Cell Cycle Flashcards
What is the overview of the cell cycle?
Interphase: cell growth, nutrient accumulation, DNA replication, transcription-translation, checks: quality control checks, DNA checks
Mitotic Phase: mitosis, Cytokinesis
G1 -> S -> G2 -> mitosis -> cytoinesis
G0 = cycle arrest (optional) = resting state
e.g nerve and muscle cells - dont replicate much, arrest in G0 phase
- varies in time taken - e.g cancer cells take 24 hrs
What is the interphase?
Interphase: G1, S, G2
G1 phase: organelle replication, growth, mRNA synthesis, translation protein synthesis
S phase: DNA replication/chromosome duplication
G2 phase: Cell growth, preparation for mitosis, checks
How does mitosis and cytokinesis occur?
- *Prophase**
- chromosomes condense and become visible
- nuclear envelope breaks down, releasing chromosomes
- centrioles migrate to opposite poles of the cell and produce mitotic spindle fibres which attach to centromeres of the chromosomes
- *Metaphase**
- the spindle has captured all the chromosomes and lined them up at the middle of the cell, ready to divide.
- All the chromosomes align at the metaphase plate (not a physical structure, just a term for the plane where the chromosomes line up).
(spindle checkpoint and helps ensure that the sister chromatids will split evenly between the two daughter cells when they separate in the next step.)
- *Anaphase**
- spindle fibres contract and shorten - driven by motor proteins
- this separates sister chromatids to opposite poles of the cell
- *Telophase**
- The mitotic spindle is broken down into its building blocks.
- Two new nuclei form, one for each set of chromosomes. Nuclear membranes and nucleoli reappear.
- The chromosomes begin to decondense and return to their “stringy” form.
- *Cytokinesis**
- the division of the cytoplasm to form two new cells
How do we know what occurs during S phase?
S Phase
- DNA synthesised
Can be identified by 3H -Thymidine autoradiography
- Tritriated Thymidine used to treat growing cells which incorporated it into its DNA (thymidine = ribose + thymine)
- If cell is in S phase, it will take up tritriated thymidine and incorporate it into newly synthesised DNA
- treat the cells with a pulse with layer of celluloid film over it - when electrons ping off, they develop the film
→ about 1/2 the cells shown are labelled, so half the cells are in S phase
What happens if we used tritriated adenine instead?
RNA is synthesised continuously and in the cytoplasm so majority of the cells would be labelled
How do we know what occurs during G1 and G2 phase?
- *G1 and G2 are harder to identify**
- bring in nutrients, grow to a particular size to support cell division, undergo replicative repair, etc.
Identify using Flow Cytometry:
- add a dye (usually fluorescent) to cells which binds DNA
- Take a suspension of exposed cells and vibrate them really quickly in this vessel so that drips only contain 1 cell
- A laser is shone through the stream of drips
- Analyser works out how much fluorescence is in each cell
Fluorescence in each cell is measurement of relative amount of DNA:
G1 cells have 1 set of DNA so will fluorescence at 100
S will be at both 100 and 200 ( and in between)
G2 and M phase will be at 200
How did cell fusion experiments show phase induction?
Cell fusion experiments in HeLa cells (human cancer cell lineage)
- allows you to take 2 cells at different stages of cell cycle and fuse them using PEG (polyethylene glycol), align membranes to fuse
→ Something in S phase cells is lacking in G1 phase, as G1 cells are rapidly induced into S phase when fused.
→ That ‘something’ in S phase which propels G1 phase into S phase is lost by the end of G2
How did injection studies in frog oocytes show maturation promoting factor? (MPF)
Injection studies in frog oocytes showed ‘maturation promoting factor’ (MPF) can induce M Phase in G2 nuclei
- G2 cells produce MPF in response to external factors (e.g growth factors, hormones) which induce M phase
- MPF = a cyclone CDK complex (cyclin dependent kinase)
What happens when a transition is blocked?
Phase Interruption
Cell division cycle mutants help us to understand cell cycle regulation
2 types of yeast (S.cerevisiae - budding yeast and S.pombe - fission yeast)
- temperature sensitive yeast mutants arrest G1 at high temperatures
1. mutations were generated in genes
2. a block was seen in the cell cycle
3. went back and identified the temperature sensitive mutation that produced this phenotype
4. allowed identification of cdc28 and cdc2
→ clearly there is a check point in G1 and mutant arrest there
→ CDK1 human homologue
What are cyclins?
