Lecture 11-13 - Cell cycle I-III

What are the requirements for a cell to grow?

• right temperature

• right environment

• food

What are the state of most adult cells and why?

• most cells in our body are in a differentiated state and don’t have the signal to proliferate or grow but rather perform a specific function (ie.a liver cell for detoxification)

• growth and division must be controlled

What are the two things that must be duplicated?

cell content must be duplicated so that the cell can grow bigger and DNA must also be duplicated

• two processes can be coupled or uncoupled

ie. nerve cells stop dividing then there is a growth factor which tells them to differentiate into a large cell (still one cell, but looks like a thousand)

Why is yeast a good model to study?

its a simple organism → its only one cell

yeast behaves like a cancer

What are the two phases of the cell cycle?

M phase and Interphase

• cells spend much more time in interphase (you cant really see anything here → its where DNA is replicated and where cell growth occurs

• M phase is much shorter → lasts around an hour

What are the 5 stages of mitosis?

• prophase

• pro-metaphase

• metaphase

• anaphase

• telophase

• cell division (cytokinesis)

What phase is mitosis apart of?

M phase

What are the 4 phases of the cell cycle?

interphase consists of 3 phases:

• S phase → DNA replication

• G1 → gap between M and S → checks outside environment → if theres enough food, correct temperature etc.

• G2 → the gap between S and M → a checkpoint where the cell makes sure that you have all the chromosomes and the exact amount of DNA required

M phase → includes mitosis and cytokinesis

What happens if temperature and conditions are not optimal?

the cells arrest in G1 and wait

What is G0?

exit from the cycle → cells that are stuck in G1 realize that there are no signals to grow and proliferate, they move here and differentiate to have a certain job in a certain organ

What is the difference in the cell cycle between fission yeast and budding yeast?

budding yeast does not have a proper G2 phase resulting in a smaller than larger cell but both have the same basic machinery

How were cell cycle genes identified?

Yeast divide at a low temperature → if you put them in a higher temperature, they stop growing and proliferating because the temperature change cases certain proteins to misfold and lose function

• this was used to find the different genes important in different parts of the cell cycle

• when you increase temp → cells get stuck dividing → take those cells (only seen in high temperature) → find out which genes are responsible for the arrest

• take the yeast with the problem → introduce wild type DNA so each cell gets a wild-type copy → see which ones overcome arrest → take those, isolate the DNA → identify gene

• cdc genes in yeast (what they are called)

How does cell proliferation and growth work in frogs (reptiles)?

• you have the oocyte which grows without dividing in the mother (100 times the size of the normal cell)

• DNA does not grow but contents increase 1000 fold

• when its mature egg → they are laid → fertilized by males → cells divide rapidly (one division cycle occurring every 30 minutes → embryonic cycle = fast)

  • no G1 or G2 → only through S and M

• no growth involved after fertilization so the tadpole is as small as the egg (make organism quickly)

• tadpole then eats and gets bigger becoming a frog

What happens if you isolate from eggs? what is seen?

• destroy the membrane to get cytoplasm which contains all the factors for S and M phase

• get uncle from frog sperm

• get ATP

• put this all in a test tube

• cell free mitotic cycles occur every 40-60 minutes

• study in test tube

how can you identify the phases of the rat fibroblasts?

interphase → spindle shape

mitosis or cytokinesis → round shape

How can we visualize cell divines in tissues?

left picture:

• see that uncles is black because radioactive nucleotide is injected (3H-thymidine)

• incorporated into DNA when cell divides

• put film on it and you ca detect the radioactivity

right picture:

• non radioactive way

• red-nuclei incorporate fluorescent nucleotide (brdU)

• incorporated into DNA when cell divides

• get antibody made against drbU so that when it recognizes the modified nucleotide, it has a florescent maker, and you can identify the cells where the antibody has attached (cells in S phase)

What is flow cytometry?

What people use now days to study the cell cycle → more precise because you can study all the phases and which are dead or dying

• you have a fluorescent dye once it binds to DNA

• machine measures fluorescence of single cell and gives it a number

• laser that activates florescence and a detector that measures it

• plotted on graph

most cells are in G1 → happy cell

peak below 1 are dying cells

0 when dead

Can the cell cycle move backwards?

NO, only forwards

Where are checkpoints located?

