Cell Cycle Regulation and Discovery of Cdks

Cell Synchronization and Measurement

Synchronizing cell populations using drugs to block cell cycle steps.
Using labels like tritiated thymidine to measure DNA synthesis.
Flow cytometry to count DNA in individual cells, visualizing differences between cells with doubled DNA vs. single copy.
Peak heights in flow cytometry used to estimate the duration of cell cycle phases.

Cytoplasm of M phase cells contains factors that trigger M phase.
Injecting M phase cytoplasm into non-M phase cells induces mitotic spindle formation.
Cdks (Cell Division Kinases):
- Turned on during specific cell cycle phases.
- M-Cdks trigger M phase when activated.
- Activation leads to activation of other proteins, triggering cellular activities.
Cyclins:
- Control when Cdks are activated.
- Cyclin concentration increases over time; reaching a threshold triggers a specific phase.
- M-Cyclin: Triggers M phase; destroyed at the end of M phase.
- S-Cyclin: Triggers S phase; remains high during G2 phase, drops during M phase.
- Timing controlled by how long it takes to synthesize enough cyclin to trigger the next phase.

M-Cyclin concentration increases linearly, drops sharply at the end of M phase, and remains low during G1.
S-Cyclin concentration rises earlier than M-Cyclin, remains high during G2, and drops at the beginning of M phase.
G1 phase occurs after M phase when M-Cyclin levels drop.
Different cyclins and Cdks control different phases, acting as internal clocks.
Daughter cells replicate the same clock machinery as parent cells, maintaining synchronization through multiple divisions.
Synchronization may break down after about 10 cell cycles.

Cells exit the cell cycle and stop replicating.
Most adult cells are in G0 phase.
Embryonic cells divide rapidly with few or no cells in G0.
Cells can re-enter the cell cycle from G0 (e.g., during wound healing).
The decision to enter or exit G0 is controlled by external and internal factors.

Checkpoints regulate cell cycle progression based on internal and external signals.
Growth factors secreted by immune cells signal neighboring cells to replicate after injury.
Environmental signals, such as mechanical stiffness and extracellular matrix proteins, also influence cell growth decisions.
Signals to inhibit growth are crucial for preventing cancer.
Checkpoints before M phase ensure DNA is fully copied and errors are repaired.
Length of cell cycle can vary due to time required for DNA repair.
Checkpoints exist during S phase to ensure DNA quality.
These checkpoints ensure that daughter cells receive a good copy of DNA.
Failure of checkpoints leads to mutations, potentially breaking growth control pathways and causing cancer.

Using mutants to understand cell cycle control.
Creating mutants that disrupt specific cell cycle phases.
Identifying genes controlling critical proteins for each phase.
Problem: Mutations that break the cell cycle can prevent cell growth and division.
Cell cycle mutations often prevent cell growth, making it difficult to study essential genes.
Conditional mutants can be turned on and off, allowing cells to divide until the mutation is activated for study.

Conditional Mutant: A mutation that causes a minor error in protein folding.
At lower temperatures, the protein folds normally and is functional.
At higher temperatures, the protein misfolds and becomes non-functional.
Example: Protein folds properly at 25°C but misfolds at 37°C, leading to loss of function.
Cells with a conditional mutant will move through the cell cycle normally at a lower temperature but get stuck at a specific stage when the temperature is increased.
Useful for studying the effects of mutations on different cell cycle stages.(e.g. G2)
Temperature-sensitive mutations affect protein folding, leading to loss of function at higher temperatures.

M phase is easily observed by visualizing cell division.
Combining conditional mutants with cell cycle arresting drugs to identify specific stages.
Using drugs that prevent S phase to synchronize cells at the beginning of S phase.
Releasing the drug while activating the conditional mutant to observe where the cells get stuck.

Step 1: Treat cells with a drug that blocks S phase at 25°C.
Step 2: Remove the drug and increase the temperature to 37°C.
Cells will move from S phase to the stage where the conditional mutant blocks them.
If the conditional mutant blocks G2, cells will accumulate in G2.
By manipulating both drug and temperature one can determine the stage at which Conditional mutants and drugs take effect.