future daughter cells.

Question 13

What happens after completion of the cell cycle

Answer 13

1. After completion of the cell cycle most cells re-enter G1

2. When embryonic cell divide they do not enter G1, they go straight into another round of division.

Question 14

What does re-entry into G1 depend on

Answer 14

1. Re-entry into G1 depends on APC/C activation by Cdh1

Question 15

Describe how re-entry into the G1 is controlled

Answer 15

1. Firstly, APC/Ccdc20 can target M-cyclins for degradation (resulting in reduced M-cdk activity) and it can also target Securin for degradation which results in the cell completing mitosis (by entering anaphase) and thereby moving to a G1 phase.
2. M-Cdk inhibits the formation of a Cdh1-APC/C complex by phosphorylating Cdh1.
3. Following the destruction (via ubiquitylation) of M-cyclin, the Cdh1 molecule is dephosphorylated and is able to bind to APC/C causing it to become active.
4. APC/C can also act in its APC/CCdh1 form by restricting S-phase entry.
5. It can do this by targeting for degradation targets such as Aurora A kinases, Aurora B kinases and the polo-like kinases (kinases which are required for mitosis to take place) and geminin (a protein which usually keeps Cdt1 inactive).
6. When Cdt1 (not to be confused with Cdh1) is active in G1 it contributes to the assembly of new pre-replicative complexes at DNA sites in preparation for S-phase.

Question 16

How does APC swap from being bound to cdc20 to Cdh1

Answer 16

1. APC/C needs to be phosphorylated by M-Cdk in order for enhanced cdc20 binding to occur (and therefore the APC-cdc20 complex to be active).
2. When m-cyclin is degraded (resulting in a decrease in active M-Cdk), APC/C phosphorylation is dramatically decreased and cdc20 dissociates.
3. M-Cdk inhibits the formation of a Cdh1-APC/C complex by phosphorylating Cdh1.
4. Upon decreased levels of active M-Cdk, the Cdh1 molecule is dephosphorylated.
5. However, Cdh1 doesn’t require APC/C to be phosphorylated in order to bind to APC/C and thereby form an active APC-Cdh1 complex.
6. The targets of APC-cdc20 and APC-Chd1 are different and this is why there needs to be two activating subunits.

Question 17

What does p27 do

Answer 17

1. p27 contributes to G1 stability
2. Acts in G1 to block G1/S-Cdk and S-Cdk activity
3. maintains G1
4. prevents cell cycle progression
5. levels elevated in mitogen-starved cells
6. p27 is a Cyclin-dependent kinase inhibitor (CKI)

Question 18

How is p27 inhibited

Answer 18

1. Internal state and external environment need to be appropriate for p27 to be inactive
2. Late G1- mitogens received by cell
3. If continued to be received then there are elevated levels of G1-CDK
4. G1-cdk Sequesters p27 from G1/S-Cdk
5. Once G1/S-cdk is active it inhibits p27 by phosphorylating
6. Involved downregulation by ubiqutitation
7. Need G1/s kinase to phosphorylate p27
8. Phosphorylation allows ubiquitin molecule to be added to p27- targeting for degradation- added by SCF

Question 19

What is SCF

Answer 19

1. E3 ubiquitin ligase- SCF complex
2. SCF activity is dependent on substrate-binding units called F-box proteins.
3. SCF activity is constant during the cell cycle.
4. Ubiquitylation by SCF is controlled by changes in the phosphorylation state of its target proteins, as F-box proteins only recognize specifically phosphorylated proteins.

Question 20

What protein family contribute to the cell entering the G0 state

Answer 20

1. INK4 family proteins (p16/p15/p18/p19) contribute to the cell entering the G0 state.

Question 21

How is entry into G0 controlled

Answer 21

1. In the absence of mitogens, INK4 proteins act as CKIs (Cyclin-dependent kinase inhibitors) which can block the function of G1-Cdk.
2. Without active G1-Cdk, levels of Rb are kept high and the cell enters a resting phase.
3. In the absence of mitogen signalling, G1-Cdk is prevented from entering the nucleus. It is in the nucleus that G1-Cdk phosphorylates the majority of its targets – a requirement for cell cycle progression.
4. In the presence of mitogens, Myc (the immediate-early gene transcribed following mitogen stimulation) is able to indirectly lower the expression of INK4 proteins resulting in G1-Cdk becoming active.
5. In the absence of a mitogenic signal to proliferate, Cyclin-dependent kinases are inhibited in G1 and cell cycle progression is blocked.
6. In some cases, cells partly disassemble their cell-cycle control system and withdraw from the cell cycle into a specialized non-dividing/resting state termed G0.
7. The molecular basis and reversibility of the G0 state varies in different cell types.