• entering M

• exiting M

• entering S

*there are more than this, these are just examples

Are checkpoints essential for the cell cycle?

they are not essential

negative signals that block something

if the cell did not have checkpoints it could still go through the cell cycle like in embryonic development

but they are useful because it allows you to make sure the cell is okay to go to the next step and reduce mistakes and prevent disease

when the checkpoints are mutated or deleted → cells can proliferate uncontrollably → you can add a factor to get it to stop but it won’t respond because theres no checkpoint

How does the cell know when its going into each phase?

it is directed by a protein complex comprised of cyclin and Cdk → the phosphorylation of these substrates activates them and then they can promote the next phase

Without cyclin Cdk is inactive

Each phase of the cell cycle has its win cyclin the drives the activity of the Cdk

what are the different types of cyclin?

• cyclin G1 → starts accumulating at G1 and gets degraded at the beginning of S

• Cyclin S → starts accumulating at the end of G1 and gets degraded at the end of anaphase, when metaphase beings

• Cyclin M → Starts accumulating at the beginning of G2 and peaks at M → degraded after metaphase-anaphase transition

*cyclin is very active in the middle of G1

*in the middle of mitosis it all goes back down to 0

How can one kinase do all these phases?

how does it become so specific? → when S cyclin binds to Cdk, it directs the kinase to the substrate → it can only find S substrates when bound to S cyclin

so if it was bound to G1 cyclin it cannot find S cyclin substrates

what cyclin is associated with which Cyclin-Cdk complex?

G1-Cdk → cyclin D and Cdk4 and Cdk6

G1/S-Cdk → cyclin E and Cdk2

S-Cdk → cyclin A and Cdk 2

M-Cdk → cyclin B and Cdk 1

What are the 3 phases of activation of Cdk?

1. Inactive state → no cyclin is bound and the active site it blocked by a region of the protein called the T-loop

2. Partially active state → cyclin binds causing the T-loop to move out of the active site

3. Active state → the phosphorylation of Cdk. By CAK at a threonine residue in the T-loop further activates the enzyme by changing the shape of the T-loop to improve the ability of the enzyme to bind its protein substrates → can phosphorylate substrates

What is the role of Wee1? and Cdc25?

Wee1 can put an inhibitory phosphate on the Cdk causing it to become inactive

Cdc25 can undo this addition and reactivate the Cdk

What is the role of a CKI p27?

it is a Cdk inhibitor

P27 binds to cyclin and Cdk → squeezes it and causes it to become inactive (distorts active site of Cdk)

how do the cells get rid of p27 inhibition?

1. the phosphorylation of the p27 allows it to be recognized by SCF, which is always active

2. With the help of two Ubiquitination enzymes E1 and E2, SCF serves as a ubiquitin ligase (E3) that transfers multiple ubiquitin ligases onto p27

3. marked for degradation

What is another ubiquitin ligase that controls the degradation of M-cyclin?

1. Inactive APC binds to always active Cdc20 → creating active APC

2. With the help of two Ubiquitination enzymes E1 and E2, APC serves as a ubiquitin ligase (E3) that transfers multiple ubiquitin ligases onto M-cyclin

3. Marked for degradation

describe the results of the 3 cell fusion experiments that were carried out?

1. S and G1 → G1 phase nucleus entered S phase and the one on S phase continued DNA replication indications that S-phase nucleus has factors that can drive the the G1 into DNA synthesis

2. S and G2 → G2 having already replicated its DNA is refractory to the factors carried by the nucleus of S

3. G1 and G2 → fusion of G1 and G2 does not drive the G1 nucleus into DNA synthesis

These indicate that the cytoplasmic factors for DNA replication that were present in S phase disappear when the cell moves from S to G2

Why is it that DNA is only replicated once in every cell cycle?

there is a stop signal that doesn’t allow re-replication

1. ORC binds to specific part of the DNA (inactive)

2. in G1, Cdc6 is produced which sits on ORC

3. Mcm, which are DNA helicase that unwinding the DNA, along with the other stuff already on the DNA, form the pre-replication complex

4. Mcm are activated in G1, but they don’t do anything until S-Cdk is there → starts S phase

5. s phase starts when Cdc6 is phosphorylated by S-Cdk → Cdc6 is degraded

6. ORC is phosphorylated

7. Mcm proteins begin unwinding DNA

8. DNA is replicated → ORC is still bound and still phosphorylated

9. Due to phosphorylation ORC cannot start the process again → barrier for re-replication

*if Cdk is not present it keeps the cell in G1 until the cell gives a signal

How is M-Cdk activated for mitosis?

