Lecture 4 - Cell Cycle Control

Question 1

characteristics of cell cycle control:

Answer 1

• each event turns off at a specific time
• events are initiated in the right order
• each event is triggered only once per cycle
• irreversible
• robust (backup mechanisms)
• adaptable

Question 2

4 phases of cell cycle:

Answer 2

M - Mitosis
G1 - Gap1
S - Synthesis
G2 - Gap 2

Question 3

studies of what two single cells eukaryotes have been crucial in understanding cell cycle control:

Answer 3

budding yeast (S.cerevisiae) & fission yeast (S.pombe)

Question 4

how were genes involved in the cell cycle by mutagenesis identified?

Answer 4

colony of cells started by one cell were plated and grown at 23C, then the colonies replicated onto two identical plates and were incubated at 2 temperatures (35 & 23 C) and the cells couldn’t divide in the warmer temperature and this allowed for the cdc & wee mutants to be identified

Question 5

master cell cycle regulator:

Answer 5

cdc2 gene is the master gene of cell cycle regulation - being required in G2, M, G2, S

Question 6

what are cdc and wee?

Answer 6

they are mutants of the master cell cycle regulator gene - cdc2

Question 7

what is cdc2?

Answer 7

it is the master controlling gene of the cell cycle crucial in nearly all stages of the cycle

Question 8

when are cycling’s made and destroyed?

Answer 8

cycling’s are made and destroyed each cell cycle

Question 9

what do mitotic nuclei have which effects all interphase nuclei?

Answer 9

mitosis promoting factors (MPF)

Question 10

when was cdc2 identified?

Answer 10

cdc2 was identified in a mutant screen in fission yeast as required for entry into mitosis

Question 11

what were cyclins identified as?

Answer 11

cyclins were identified as proteins that varied in a cyclical fashion during the cell cycle. Expressed and destroyed each cell cycle.

Question 12

what is mitosis-promoting factor?

Answer 12

mitosis-promoting factor (MPF) is a cell cycle control element able to cause metaphase when injected into amphibian oocytes or when incubated with nuclei in a cell-free system

Question 13

what type of protein is cdc2?

Answer 13

cdc2 is a cyclin-dependent protein kinase (CDK)

Question 14

MPF is a complex between _________, MPF therefore being a _________:

Answer 14

1) a 34kD serine/threonine protein kinase, identified as a Xenopus homolog of the cdc2 gene product, p34cdc2, and a 45kD substrate, identified as a Xenopus B-type cyclin.

2) mitotic CDK cyclin complex

Question 15

MPF controls entry into mitosis by phosphorylation of key targets:

Answer 15

1.compaction of chromosomes (condensin loading)

2.assembly of the mitotic spindle

3.nuclear envelope breakdown (Lamins and nuclear pore proteins)

4.activation of the APC (anaphase promoting complex) – targets proteins for destruction
•Cohesion between sister chromatids must be removed: must degrade “glue“ for metaphase to anaphase transition
•Degradation of cyclin is essential to exit mitosis and keep cell cycle moving forward.

Question 16

what does the difference between cyclin concentration of mitotic CDK activity show?

Answer 16

the difference between cyclin concentration of Mitotic CDK activity shows there are more levels of control than just cyclin concentration

Question 17

Cdk regulation ensures ordered progression through the cell cycle:

Answer 17

Inhibitory phosphorylation by Wee1 keeps M-CDK inactive until a critical threshold of cyclin-CDK is passed. Then the rapid removal of phosphorylation by Cdc25 promotes a “switch-like” change

Question 18

APC promotes the destruction of cyclin and mitotic exit:

Answer 18

When M-CDK activity has successfully brought about the cell state required for cell division it helps trigger its own destruction by promoting the activation the APC which ubiquitylates cyclins leading to their destruction

Question 19

licensing:

Answer 19

loading of the inactive core of the replication machinery, needs low S-CDK

S-CDK activates replication machinery initiating DNA replication

Question 20

what controls progression through mitosis?

Answer 20

MPF/Mitotic CDK controls progression through mitosis.

Question 21

progression from G1 → S:

Answer 21

major commitment point

Cells exit cell cycle in G1

Most cells in body in G0, post mitotic, specialised
Have to be stimulated to re-enter cell cycle

Cancer cells – cells growing in wrong place at wrong time

Question 22

different cyclin-CDK complexes have different:

Answer 22

target specificities

Question 23

one of the most important events during G1/S:

Answer 23

the transition between preparing for DNA replication in G1 and performing DNA replication in S – making sure the cell does not try to re-replicate already replicated DNA

Question 24

how do cells only go through the S phase once per cycle?

Answer 24

cells are ‘licensed’ to go through S phase once in a cell cycle. – Licencing of unreplicated DNA occurs in G1 and is inhibited in S phase when the already licenced DNA is replicated

Question 25

cell cycle is a CDK cycle:

Answer 25

1) [G1] low CDK activity, loading of licensing factor at origins

2) [G1] S-cyclin increase

3) [Enter S] S-CDK activates replication

4) [S-CDK activity high - no re-licensing]

5) [G2] M-Cyclin increases

6) [Enter M] M-CDK activated triggers mitosis

7) [Exit M] after spindle checkpoint is satisfied, APC is activated and cyclins are destroyed - re-setting the cycle

Question 26

checkpoints:

Answer 26

cells delay cell cycle progression in response to external signals, DNA damage etc

Question 27

major transition points or checkpoints in interphase:

Answer 27

1) start cells are licensed in G1 to enter S phase

2) G2 → M transition

Question 28

cell cycle proceeds through cell in one direction which is controlled by:

Answer 28

cyclin-dependent kinases, Cdks (Cdc2 = CDK1)

Question 29

Cdks form a complex with cyclins which:

Answer 29

are periodically expressed and destroyed

Question 30

the CDK/cyclin complex is kept inactive by:

Answer 30

Wee1 until it is activated by Cdc25

Question 31

what does spindle checkpoints monitor?

Answer 31

whether cells assemble their chromosomes and spindles correctly by checking occupancy of kinetochores and tension - ensuring all chromosomes are lined up of metaphase plate and attached to microtubules to both poles before anaphase is triggered

Question 32

Mad2 proteins:

Answer 32

MAD proteins at unattached kinetochores inhibits separation, MAD proteins at unattached kinetochores prevent activation of APC

Question 33

summary:

Answer 33

•Cells progress through the cell cycle in one direction

•Progression through the cell cycle is controlled by CDK kinase complexes

•Cyclins increase in concentration through the cell cycle and interact and regulate CDK activity.

•CDK-cyclin activity is fine tuned by other regulators

•Cyclin destruction at the end of mitosis resets the cell cycle

•Progression through the cell cycle can be paused by the action of cell cycle checkpoints which ensure that essential events are completed before the next stage of the cell cycle begins.