Mitosis Flashcards
What are the phases of the cell cycle?
-
Interphase
- G1 phase
- S phase (DNA replication)
- G2 phase
-
Mitosis
- Mitosis (nuclear division)
- Cytokinesis (cytoplasmic division)
What controls the cell cycle?
The cell cycle is critically controlled by cyclin dependent kinases.
Describe CDK.
- CdK has 2 units.
- Cyclin + cyclin-dependent kinase.
- Cyclin does not actively participate in the cataylic reaction but it helps the enzyme.
Describe the activity of CDK during the cell cycle.
- Activity is cyclical
Describe the regulation by phosphorylation / dephosphorylation of CDK.
- Cyclin binds to CDK and it becomes an inactive cyclin-CDK complex.
- Protein kinases cause the CDK to bind an inhibitory phosphate and an activating phosphate.
- In this form it is still an inactive cyclin-CDK complex.
- An activating protein phosphatase changes this to an active cyclin-CDK complex.
Where do the 2 distinct cyclin:CDK complex control events occur in cell division?
- Active S-Cdk between G1 phase and S phase.
- Active M-Cdk between G2 phase and M phase.
- Mitotic cyclins accumulate late in the cell cycle and are active for a short period during mitotic division.
- Other cyclin:Cdk activity much earlier in the cell cycle - S-Cdk.
What is the main cyclin in the early cell cycle (G1 phase)?
What does it interact with?
- Cyclin D
- Interacts with either Cdk4 or Cdk6
What is the main cyclin during late G1 and early S phase?
What does it interact with?
- Cyclin E
- Interacts with Cdk2
Cdk2 can bind to 2 different cyclins during G1 - S phase.
What are these 2 cyclins?
- Cyclin E
- Cyclin A
What is the main cyclin during S-G2 phase?
What does it interact with?
- Cyclin A
- Interacts with either Cdk1 or Cdk2.
What is the main cyclinduring mitotic division?
What does it interact with?
- Cyclin B
- Interacts with Cdk1
How do different cyclins become destroyed?
- Cyclins become destroyed by the addition of ubiquitin, which is a small protein (76 amino acid) onto the cyclin subunit.
- It DOES NOT attach to the cyclin-dependent kinase.
- When cyclin is targeted by ubiquitin it is targeted to the proteasome and destroyed.
- The enzyme cannot work without the cyclin co-factor.
- The destruction of the cyclin conributes to the inactivation of the cyclin-dependent kinase.
Describe ubiquitination.
- 3-step process
- Initially, ubiquitin is attached to a ubiquitin-activating enzyme (E1).
- There are just 2 E1 enzymes.
- The attachment of ubiquitin is consuming energy, so there is a high energy bond between ubiquitin and E1.
- The ubiquitin is then transferred onto E2.
- There are ~35 E2 enzymes. They are known as ubiquitin-conjugating enzymes.
- Ubiquitin has many different substrates, so the Cdk:cyclin breakdown reaction has to be specific.
- The specificity comes from the E3 enzymes. These are known as ubiquitin ligases.
- These facilitate the transfer of the activated ubiquitin onto the substrate.
- Often, ubiquitination is through a lysine residue of the substrate (but not exclusively).
- Once the cyclin is ubiquitinated it can be recognised by the proteasome.
Describe the form of a proteasome.
- When a protein has been ubiquitinated, it can be recognised by a proteasome.
- Proteasome is a big complex.
- Core particle with a regulatory particle on top (regulatory particle is like a lid).
- There is a hole in the bottom.
- When the conditions are right and the ubiquitin is recognised, the ‘lid’ is opened (regulatory particle) and the protein for destruction moves inside the proteasome.
- Inside, the protein is chopped up into its composite amino acids, and discharged out through the hole in the bottom.
Describe the 2 checkpoints in G1 phase.
- Damaged DNA - the cell cannot proceed to replication if there is damaged DNA. This must be repaired before the cell can proceed to repication.
- Unfavourable extracellular environment - the cell cannot proceed if the conditions are not right.
Describe the checkpoint in S phase.
- Damaged or incompletely replicated DNA - the cell cycle must halt until this has been ressolved.
Describe the checkpoint in G2 phase.
- Damaged or incompletely replicated DNA - the cell cycle must stop until the DNA is fit to proceed to mitotic divisions.
Describe the checkpoint in mitosis.
