Lecture 17 - Spindle Assembly Checkpoint Cytokinesis

Question 1

Treatment of what drugs leads to the mitotic arrest of cells?

Answer 1

Colchicine or vinblastine (microtubule destabilizing drugs) or with taxol (microtubule stabilizing drug)
- Cdk activity is high, cyclin B and securin are stable

Question 2

What mutants that were screened for overcome the mitotic arrest?

Answer 2

Spindle assembly checkpoint genes - Mad2 and others

Question 3

What do Mad2 mutants do?

Answer 3

Enter anaphase prematurely, before all kinetochores have been attached to spindles

Question 4

How do Mad2/APC double mutants behave?

Answer 4

Like APC mutants
- Arrest at metaphase

Question 5

Where do Mad2 and SAC function genetically?

Answer 5

Upstream of the APC

Question 6

When does the inactive APC become active?

Answer 6

When all kinetochores have made stable connections to spindle microtubules from both poles

Question 7

Where is the Spindle Assembly Checkpoint (SAC), a complex of proteins, active?

Answer 7

At unoccupied kinetochores (that are not attached to spindle microtubules)

Question 8

What SAC component is activated at these unoccupied kinetochores?

Answer 8

Mad2

Question 9

What does Mad2 do?

Answer 9

• Diffuses away and binds to Cdc20
• Inhibits the activity of the APC/Cdc20 complex, preventing cyclin B and securin destruction

Question 10

What inactivates the SAC?

Answer 10

Kinetochore/spindle attachment leading to APC activation

Question 11

How does Mad2 inhibit APC/Cdc20?

Answer 11

1) Mad1 is recruited to unattached kinetochores
2) Mad2 binds to Mad1, and Mad2 changes conformation and becomes active and releases
3) Activated Mad2 diffuses from the kinetochores and binds and inhibits APC/Cdc20

Question 12

In prometaphase, how are chromosomes attached?

Answer 12

Some are properly attached while others are still unattached or have monotelic attachments

Question 13

In metaphase, how are the chromosomes attached?

Answer 13

Properly, leading to the inactivation of the SAC, leading to APC/C activation

Question 14

In anaphase, what does the APC/C cause?

Answer 14

• Destruction of cyclin B —> Inactivation of Cdk1 —> changes in spindle behaviour that allow completion of anaphase
• Destruction of securin —> loss of sister chromatid cohesion

Question 15

What happens in anaphase A?

Answer 15

• Loss of cohesion
• Shortening of kinetochore microtubules
• Movement of daughter chromosomes to poles
• Forces generated mainly at kinetochores (pulling forces partially separates sister chromatids)

Question 16

What happens in anaphase B?

Answer 16

1) A sliding force is generated between interpolar microtubules from opposite poles to push the poles apart; the interpolar microtubules also elongate
2) A pulling force acts directly on the poles to move them apart (further separates sister chromatids)

Question 17

Where does microtubule growth occur?

Answer 17

At the plus end of polar microtubules

Question 18

What initiates anapahase?

Answer 18

APC/C mediated destruction of securin (anaphase A) and CycB (anaphase B)

Question 19

What filaments are involved in the cytokinesis contractile ring?

Answer 19

Actin and myosin

Question 20

What is essential for the assembly of the actin/myosin contractile ring?

Answer 20

RhoA

Question 21

Where is RhoA activated?

Answer 21

Near central spindle (interpolar microtubules)

Question 22

What does active RhoA do?

Answer 22

• Associates with cell membrane near the central spindle
• Stimulates myosin II activation and actin filament formation at that site

Question 23

What experiment was done to demonstrate that RhoA was necessary for cytokinesis?

Answer 23

Latrunculin A
- Depolymerization of actin –> RhoA accumulation is not affected by MHC is
- RNAi knockdown of MHC and RhoA still accumulates

Question 24

What was done to demonstrate that ECT2 was necessary for contractile ring accumulation of actin and myosin?

Answer 24

ECT2 RNAi cells fail to undergo cytokinesis
- ECT2 localizes to the central spindle (spindle midzone)
- ECT2 is necessary for RhoA accumulation at the contractile ring

Question 25

What was ECT2 previously found to interact with?

Answer 25

A spindle midzone protein, CYK-4

Question 26

What is CYK-4 necessary for?

Answer 26

• Cytokinesis
• Localization of MHC, actin, ECT2, and RhoA

Question 27

What is the model for mitotic spindle-dependent contractile ring assembly?

Answer 27

1) CYK-4 associates with the mitotic central spindle
2) CYK-4 recruits ECT2
3) CYK-4 interaction activates RhoAGEF activity of ECT2
4) RhoAGDP exchange for GTP activates RhoA
5) RhoA recruits actin/myosin

Question 28

How is RhoA activation regulated so it’s active only after mitosis?

Answer 28

a) Cells synchronized in metaphase at time 0
- 0-40: Metaphase; 60-120: Anaphase, G1
- ECT2 immunoprecipitated CYK-4 in anaphase
- Phosphorylation of ECT2 (pTP) correlates with the inability to bind
b) Metaphase arrest cells
- Add Roscovitine to inhibit Cdk1 activity and allows CYK-4 to bind ETC2
*Therefore, Cdk1 phosphorylation of ECT2 prevents recruitment of ECT2 to the spindle midzone and anaphase cyclin destruction permits ETC2 recruitment

Question 29

What is the model for RhoA activation at the contractile ring?

Answer 29

1) CYK-4 associates with the mitotic central spindle
- During mitosis, CYK-4 is unable to recruit ECT2 because of Cdk1 phosphorylation of a site on ECT2
2) APC inactivation of Cdk1 results in dephosphorylation of ECT2 allowing it to interact with CYK-4 at the central spindle
3) RhoGEF activity of ECT2 on the central spindle leads to RhoA activation on the overlying cell membrane
4) RhoAGDP exchange for GTP activates RhoA
5) RhoA recruits actin/myosin