Cell Division Lecture Sep 23

Question 1

What are the 4 phases of the cell cycle?

Answer 1

G1, S (synthesis), G2, M (mitosis)

Question 2

If a cell determines that it doesn’t want to divide anymore, will it arrest in G1?

Answer 2

No. It will remove itself from the cell cycle entirely by entering G0.

It can reenter the cell cycle from G0 into G1 if necessary.

Question 3

What is interphase?

Answer 3

Interphase includes G1, S, and G2

It’s hard to see what’s going on under a microscope during interphase, but in Gi, the cell is preparing t oreplicat it’s genome. In S phase, the DNA is synthesized. In G2 phase the cell is preparing to divide.

Question 4

What are the stages of mitosis/M-phase?

Answer 4

Prophase

Prometaphase

Metaphase

Anaphase

Telophase

(cytokinesis)

Question 5

WHat happens during prophase?

Answer 5

In prophase, the chromatin begins to condense and the nuclear envelope breaks down.

Question 6

WHat happens in prometaphase?

Answer 6

Microtubules from the centrioles begin to bind to kinetochores on the chromatid pairs. During prometaphase, typically only one side of the kinetochdrome is bound and there’s a sort of tug of war that goes on between the microtubules once both side are bound.

Question 7

What happens in metaphase?

Answer 7

The microubules from the spindle poles have now alligned the sister chromatids right in the middle - along the metaphse plate.

Question 8

What happens in anaphase?

Answer 8

The microtubules pull the sister chromatids apart onces separates breaks down the connections.

Question 9

What happens in telophase?

Answer 9

The daughter cells start to pinch apart and the chromatids beging to uncondensce. THe nuclear envelop beings to reform. The centrioles becomes less active.

Question 10

What are the three models for how microtubules will push and pull chromatids in order to get them aligned in the metaphase plate?

Answer 10

1. Polymerization/depolymerization of the microtubule
2. Associationw ith motor proteins: kinesins and dynesins
3. Interactions with actin and myosin filaments.

Question 11

What major checkpoint occurs during M phase?

What protein mediates this?

Answer 11

the spindle formation chekcpoint

If there is a chromatid that isn’t aligned along the center (in other words, if there is a naked kinetochore), Bub1 will be activated and will inhibit mitotic progression resulting in a metaphase arrest. If the spindles can then appropriately align the chromatics, Bub1 will be inactivated and division will continue

Question 12

How do separase and securin ensure that sister chromatids won’t separate too early?

Answer 12

Separase, as it is named, is the protein that will break down the bonds between the sister chromatids.

However, separase is inhibited thorugh most of hthe cell cycle by securin.

However, between the metaphse-to-anaphase transition, APC will degrade securin thorugh proteolysis, allowing for separase to do it’s job and separate the chromatids.

Once the bonds are broken, the tension placed on the chromatids by the microtubules will cause rapid separation

Question 13

What proteins mediate cytokinesis?

Answer 13

Actin filemtns come together and form a cleavage furrow

This is essentially a contractile ring that pinches tigher and tighter until the membrane ends are joined and the two duaghter cells divide.

Question 14

In order to divide, a cell must first do what?

Answer 14

Grow: it needs to approximately double its size in order to divide, otherwise cells would shrink with each division

Question 15

What point do cells cross in G1 that commites them to division?

Answer 15

The restriction point or start

Question 16

What proteins hold the sister chromatids togehter before separase separates them?

Answer 16

Cohesins hold them together- they are complexes of Scc and Smc proteins - they essentially form a ring with a hinge around the two chromatids.

Question 17

What proteins condense and decatenate the chromosome during mitosis?

Answer 17

Condensins are responsible for condensing the chromatids and decatenating any tangles.

They are made of Smc and Cap proteins.

Question 18

When are is the centriole replicated?

When are the centrosomes moved to the poles to become spindle poles?

Answer 18

The daughter centrioles are made in S phase.

During G2 the two centrioles move to the poles and become spindle poles/centrosomes

Question 19

What general class of proteins drive the cell cycle?

Answer 19

kinases - they phosphorylate things

In particular: the Cdks (cyclin dependent kinases) and the cyclins.

Question 20

WHat is required for a Cdk to be active?

Answer 20

1. It’s cyclin needs to be bound
2. It needs to be phosphorylated in the activation site (and NOT phosphorylated in the inactivation site)

Question 21

What enzyme will phosphorylate Cdk in the activating site, turning it on?

What enzyme will phosphorylate Cdk in the inactivating site, turning it off?

What enzyme will remove the phosphate group in the inactivatine site, turning Cdk back on?

Answer 21

CAK will add the activating phosphate group.

Wee1 will add the inactivating phosphate group.

Cdc25 will remove the inactivating phosphate group.

Question 22

In what form do cells usually hold their Cdk?

Answer 22

Cells will usually maintain their Cdk in the bound Cdk-cyclin form with both the activating and inactivating phosphate groups in place. This means that only one step is required to activate the Cdk quickly–removal of the inactivating phosphate with Cdc25

Question 23

Besides the inactivating phosphate group, what else can bind to the Cdk/cyclin combo to inactivate its kinase activity?

Answer 23

Ckis!

These are the cyclin dependent kinase inhibitors. They will bind to the cyclin to block kinase activity.

Question 24

Besides the binding of the inactivating phosphate group and the Cki, what else can the cell do to turn off the Cdk?

Answer 24

APC will ubiquinate the cyclin, which makes the cyclin for prosteosomal destruction. Without the cyclin, the Cdk is inactive.

Question 25

Why is it important that Cdk activity be low at the beginning of G1?

Answer 25

Generally speaking, all the Cdks are destroyed in the M phase previous oto G1, to Cdk activity is very low.

