IT2: Cell cycle Flashcards
What are the roles of Cdc2, Cdc25 and Wee1?
CDC2, also known as CDK1, is a protein kinase that forms a complex with cyclins, which leads to phosphorylation of target proteins that promote cell cycle progression.
Wee1 is a protein kinase that plays a critical role in the negative regulation of CDC2 activity. It phosphorylates a specific site on CDC2, which inhibits its activity and prevents the cell from entering mitosis.
CDC25 is a phosphatase that activates CDC2 by removing the inhibitory phosphorylation.
How have budding and fission yeast been used to study the cell cycle?
Budding:
- Genetic screens identified cell cycle mutants.
- Pheromones were used to synchronise cell cycle
- Identified different cell cycle proteins
Fission:
- Paul Nurse discovered the cdc2 gene from mutants and complementation experiments
- cdc2 gene encodes a CDK that was shown to require phosphorylation for activation
- Identified Wee1
How have frogs been used to study the cell cycle?
Frogs have large oocytes that makes them easier to handle for studying the cell cycle.
- Found cytoplasmic activity in metaphase eggs that could promote maturation of G2-arrested oocytes –> maturation promoting factor (MPF).
- Egg extraction experiments identified MPF to contain CDC2 and cyclin B. - Found cytoplasmic activity in mature oocytes that prevented the cell cycle and arrested early embryonic divisions –> cytoplasmic factor (CSF).
How have sea urchins been used to study the cell cycle?
Metabolic labeling of sea urchin eggs showed cyclical synthesis of proteins, coordinated with CDK activity.
Led to identification of cyclins and that their synthesis drives the cell cycle.
Further research showed CDK-cyclin complexes are conserved amongst many animals.
How have human cell lines been used to study the cell cycle?
Genetic studies showed that CDK-cyclin activation requires phosphorylation:
- Wee1 found to inhibit CDK1
- CDC25 found to activate CDK1
How is mitotic entry regulated at the level of CDK1-cyclin B?
- Cyclin B is synthesized in late S/G2 phases and accumulates in the nucleus.
- Cyclin B binds CDK1 in the inactive form.
- CDK1 is phosphorylated by the CDK-activating kinase (CAK).
- CDC25 removes the inhibitory phosphates on CDK1, placed there by Wee1.
- CDK1- cyclin B is now fully active and phosphorylates substrates for mitotic entry.
Describe the bistable switch mechanism of CDK1-cyclin B complex activation.
Initially, when cyclin B is absent and CDK1 activity is low, Wee1 activity is high and Cdc25 activity is low.
As the concentration of cyclin B increases, Wee1 phosphorylates and inactivates the CDK1-cyclin B complexes.
The complex concentration reaches a threshold that triggers the bistable switch, where CDK1 can now inactivate Wee1 whilst activating CDC25. This results in a rapid activation of CDK1 activity.
Cyclin B is rapidly destroyed by the APC/C, resulting in a loss of CDK1 activity. This also reduces CDC25 activity, and increases Wee1 activity.
What are the advantages and disadvantages of using a bistable system for CDK1-cyclin B activation?
Advantages:
- The complex can stabilize its own activity
- Process is rapid
- Kinase activity used is robust to temperature and oxidative stress fluctuations
- Allows further regulation by other stimuli
Disadvantages:
- The complexes’ active state is highly stable so requires a special mechanism to reverse this.
How was tubulin discovered? Describe its structure.
Colchicine is a drug known to block mitosis, and it was shown using radioactive drug binding assays that it does so through blocking microtubule proteins.
Two subunits (alpha and beta) that form a heterodimer. These bind to GTP and polmerise into a filament with defined polarity.
How were centromere and kinetochore proteins discovered?
Using antibodies that recognized specific proteins, researchers were able to identify and purify centromere and kinetochore proteins from cells. For example, one key protein, CENP-A, was identified in human cells based on its association with the centromere and its unique sequence compared to other histone proteins.
Advances in microscopy techniques, including super-resolution microscopy, allowed researchers to visualize the organization and dynamics of centromere and kinetochore proteins in living cells.
What are microtubules? What are they composed of?
Dynamic polymers with defined polarity.
They’re composed of alpha and beta tubulin heterodimers that bind GTP to grow at the + end.
What are centrosomes? What are they composed of?
How are microtubules anchored to centrosomes?
Organelles that function as the microtubule organising centers.
They’re composed of a pair of centrioles that are embedded in a meshwork of proteins (PCM).
When cells enter mitosis, the PCM recruits the microtubule - ends and anchors them at the centrosome.
What are dynein and kinesin proteins?
Force-generating ATPases that can move chromosomes and slide microtubules past one another.
Dynein = moves towards -
Kinesin = moves towards +
What is the difference between astral microtubules and kinetochore fibres?
These are the 2 main populations of microtubules.
Astral microtubules position the spindles along the midline of cells. Kinetochore fibers capture and pull chromosomes into position.
What is the kinetochore network?
A network of proteins, such as KNL1 and NDC80, that form the major site of microtubule capture at kinetochores. This sits on a platform created by CENP-A (centromere) nucleosomes.
What is the Ran-Importin pathway? How is it used in mitotic spindle assembly?
It’s responsible for the transport of proteins between the nucleus and cytoplasm. Importin binds nuclear localization sequences and brings them into the nucleus where Ran, a small GTPase, binds to Importin and induces cargo release.
TPX2 is transported through this pathway, being released into the cytoplasm to activate aurora kinase A. Aurora kinase A then phosphorylates different targets involved in spindle assembly.
