Lecture 1: Introduction Flashcards
How do meiosis and mitosis work during vertebrate development?
Meiosis and mitosis both play an important part in early vertebrate development.
1) Immature oocytes are 4N after DNA replication occurs.
2) Metaphase I creates 2N cells. This is triggered by progesterone.
3) Metaphase II creates 1N cells (mature oocyte).
4) The oocyte is fertilised.
5) Maternal and paternal genomes combine.
6) DNA is replicated to form a 4N cell.
7) 2N daughter cells are formed by mitosis.
Give an overview of the cell cycle
The cell cycle is a series of phases that the cell goes through during its life cycle. Most broadly it is split into interphase (when the cell is acting normally and performing its function) and mitosis (when the cell is dividing).
There are also gap phases which provide time for organelle/protein replication and synthesis. They also provide time to monitor internal and external signals to see if it is possible to pass from G1 to S or from G2 to M. Checkpoint requirements need to be met.
1 – G1: In G1, the cell grows after the previous mitosis.
• Organelle duplication.
• Initiation of centrosome replication.
• The length of it is highly variable, it depends on paracrine and endocrine signalling.
• The cell can enter G0 if signals indicate unfavourable conditions.
• Increase the protein supply.
• The restriction point is regulated by G1/S cyclins. The cell has 3 options: to continue the cell cycle, stop the cell cycle and enter G0 or become arrested in G1 (it can either continue or enter G0).
2 – S: In S phase the DNA is replicated.
• Rate of transcription and translation are very low.
• The exception is histone production.
3 – G2: The cell grows again after DNA replication.
• Preparation for mitosis.
• Microtubules being to reorganise to form a spindle.
• Protein synthesis.
• Rapid cell growth
4 – M: Mitosis is when the chromosomes separate.
• It is split into different parts: prophase, prometaphase, metaphase, anaphase and telophase.
• Time varies between organisms.
• In many cells, cytokinesis is guided by a contractile ring of actin filaments and myosin motor proteins.
What do cell fusion experiments show?
Experiments with virus-induced cell fusion proved that cell cycle state and DNA replication are coupled. These were first performed in 1970 by Rao and Johnson.
• Cells were fused at different cell cycle stages.
• If S and G1 cells were fused, then the G1 cell would be triggered into S phase.
• If S and G2 cells were fused, then G2 would not re-replicate. They didn’t enter mitosis until S-phase had finished.
• Fusion of G1, S or G2 cells with M-phase cells results in entry into mitosis. M-phase cells have dominant activity that promotes mitosis.
What are cyclins and CDKs?
Cyclins and CDKs are vital for normal cellular function.
• CDKs stay in the cell constantly.
• Cyclins are synthesised and degraded for different sections of the cell cycle.
• Cyclins and CDKs form a complex.
Phosphorylation of the CDK by CDK-activating kinase (CAK) is required for full activation. There is a consensus sequence where phosphorylation occurs: [S/T*]PX[K/R].CAK levels remain constant and they are not regulated.
Different cyclins and Cdks are used at different stages of the cell cycle.
• G1 and G0 exit uses Cdk4/6 and cycD.
• G1/S uses cdk2 and cycE.
• S uses Cdk2 and cycA.
• S/G2 uses Cdk1 and cycA.
• G2/M and M phase use Cdk1 and cycB.
What do budding and fission yeast genetic experiments show?
Nurse and Hartwell groups pioneered cell cycle genetics. They isolated cell division cycle (CDC) mutants mapped to single loci (single genes).
• Budding yeast (Hartwell) were used to find cdc4, cdc28 and cks1.
• Fission yeast (Nurse) were used to find cdc2, cdc13, cdc25, wee1 and suc1.
• The yeast can be grown as haploid mutants.
• Search for temperature sensitive growth mutants.
The following technique was used:
1) Mutagenize yeast cell culture.
2) Plate out and grow at 23-25 C.
3) Replicate and grow at 35-36 C.
4) A cdc mutant will not grow at this temperature. Isolate the mutants.
5) Culture the mutants at a permissive temperature and with wild type genomic DNA library plasmids.
6) Plate out at 23-25 C.
7) Make a replica at 35-36 C.
8) Some yeast will complement. Recover the plasmid and look at the gene sequence.
What is CDC2?
CDC2 in fission yeast encodes for a protein kinase. It is CDC28 in budding yeast.
• S. pombe cdc2 mutants can be rescued by a gene conserved to humans. This shows that the cell cycle is controlled by conserved genes and gene products (proteins).
• Nurse showed that CDC2 acted at multiple points in the cell. It regulates G1 to S and G2 to M phase transitions.
• CDC2 levels don’t change.
What are cyclin dependent kinases?
Cdks are kinases involved in the cell cycle. They are activated by cyclin molecules.
• They are a group of serine/threonine kinases.
• 34-40 kDa in size.
• They phosphorylate at a consensus sequence of (S/T) P X (K/R).
• Full activation requires phosphorylation on a threonine by a CAK (Cdk activating kinases).
• Conserved enough that Cdk1 can rescue cdc28 in yeast. Sequence alignments also show that there is a high level of conservation.
• Cdks also have a conserved tertiary structure.
• They have a small N-terminal lobe, a larger C-terminal lobe and an ATP binding site between these.
• Without cyclin binding and CAK activity, the T-loop blocks the active site.
• The L12 helix comes just before the T-loop in primary sequence. When cyclin binds, the helix turns into a β strand and it helps to rearrange the T-loop.
