Chapter 8: 8.5 Cell Cycle Checkpoints Flashcards
Cell Cycle
Cycle of events by which a cell duplicates its chromosomes + other cellular components, and divides into 2 daughter cells
What must happen in order for a cell to proceed to the next phases in the cell cycle?
It must pass checkpoints
* Confirms that the cell is prepared to enter the next phase
What are the phases of the cell cycle and what happens during them?
- M Phase: Mitosis + Cytokinesis
- Interphase: Period between M phases, cell grows and replicates its DNA
- G1 phase: 1st gap phase, Cell is growing
- S phase: DNA is replicated
- G2 phase: 2nd gap phase, Cell is growing
What are the checkpoints in the cell cycle?
- G1 to S
- G2 to M
- M phase
What does the G1 to S checkpoint check for?
Confirms that the environment is suitable for replication
G1 to S Checkpoint:
What is an ideal environment that is suitable for replication?
- Sufficient nutrients
- Extracellular signals
- Other
G1 to S Checkpoint:
If the environment is unsuitable for replication, what happens?
Cell remains in G1 (also known G0)
Is G0 temporary or permanent?
Can be temporary or permanent
What is temporary G0 known as? What happens to cells in this condition?
Quiescence
* Cells retain the ability to re-enter the cell cycle
What is permanent G0 known as? When does this occur in cells?
Senescence
* Occurs in cells which are terminally differentiated (e.x. nerve cells)
G2 to M Checkpoint
Confirms that the DNA is fully replicated and undamaged
M phase Checkpoint
Known as the Spindle Checkpoint
* Confirms that duplicated chromosomes are properly attached to the mitotic spindle
The control of the cell cycle depends on how many proteins?
Mainly 2 types of proteins
What two types of proteins does the control of the cell cycle mainly depend on?
- Cyclins
- Cyclin-dependent kinases (Cdks)
Cyclins
Proteins that bind to and activate Cdks
Describe:
The concentration of cyclins
Rise and fall in a cyclical fashion
What causes increases and decreases of concentration of cyclins?
- Increased transcription = Increase in concentration
- Targeted proteolysis = Decrease in concentration
Cyclin-dependent kinases (Cdks)
Enzymes that phosphorylate other proteins involved in the cell cycle
What is the significance of phosphorylation status?
Regulates the activity of various proteins
True or False:
There is one cyclin-Cdk pair for the cell cycle
False, there is one cyclin-Cdk pair PER PHASE of the cell cycle
G1 cyclins
Control the activity of G1/S cyclins
G1/S cyclins
Activate Cdks in late G1, triggering progression to S phase
S-cyclins
Activate Cdks at the beginning of S phase
* Remain active through S, G2, and early M phases
M-cyclins
Activate Cdks that stimulate entry into mitosis at the G2/M checkpoint
In regards to cyclin-Cdk activity, what happens if a checkpoint is not met?
The cell cycle can be paused by modulating cyclin-Cdk activity
What is the transition for G1 to S phase blocked by?
Cdk inhibitor proteins
What do Cdk inhibitor proteins do?
Block assembly of cyclin-CDK complexes
What must the cell do in order to enter S phase?
Degrade the Cdk inhibitor proteins
True or False:
Once the G1 to S checkpoint is passed, the cell generally completes the cell cycle
True
What are Cdk inhibitor proteins also involved in besides the G1 to S checkpoint?
Involved in cells enter G0
Mitogens
Extracellular signals that promote progression from G1 to S phase by stimulating synthesis G1 cyclins, G1/S cyclins, and other proteins associated with DNA replication
Retinoblastoma (RB) protein
A regulatory protein that binds and inhibits transcriptional regulators required for cell proliferation
What is Rb phosphorylated by?
G1-Cdk-cyclin and G1/S-Cdk-cyclin complexes
What does the Rb phosphorylation trigger?
Conformational change that leads to dissociation of Rb from transcriptional regulators
What is the end result of phosphorylation of Rb?
Promotes cell proliferation
DNA damage causes the cell to temporarily halt progression through…
G1
What does DNA damage that temporarily halts progression through G1 cause?
- Increase in concentration and activity of protein p53
- Increase in protein p21
p53
Transcriptional regulator that activates p21
p21
Cdk-inhibitor protein, binds G1/S-Cdk and S-Cdk
What does temporarily halting in G1 allow for?
Cell to repair the damage before proceeding
What happens to cell if DNA damage is irreparable?
p53 induces apoptosis
Apoptosis
Programmed cell death
Mitogen effects of G1-Cdk and G1/S-Cdk
In G1, the cell is prepared for replication by forming the…
Pre-replicative complex
What is the pre-replicative complex comprised of?
