2. Cell Cycle Flashcards
Outline the significance and relevance of cell division
Cell division in specific cells
- Different cells divide at different rates
- Embryonic vs adult cells (early from embryo cells divide every 30 mins)
- Complexity of system (e.g. yeast cell divides every 1.5-‐3 hours)
- Necessity for renewal (intestinal epithelium -‐ every 20 hours, hepatocytes -‐ every 1 year)
- State of differentiation (some cells never divide i.e. neurons and cardiac myocytes)
- Tumour cells have an inability to regulate the cell cycle, lose ability to control differentiation
Relevance of the appropriate regulation of the cell cycle
- Cell death – when there is premature/aberrant mitosis.
- Aneuploidy – due to mutations in oncogenes and tumour suppressor genes (abnormal chromosome number and content)
- Chromosome instability (lose and gain whole chromosomes during cell division)
- Contact inhibition of growth:
- Contact inhibition of growth -‐ cells normally grow by sensing neighbouring cells
- Tumours usually lack contact inhibition so they don’t stop growing
- Anti-cancer strategies – aimed at attacking machinery that regulates chromosome segregation.
Attacking the machinery that regulates chromosome segregation is one of the most successful anti-cancer strategies in clinical use
Mitosis: identify the named stages of mitosis
Mitosis - most vulnerable period of cell cycle:
- Cells are more easily killed (irradiation, heat shock, chemicals)
- DNA damage can not be repaired
- Gene transcription silenced
- Metabolism
Mitosis = nuclear division and cytokinesis.
Interphase = duplication of DNA, organelles and protein synthesis.
Named stages
- Prophase
- Prometaphase
- Metaphase
- Anaphase
- Telophase
- Cytokinesis
Cell cycle: recall the various cell cycle phases, and demonstrate the ability to label and annotate a diagram of the cycle.
Cell cycle: Orderly sequence of events in which a cell duplicates its contents and divides in two
- Duplication
- Division
- Co-ordination
Regulated progression through the cell cycle:
M-phase: Mitosis (Division)
- Nuclear division
- Cell division (cytokinesis)
Interphase (Duplication)
- DNA
- organelles
- protein synthesis
The Eukaryotic Cell Cycle Overview
- Cells are normally resting in G0
- The mitosis itself happens very fast (roughly 5 mins relative to the 24 hour clock model) because it is a dangerous time for the cell
- When a cell decides to enter the cell cycle, they go into Gap phase 1 (G1)
- Following G1 is the S phase (synthesis) where duplication takes place
- Once all the duplication has taken place, it enters G2 (decision point) where the cell checks that everything is OK and ready to go into mitosis
Outline the events that occur during mitosis
Mitosis - S Phase – Replication for devision
- DNA replication
- Protein synthesis: initiation of translation and elongation increased; capacity is also increased
- Replication of organelles (centrosomes, mitochondria, Golgi, etc)
- In case of mitochondria, needs to coordinate with replication of mitochondrial DNA
Prophase
Prophase -‐ Condensation of Chromatin
- Condensed chromosomes consist of 2 sister chromatids, each with a kinetochore -> They need to be condensed so that you can minimise DNA damage during mitosis
- The centromere acts like a belt -‐ it is a constriction around the chromosomes
- At the centromere there are a load of protein complexes that forms the kinetochore
The kinetochore is a complex of proteins and it is a key regulator of the processes around chromosomes in the cell cycle - Provides site of attachment for spindles.
Actions in prophase:
- Chromosomes condense.
- Duplicated centrosomes migrate to opposite sides of cell and become MTOC.
- Mitotic spindle forms between 2 centrosomes - The spindle is like a highway that guides the chromosomes to where they have to go
Prophase – Condensation of chromatin:
2nm DNA -> 11nm chromatin string -> 30nm chromatin fibre -> 300-700nm scaffold-associated form -> 1400nm chromosome.
Spindle Formation:
- Radial microtubule arrays (ASTERS) form around each centrosome -> MTOC.
- Radial arrays meet.
- Polar microtubules form.
This is a DYNAMIC environment with polar microtubules constantly forming and breaking.
Metaphase/ Prometaphase
Metaphase
The chromosomes that have leaked into the cytoplasm, following the breakdown of the nuclear envelope, go with their pairs to the centre of the cells
Prometaphase
Early Prometaphase
- Chromosomes align at the equator of the spindle.
- Early prometaphase:
- Breakdown of nuclear membrane.