1. M-cyclin binds to Cdk 1 → still inactive

2. CAK adds an activating phosphate but wee1 adds inhibitory phosphate

3. Phosphotase Cdc25 removes inactivating phosphate from the inactive M-Cdk complex

4. Active M-Cdk complex formed

Positive feedback loop of M-Cdk phosphorylating Cdc25

That feedback is enhanced by ability of M-Cdk to inhibit wee1

What were the two experiments that showed that protein must be degraded to exit from mitosis?

1. if you inhibit APC ubiquitin ligase which we know is important for M-cyclin degradation after mitosis → metaphase is arrested → kinetochores are not able to separate the chromatids (there is a protein glue that holds them together → APC must target a protein that is responsible for holding the chromatids together

2. in another case there was a non-degradable M-cyclin which is usually degraded by APC complex → now mutated so it cant be degraded by APC → causes arrest in anaphase → M-Cyclin must be degraded for anaphase and cytokinesis to occur

What is the glue that holds chromatids together?

condensin compacts chromatin into chromosomes

cohesin which binds the two sister chromatids and holds them together

• has an ATP binding site

• hinge

• antiparallel coil

How is sister chromatid separation by APC regulated?

1. MCDK activates CDC20

2. CDC20 activates APC

3. active APC removes cohesin molecules → there is a protease called separatease (which is held in an inactive state by an inhibitor called securin

4. securin is degraded by APC

5. separatease can then destroy cohesins and separate chromatids

What happens if a chromosome doesn’t align properly?

there is a checkpoint involving the protein called Mad2

• Mad2 binds to the unattached chromosome and tells APC not to destroy the glue

• otherwise the cell may have too many chromosomes or not enough chromosomes

How is M-Cyclin activity kept low?

two types of cells:

• embryonic (no G1 or G2)

  • cells in M phase have high M-Cyclin levels which eventually induces CDC20 → activates APC and that leads to the degradation of M-Cyclin

• cells that have G1 and G2

  • there is a second HCT1 protein that replaces CDC20 keeping APC active to M-Cyclin levels are low in G1 (happens from M to G1)

How are cells reactivated to go into another round of cell division?

• as cells exit from mitosis ad inactivate M-cyclin it results in the increase of two inhibitors of M-Cdk

  • Sic1 which just binds to M-Cdk and inactivates it

  • Hct1 which binds to APC and activates it → degrades M-Cyclin

• both become active when they are dephosphorylated

• then in G1 all the M-Cyclin activity goes down

• from there cells can then decide if they want to stay in G1 or go into G0

to get new cells to proliferate, G1 cyclin is required and is independent of the two inhibitors

• G1-Cdk doesn’t get targeted

• if cell is singled to get out of G1 and go into another round of cell cycle its initiated by G1-Cdk → move to S by activating S-Cdk → G1/S Cdc can phosphorylate the two inhibitors and inactivate them again to that M-Cyclin can be reactivated instead of degraded

What is the mechanism to controlling S phase initiation?

1. G1-Cdk complex initiates Rb phosphorylation

2. that causes Rb to dissociate from E2F protein (a transcription factor specific to S phase gene transcription)

3. once E2F is active it transcribes lots of genes and proteins required for cells to go into S phase

4. G1-S and S-Cyclin is increased

5. we get active S-Cdk

*3 positive feedback loops

How is cell size controlled in yeast?

• cell growth is when the cell just gets bigger → number of proteins and other contents increase BUT the DNA stays the same

• cells without nutrition:

  • the growth part slows down because theres not enough energy to grow → cells become smaller with each cell division eventually the cell becomes a tumour or is no longer viable

• cells with nutrition:

  • if the cells are initially provided with nutrition but they have time to make all the required contents needed, then once nutrition is reduced it will just take longer of the cell to reach the same size

How is sensing of cell growth regulated? when is it big enough to go into S phase?

• there are DNA binding proteins that bind to the DNA and they sit there

• they are also G1-cyclin binding proteins → G1-cyclin is produced while the cell is growing

• as the cell reaches a certain size, ALL the DNA binding proteins are bound by G1-Cyclin

• as free G1 cyclin accumulates as the cell gets even bigger it activates Cdk and activates S phase

How does DNA damage arrest the cell cycle in G1?

• if a cell is damaged this checkpoint decides whether or not the cell moves into S phase

1. DNA damaged by x-rays, UV

2. protein kinase binds to damaged regions → it becomes active and phosphorylates P53 a transcription factor and tumour suppressor (in normal cells P53 is degraded by Mdm2 E3 ligase)

3. P53 accumulates due to phosphorylation

4. induces many genes linked to the CKI protein p21

5. p21 then binds to G1/S-Cdk and inactivates them → can’t go into S-phase until its repaired or if damage is too much then cell will be single for degradation