- ORC
- DNA helicase
ORC
Origin recognition complex
* A protein complex that binds to origins of replication on DNA throughout the entire cell cycle
Cdc6
A protein recruited by ORC to help load DNA helicase onto DNA
What does DNA helicase do?
Unwinds DNA
DNA Damage, p53 and p21 processes
What happens in S phase with DNA helicase?
- S-Cdk activates DNA helicase
- Promotes assembly of the proteins required for the replication fork
- Initiates DNA synthesis
How is it ensured the DNA is replicated only once?
S-Cdk phosphorylates (and inactivates) Cdc6
What is the transition from G2 to M phase blocked by?
Inhibition of Cdc25
* A phosphatase needed to activate M-cyclin-Cdk complexes
What may result in inhibition of Cdc25?
Incomplete or erroneous DNA replication
What does M-Cdks contain that determines if it is active or not?
Contain an inhibitory phosphate group
* Must be removed before it can become active
Wee1
Inhibitory kinase that phosphorylates M-Cdk
What are M-Cdk levels like during G2? Is it activated?
M-Cdk levels increase throughout G2, but remains inactivated until the end of G2
Why does M-Cdk become active at the end of G2?
Cdc25 removes the inhibitory phosphate group
What type of feedback loop is M-Cdk activation?
Positive feedback loop
* Activated M-Cdk phosphorylates more Cdc25, which activates more M-Cdk
ORC, DNA helicase, and Cdc6 mechanism
M-Cdk, Cdc25, Wee1 mechanisms
Metaphase plate
Halfway point between the two opposite spindle poles
* Chromosomes align bound by microtubules align here
How do chromosomes ensure that they are properly aligned at the metaphase plate?
Dynamic instability of microtubules (growth and shrinkage) helps adjust
Spindle Assembly Checkpoint
Unattached chromosomes send a “stop” signal which inhibits activation of the APC
APC
Anaphase-promoting complex
What happens APC is activated?
- APC ubiquitylates M-cyclins and S-cyclins (targets them for degredation)
- Inactivates the associated Cdks
What will prevent the APC from being activated?
Chromosomes are not correctly attached
What is the result of chromosomes not correctly attached?
APC is not activated, thus cyclins will not be degraded
When APC is activated, what can the cell do?
Progress to anaphase
What is cleaved in anaphase? What is the purpose of the cleaved things?
Cohesins
* Proteins that bind sister chromatids to each other
What does the cleavage of cohesins trigger?
Separation of sister chromatids to opposite poles of the cell
Separase
Enzyme that catalyses the degradation of cohesins
APC promotes the degradation of…
Securin
What does securin do?
Normally inhibits the action of separase
Describe the process of separation of the sister chromatids to their respective poles
A result of two processes affecting the mitotic spindle
* Anaphase A
* Anaphase B
What happens in:
* Anaphase A?
* Anaphase B?
Anaphase A
* Kinetochore microtubules shorten
* Tubulin subunits lost on both ends
Anaphase B
* Spindle poles move apart
Dynein
Achored to the cell cortex, pulls apart the spindle poles
Kinesins
Act on (growing) interpolar microtubules, causing them to slide past each other
APC, securin, Anaphase mechanisms
What experimental techniques are used in cell biology?
- Labelled nucleotides
- Fluorescence-activated cell sorting (FACS)
- Cells with synchronised division
How are labelled nucleotides used in cell biology?
Radioactive/fluorescent signal can be observed in the nucleus when labelled nucleotides are used during DNA replication in S phase
How is FACS used in cell biology?
- Cells are stained with dye that fluoresces when bound to DNA
- Amount of DNA = Amount of fluorescence emitted
- Cell sorter used to separate cells with different amounts of fluorescence
How are cells sorted in FACS based on amounts of fluorescence?
G2 cells have 2x the amount of DNA than G1 cells (double the fluorescence)
S cells have somehwere between G1 and G2 cells as replication wouldn’t be complete
What is used in cells with synchronised division?
Xenopus (frog) eggs
Why are large cells used in cell synchronization?
Provide a relatively large amount of cell extract, which can be used to purify proteins involved in the cell cycle
What happens in immature (unfertilized) eggs in the cell cycle?
Arrest in G2
How do we ensure eggs (cells) divide synchronized?
Fertilize the eggs at the same time
What cytoplasm injection experiments have been done in cell synchronization?
Cytoplasm of M phase cell injected into a cell in G2
* G2 cell goes to M phase
Ctyoplasm of S phase cell injected into a cell in G1
* G1 cell goes to S phase