- Spindle formation complete.
- Chromosome attachment via spindles to kinetochores (centromere region of chromosome)
- Each of the microtubules meeting in the middle needs to find a chromosome
- The chromosomes attach to the spindles via the kinetochores
- One microtubule array will attach to the kinetochore on one side, and another microtubule array will attach on the other side
Late prometaphase
- Microtubule from opposite pole is captured by sister kinetochore
- Chromosomes attached to each pole congress to the middle
- Chromosome slides rapidly towards center along microtubules
In late prometaphase, the sister chromatids have been captured by the microtubule arrays
Once captured, the chromosomes slide rapidly towards the middle of the cell
In the kinetochores there are specialised proteins, which sense the attachment of microtubules e.g. CENP-‐E -‐ this senses whether the kinetochore is attached to microtubules or not
There are three types of half-‐spindle:
- Kinetochore microtubule -‐ bound to the kinetochore
- Polar microtubule -‐ a microtubule that has met and connected with a microtubule from the other centrosome
- Astral microtubule -‐ a microtubule that is originating from the centrosome that does not connect to a kinetochore
Anaphase
Paired chromatids separate to form two daughter chromatids
Cohesin is a protein complex that holds the sister chromatids tightly bound together
Anaphase can be split into Anaphase A and Anaphase B
Anaphase A:
- Cohesin is broken down and the microtubules get shorter, and the chromatids start moving towards the centrosomes
- The daughter chromatids are pulled towards opposite spindle poles
Anaphase B:
1-Daughter chromosomes migrate towards poles
2-Spindle poles (centrosomes) migrate apart
The daughter chromosomes can reach the opposite poles either because of the shortening of the microtubules that form the spindles or by the pulling apart of the spindle poles
These two movements really segregate – since they split in two they have to be really empty towards the edges
Telophase
- Daughter chromosomes arrive at the pole
- Nuclear envelope reassembles at each pole
- Contractile ring forms – made of actin and myosin.
- This contracts to leave a mid-body.
- The centrosomes are moved apart and the cells try to revert to their normal size
- There is a condensation of material where the cells are going to split and you get the assembly of a contractile ring of actin and myosin filaments
- The contractile ring then squeezes the cell so that it divides into two daughter cells
- The cleavage furrow is where the cells are going to be cleaved
Cytokinesis
- This is the last phase of mitosis
- You get insertion of the new membrane at the cleavage furrow
- Midbody = where the actin-‐myosin ring is formed
Explain the role of the centrosome in the cell cycle
The Centrosome
Consists of two centrioles (barrels of nine triplet microtubules)
Functions: microtubule organizing center (MTOC) and mitotic spindle
- Centrosome -‐ an organelle near the nucleus of a cell which contains the centrioles, and from which the spindle fibres develop in cell division
- They are referred to as mother and daughter centrioles
- They regulate the microtubule network to orchestrate cell division
- The centrioles themselves are made of microtubules
- Appear as a double barrel – triple filaments with protein structures holding them together
- Headquarters that will coordinate chromosome movement
Duplication of Centrosomes and DNA
- When the cell initiates duplication and enters the cell cycle, it needs to duplicate the centrosomes
- In G1 phase there is separation of the mother and daughter centrioles (they are normally stuck together)
- When they separate they start to duplicate -‐ the mother centriole will produce a daughter and the daughter centriole will produce a mother
- This duplication takes place in the S phase
- There is a cloud of protein complexes around them and there are points where they make nucleating sites for the microtubules
- It is kind of like lego, you put monomers together and make a long filament
- When you put microtubules together it is called nucleation
- As the cell encounters a need for mitosis, the microtubules start to grow from these points and form an array of microtubules (looks like a sea urchin)
Spindle Formation
- Radial microtubule arrays (asters) form around each centrosome
- The radial arrays from the two centrosomes meet in the middle and when they meet each other they are then called polar microtubules
- These form highways telling the chromosomes which way to go
Cell cycle checkpoints: Explain the effects of internal checkpoints on the regulation of the cell cycle and recall examples.