- Chromosomes improperly attached to the mitotic spindle - the stages of mitosis cannot proceed until this is rectified.
How are the checkpoints during cell division controlled?
More specifically, based on the conditions in the cell, what makes the cell decide either to stop or to proceed through a checkpoint?
Cyclin dependent kinase inhibitors
Describe how cyclin dependent kinase inhibitors work.
- If the conditions in a cell are not right (DNA damage or something), the transcription of cyclin dependent kinase inhibitors can be stimulated.
- They function in different ways but essentially they convert an active cyclin dependent kinase to an inactive form and hold it ready but not active.
What is the key target at the checkpoint before cell division; the restriction site/the ‘commitment’ stage where it commits to replicate its DNA?
A tumour suppressor protein - retinoblastoma protein.
What is the role of the retinoblastoma protein in its active form and in its inactive form?
-
Active form - In a quiescent cell, the protein is in an active form telling the cell not to divide.
- It works to repress transcription factors that would be required for the expression of genes responsible for S phase. Eg. DNA polymerase.
-
Inactive form - once the cell has the right conditions for replication, a key target for Cdk is the tumour supressor protein. It becomes phosphorylated by Cdk. The phosphorylated form of the tumour suppressor protein is inactive.
- This relieves the inhibition of the transcription factors and this allows expression of the genes required for S-phase.
What is the G1-S phase transition?
Why is this clinically important?
- The R point - restriction point where replication is stopped if there is a problem.
- Failure of this checkpoint due to changes in genes which code for proteins at this checkpoint is implicated in many tumours.
How does a cell know that it should only replicate its DNA once?
It is controlled by Cdc6
Explain the role of Cdc6.
- Cdc6 sits at the origins of replication in the pre-replicatie complex.
- Once the cell is moving into S phase, Cdk can phosphorylate the Cdc6, Cdc6 falls off the pre-replicative complex, allowing the replication fork to assemble and DNA repliation to start from that site.
What prevents Cdc6 from binding to the newly replicated replication forks and origins of replication?
The cell targets the Cdc6 for ubiquitination and degradation.
This is the crux of how DNA replication is only allowed to occur once per cycle.
What are the 2 key proteins involved in DNA damage?
- p53 - directs transcription of the Cdk inhibitors.
- Frequently changed in many cancers.
- CHK2 - inhibits Cdc25.
- Cdc25 is the last protein that works in the activation of the Cdk.
- It is the phosphatase that removes the inhibitory phosphate.
- If Cdc25 is inhibited, there can’t be an activated Cdk.
What is the first thing that has to happen in mitosis?
How does this happen?
- DNA must condense.
- Specific proteins, condensins, form the condensed DNA structures.
Describe the centrosome.
- Comprised of 2 centrioles.
- The ‘organising centre’ for the spindle fibres.
What allows the spindle to form?
Favouring microtubule polymerisation over depolymerisation.
What does the mitotic Cdk do?
- Phosphorylates the nuclear lamin.
- The nuclear lamin, once phosphorylated, contributes to the destruction of the nuclear envelope.
- The nuclear lamin is on the inside of the nuclear envelope.
- When the Cdks are active, this disrupts the nuclear envelope.
- THIS IS ALL IN PROMETAPHASE.
Where is the spindle checkpoint in mitosis?
- The beginning of metaphase.
- Mitosis cannot proceed until chromosomes are properly attached and under tension.
How are the replicated chromosomes held together?
A protein complex known as a cohesion complex which pulls the sister chromatids together until the right stage of the cel cycle is reached, when the choesion rings are removed and allow the chromatids to separate.
Describe what happens to cause Roberts syndrome.
- The chromosomes are not held together properly due to a defect in the cohesion complex.
- Child is unlikely to survive.
What happens if separase becomes active prematurely?
- Mitotic catastrophe.
- The chromosomes would start moving to the opposite poles when the cell was not ready / spindle was not complete.
- So, the activity of separase must be exquisitely controlled.
Which protein controlls the activity of separase?
- A protein called securin.
- This protein is normally bound to separase and keeps it in its inactive form.
Which protein is most tightly controlled at the metaphase-anaphase transition?
The anaphase promoting complex.
What factors are instrumental in cytokinesis?
- A contractile ring of actin and myosin filaments in the cleavage furrow.
- Ongoing microtubule polymerisaton and depolymerisation results in the separation of the 2 cells.