• The PSTAIRE helix rearranges and helps to change the position of key amino acid residues in the active site.
What are cyclins?
Cyclins bind and activate Cdks. Each cyclin targets the previous one for degradation and activates the expression of the next one.
1) In G1 cyclin D causes entry into the cell cycle. It integrates external and internal growth signals, slowly accumulating over G1 to reach a threshold).
2) In G1/S cyclin E causes progression past the restriction point.
3) In S cyclin A causes progression and stimulates DNA replication.
4) In M cyclin B causes assembly of the mitotic spindle and alignment of chromosomes. They are destroyed to exit mitosis.
Cyclins also have a role in Cdk action targeting. They confer substrate and regulatory specificity.
How were cyclins discovered?
Cyclins were discovered following two experimental observations.
1) Ruderman (1978-1980) found that translation of proteins was important to activate unfertilised clam eggs.
2) Hunt (1983) found that cyclical protein synthesis fertilisation follows fertilisation in sea urchin eggs.
The graph shows that A has cyclical synthesis while B has continuous synthesis.
It was later discovered in 1984 that MPF also follows this cyclic activity, just like cyclin.
• Activity peaked and this was followed by cell cleavage.
• Protein synthesis inhibition with cycloheximide in G2 prevents MPF from appearing.
• MPF disappearance is blocked by CSF.
How do cdk inhibitors function?
Cdks are activated by CAKs and cyclins, but it is also important that they can be controlled through inhibitory mechanisms.
• Inhibitory kinases repress activity (for example Wee1 for cycB-cdk1).
• They are often involved in cell cycle arrest.
• Several CKIs are tumour suppressor genes.
• CKIs bind to Cdks and inactivate them.
• Some CKIs inhibit Cdks through phosphorylation.
• CKIs were originally discovered with yeast two hybrid screens for interacts with Cdk proteins. They were also identified in cyclin Cdk complexes which were purified from cells.
• During G1 Cdk activity is suppressed, CKIs are one of the mechanisms used to do this.
• There are different CKI families. They can both inhibit and promote cell cycle transitions.
• p21, p27 and p57 can inhibit cdk1 and cdk2.
• p15, p15, p18 and p19 can inhibit cdk 4/6.
• CKIs are broken down at different points. p27 is broken down by cdk4 once enough cyclin D accumulates. It is then targeted for destruction.
What is wee1?
Wee1 encodes for a protein kinase.
• Nurse wanted to find the rate limiting components of the cell cycle control pathway.
• Fission yeast grow like a rod and divide when they reach a characteristic length.
• Nurse looked for mutants that divided at smaller sizes.
• Wee1 (cdc9) had LOF mutants which entered mitosis and divided at a smaller size.
• Overproduction had the opposite effect.
• Wee1 determines when mitosis commences.
• Wee1 mediates inhibitory phosphorylation.
• Wee1 inhibits Cdk1 by phosphorylating it on two different sites: tyr15 and thr14.
• This is opposed by Cdc25, which removes the phosphate groups added by Wee1.
What is ubiquitin dependent proteolysis?
Ubiquitin is important for proteolysis, which is necessary during the cell cycle for controlling the levels of various proteins.
• Cyclin B has a cis acting destruction signal in its first 90 amino aicds. CycB which lacks this cannot be destroyed.
• Cyclin is stable in interphase xenopus extract.
• Cdk1 addition triggers cyclin destruction.
• Cyclin B involves ubiquitin modification. Cyclin B was labelled with iodine-125 and added to metaphase xenopus extract to perform SDS page autoradiography. Long exposure revealed ladders of bands 7kDa apart which is a sign of ubiquitin.
• Repeated experiments with unlabelled cyclin B and labelled ubiquitin observed the same ladder in metaphase, not interphase.
Ubiquitination involves 3 steps.
1) Activation: Ubiquitin is activated by an E1 ubiquitin activating enzyme, which is dependent on ATP. The result is E1 being linked to the C-terminal carboxyl of ubiquitin by an E1 cysteine sulfhydryl group.
E1 + Ub + ATP E1-cys-ub + AMP + PPi
2) Conjugation: E2 ubiquitin-conjugating enzymes catalyse the transfer of ubiquitin from E1 to the active site cysteine of the E2.
E1-cys-ub + E2 E1 + E2-cys-ub (transferred to E2)
3) Ligation: a bond is created between a lysine of the target and the ubiquitin. E3 ligases bind to both the substrate and E2 to catalyse this reaction.
E2-cys-ub + target-lys-E3 target-lys-ub
What is the APC/C?
APC/C stands for anaphase promoting complex. It is an E3 ubiquitin ligase which is involved in targeting cell cycle proteins for degradation.
• It was purified by Hersko and then Kirschner.
• It acts on cyclin A/B.
• It is active when isolated from mitotic cells but not interphase cells.
• Candidate E1 and E2 enzymes were found as well (UBCU enzymes).
• Subunits of this complex were found to be encoded by cdc16, cdc23 and cdc27.
• They were shown to be required for cyclin destruction at the metaphase to anaphase transition in budding yeast. They aren’t required for other proteolytic events.
APC/C is active in mitotic cells.
• The main targets are securing, cyclin A, cyclin B, aurora and plk.
• Cdc20 and Cdh1s are activators of APC/C.
Cdc20 acts at the metaphase to anaphase transition to target securing, cyclin A/B and cdc20.
• Activated by cyclin B-cdk1 in metaphase.
• Cdh1 acts at late mitosis to target aurora kinases and plks. Cdh1 is inactivated by phosphorylation before metaphase.