Mitotic Checkpoint: Spindle Assembly Checkpoint
- The cell cycle has checkpoints which allow the cells to check that everything is in place so that they can move onto the next phase of the cycle
- One of the checkpoints is when the cell wants to exit metaphase and enter anaphase -‐ this is the spindle assembly checkpoint (aka anaphase checkpoint)
- This checkpoint sense the completion of chromosome alignment and also checks for spindle assembly
- The kinetochore has proteins that emit a signal when the kinetochore is NOT attached to microtubules
- Once the kinetochore attaches to microtubules, it stops emitting the signal
- Analogy: when a formula 1 car comes in for a pit stop, several technicians change the tyres. When one of the tyres is changed successfully, the technician will signal to say that they’re finished. In effect, each chromosome has a flag and when hooked to a microtubule, it stops sending the signals thus saying that they are good to go
- At metaphase, you’re hoping that all of the all the kinetochores will stop sending signals so that they can proceed to anaphase
There are many proteins involved in this signalling process but two important ones are:
- CENP-‐E
- BUB Protein Kinase
- BUBs dissociate from the kinetochore when chromatids are properly attached to the spindle
- They then go on to signal progression to anaphase
Outline disturbances of the cell cycle that cause aneuploidity
Road to Aneuploidy: Mitotic Checkpoint Defect
- This happens if anaphase initiates before the spindles attach properly
- It results in abnormal division of the chromosomes between the daughter cells
Road to Aneuploidy: Mis-‐attachment of Microtubules to Kinetochores
- Normally, you have a chromosome made up of two sister chromatids
- There is normal attachment, where a microtubule array one centrosome is attached to the kinetochore of one sister chromatid, and the microtubule array from another centrosome is attached to the kinetochore of the other chromatid
- This type of normal attachment will allow the sister chromatids to be split apart and go to opposite poles
- Syntelic Attachment -‐ both the kinetochores are hooked by two microtubule arrays from the SAME centrosome
- Merotelic Attachment -‐ there is more than one microtubule array attached to the same kinetochore -‐ this means that one of the chromatids is being pulled in two different directions
- Monotelic Attachment -‐ only one of the kinetochores of one chromatid is attached to a microtubule array, the other kinetochore is unattached
This is how mis-‐attachment can lead to aneuploidy
Road to Aneuploidy: Aberrant Mitosis
- If the centrosomes are not duplicated properly you could end up with 4 centrosomes in one cell
- This can lead to very abnormal attachment of the microtubule arrays to the kinetochores leading to abnormal cytokinesis
Checkpoint control use for cancer therapy
Anti-‐cancer therapy by inducing gross chromosome mis-‐segregations
- You can slow down cancer by inhibiting the proliferation of tumour cells
- One mechanism of cancer therapy is exploiting checkpoint control
- The kinetochore signalling tells the cell when metaphase is complete so if you have an inhibitor for this checkpoint, then you can make the nucleus think that it is correctly hooked onto microtubules
Taxanes and vinca alkaloids (breast and ovarian cancers)
- Alters microtubule dynamics
- Produces unattached kinetochores
- Causes long-term mitotic arrest.
- By altering the microtubule dynamics you can cause long-‐term mitotic arrest
- If you keep the cells in this disorganised and vulnerable position for a prolonged period, they are more easily killed
- These are checkpoint kinase inhibitors.
i.e. BUB protein kinase inhibitors.
Points at which DNA can be damaged:
1. Cell cycle arrest
- at check points (G1 and spindle check point)
- can be temporary (i.e. following DNA repair)
- Programmed cell death (apoptosis)
2. DNA damage too great and cannot be repaired
- Chromosomal abnormalities
- Toxic agents
At certain checkpoints the cell cycle could be arrested if something is broken
- This could be temporary if the damage is reparable
- If the problem is irreversible, then you will get apoptosis
Checkpoints and tumour progression
Cell Cycle Checkpoints and Tumour Progression
- The first checkpoint is during G1
- Then the next checkpoint is just before mitosis, to check for DNA damage before entering mitosis
- Then there is also a metaphase-‐anaphase checkpoint
- Tumours ignore these checkpoints
De-‐regulation of the cell cycle during tumorigenesis
Normally, when cells come out of G1 they enter G0 (during this phase they are still being very active)
Tumours block the ability of the cells to leave the cell cycle and enter G0 -‐ once they finish mitosis, they enter another cell cycle
Recall how the cell is directed to divide
What triggers a cell to enter the cell cycle and divide?
- In the absence of stimulus, cells go into Go (quiescent phase)
- Most cells in the body which are differentiated to perform specific functions
- Cells are not dormant, but are non-dividing
- Exit from G0 highly regulated - requires growth factors and intracellular signalling cascades