This is a good thing because there are proteins called Cdc6 and Ctd that need to bind to the origins (ORC) on the DNA. The tricky part is that Cdc6 and Ctd are substrates for inactivation via Cdk activity. Thus, Cdk activity needs to be turned off in order for Cdc6 and Ctd to be active/able to bind to the ORC.

Once you have Cdc6 and Ctd binding, they bind other scaffolding proteins called MCM helicases. THis completes the pre-replication complex.

More proteins will bind on top of that to make the pre-initiation complex. THe pre-initiation complex is then activated by Cdk-cyclin activity. Once this is phosphorylated, all the proteins but the proteins that denote the ORC itself (so Cdc6, Ctd, and others) will fall off and DNA replication can begin.

Question 26

What two proteins bind to the ORC first?

Answer 26

Cdc6 and Ctd.

Question 27

What creates the re-replication block after S phase?

Answer 27

A cell would never re-replicate its genome after S phase because the proteins required to form the pre-replication complex, Cdc6 and Ctd will be inactivated by Cdk/cyclin until the end of M phase when Cdk/cyclin activity is low.

Question 28

How do growth factors promote cell division and proliferation?

Answer 28

The growth factor will bind it’s receptor, which is an enzyme-linked receptor.

The enzyme receptor will phosphorylate and activate a small G-protein called Ras.

Ras-GTP will activatate MAP kinase (through a cascade of MAP kinases MAPKKK, MAPKK, etc).

Phosphorylated MAPK will go to the nucleus and phosphorylate transcription factors that induces them to express the gene for C-myc. C-myc is the immedate/early gene product.

C-myc is a very powerful transcription factor that will be brought back intot he nucleus after translation and will activate genes for the delayed response genes, which includes the D cyclins - the cyclins in G1.

The Gi cyclin will activate the G1 Cdk

Question 29

What will an activated G1 Cdk/cyclin combo do?

Answer 29

It phosphorylates the retinal blastoma (Rb) protein that’s holding the transcrition factor E2F in an inactive form

Phosphorylated Rb releases the E2F transcription factor

E2F will therefore go to the nucleus to initiate the expression of genes for replication proteins, including the G1,S cyclins and the S cyclin!

These cyclins will do the phosphorylating/activating required for DNA synthesis.

Question 30

How is Cdc25 activated?

Answer 30

Through pospitive feedback by the activated Cdk/cyclin complex. It will activate Cdc25 to make more activated Cdk/cyclin.

It will also inhibit Wee1.

Question 31

What do the active Cdk/cyclin pairs in M phase activate through positive feedback. What does it inhibit through negative feedback?

Answer 31

THe Cdk/cyclin in the active form will activate Cdc25 through positive feedbak, promoting th eformation of more active Cdk/cyclin

It inhibits Wee1 through the negatibe feedback loop, shutting off the inactiavtion via phosphorylation

Question 32

What 5 things does active M Cdk/cyclin do in order to promote cell division?

Answer 32

1. phosphorylates histone complexes to promote chromatin condensation that marks prophase
2. activates the centrosomal proteins to initiate spindle formation.
3. It phosphorylates lamins, promoting the breakdown of the nuclear envelope
4. It phosphorylates the membrane proteins of the ER and golgi so that they will fragment appropriately in order to provide these organelles for the daughter cells.
5. It activates the anaphase promoting complex (APC) which will ubiquinate the cyclin, thus turning off the M Cdk activity. So in addition to promotion cell division, it also shuts itself off!!

Question 33

Explain the steps of the G1 DNA damage checkpoint.

Answer 33

1. DNA damage is sense by ATM and ATR, which bind to the damaged strand.
2. Bound ATM and ATR have kinase activity and phosphorylate the checkpoint kinases: ChK1 and ChK2

Side note: p53 is constitutively expressed, by in times of no DNA damage, it is bound to a protein called MDH2, which marks it for desctruction in the proteosome.

3. ChK1 and ChK2 phosphorylate p53, making MDH2 release.
4. The phosphorylated and free p53 will dramatically increase in concentration in response to DNA damage.
5. the p53 will move to the nucleus where it will bind DNA and increase expression of the CKis and p21 and p27.
6. The Ckis and p21 and p27 will bind to the cyclin and inactivate the S Cdk/cyclin pair.
7. This will block DNA replication

Question 34

In the G1 DNA damage checkpoint, which proteins recognized the DNA damage?

Answer 34

ATM and ATR

Question 35

In the G1 checkpoint, what proteins do ATM and ATR activate?

Answer 35

The checkpoint kinases

ChK1 and ChK2

Question 36

What is the fate of p53 in a cell that doesn’t have damaged DNA?

Answer 36

It will be expressed constnatly, but bound to MDH2, which tags it for destruction in the proteosome.

Question 37

What must happen for p53 levels to build up in response to DNA damage?

How does p53 deal with DNA damage?

Answer 37

ChK1 and CdK2 must phosphorylate the p53 to make MDH2 let go.

free p53 will go to the nucleus and increase expression of CKIs that will inactivate the S Cdks, thus turning off DNA replication

Question 38

The G2 DNA damage checkpoint includes the p53 pathway, plus a more direct route of shutting down division. What is it?

Answer 38

In the G2 DNA damage checkpoint, bound ATMP will directly inhibit Cdc25, which means you won’t get the activation of the M Cdk/cyclin and the cell doesn’t divide.

Question 39

What is the mechanism of the spindle assembly checkpoint?

Answer 39

If a chromatid isn’t aligned appropriately, then there will be a free kinetochore where the microtubule was supposed to bind and didn’t

This kinetochore will activate the protein BubR1.

BubR1 inhibits APC, so you don’t get the ubiquination of the cyclins and you don’t get entrance from metaphase to anaphase and the cells don’t divide.