Cyclins are proteins that cycle in concentration over the cell cycle
Cyclins, when bound with the cyclin dependent kinases, such as the p34/cdc2/cdk1 protein, become activated (after CAK binds)
Cyclin B bound to CDK1 forms Maturation promoting factor
Cyclins activate Cyclin-dependent kinases (CDKs)
- MPFs activate other proteins through phosphorylation.
→ Cyclins determine the specificity of the CDK for its protein targetsact as ALLOSTERIC ACTIVATORS
- shift CDK into active conformation
act as ADAPTORS which bind to specific substrates → control what specific substrates CDK phosphorylates for given phase at cell cycle
Where do the cyclins and CDKs act during cell cycle?
yeast have 1 type of CDK → cdc28/cdc2
Mammals have many types of CDK
3 checkpoints: between G1 and S Phase, between G2 and M phase (between metaphase and anaphase)
G1 Phase**- 2 cyclins:
(G1 cyclin expression regulated by MAPK/K/K)
G1 cyclin and G1/S cyclin (cyclin D) bind with CDK to promote passage through start phase by phosphorylating substrates:
- the E2F transcription factorisbound to Rb
- CDK-cyclin complex phosphorylates Rb, allowing E2F to be released.
- Once released, E2F is able to activate genes for S phase.
- Mutation in Rb (the tumoursuppressorgene) means E2F isalways unbound and thus constantly drives S phase which causes cancer. (blastoma in retina of small children)
- E2F switches on S Cyclin
S Phase:
S cyclin binds to CDK which initiates DNA synthesis by phosphorylating:
- Histone mRNA stem-loop binding protein → stabilises histone mRNA
**G2 Phase:
M cyclin binds to CDK (forming MPF (from frog oocytes) which promotes passage through to M phase by phosphorylating:
- Nuclear lamins → cytoskeletal elements which surround inner surface of nuclear envelope, holding it together - when phosphorylated, dissociate from each other during prophase of M phase
- Myosin → is a protein motor associated with actin and is involved in cell motility and in contracting an actin ring to separate cells during cytokinesis. - phosphorylated to prevent cytokinesis until end of M phase
- Condesin → condenses chromsomes - important for packaging of DNA into chromatids so they’re easy to transport
- *M Phase**
- end of M phase associated with destruction of M cyclin by APC (anaphase promoting complex)
How does cyclin regulation occur?
CDK are protein kinases that are regulated by phosphorylation
- when CDK associated with a cyclin, they gain some kinase activity
- from this position, can be phosphorylated by CAKs (CDK activating kinases) to promote activity or by CDK inactivating kinases (Wee1) to diminish activity
- CDC 25 (phosphatase) removes Wee1 so that CDK/cyclin complex is fully active
How is cyclin regulation balanced?
Cyclins are regulated by a balance of synthesis and degradation
→ degradation by proteosome when they are tagged by ubiquitous residues
→ Ubiquitination can be done by APC (anaphase promoting complex) → target cyclins that have come off CDK’s for degradation
How are cyclin/CDK complexes inhibited? (CKIs, p53)
Cyclin/CDK complexes are inhibited by CDK inhibitor proteins (CKIs)
e. g p21 (Cip1)
- p53 (protein 53 - transcription factor) is activated in response to DNA damage and promotes expression of p21 which binds to CDK S cyclin complexes and inactivates it -> cell cycle inhibited
* Mutation in p53 will allow damaged cells to divide which can lead to cancer causing mutations to be propagated
*p53 = tumour suppressor gene - most cells have 2 functional copies
- if one mutates, heterozygotes are usually fine
- if some of cells become recessive double mutant, they are likely to become cancerous
What are protooncogenes and tumour suppressor genes?
Alteration in cell cycle regulatory proteins can lead to cancer
Proto-oncogenes
- dominant mutations (1 broken copy will do) → onco genes
- accelerators of cell cycle progression
→ e.g genes encoding Ras, RTK, c-Myc
Tumour Suppressor genes
- recessive mutations (need both copies to be broken)
- inhibitors of cell cycle progression
→ e.g genes encoding p53 (produce CKI), retinoblastoma (Rb) protein
