AG

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.

Regulation of the Cell Cycle

  • 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.

Cyclin Concentration and Cell Cycle Phases

  • 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.

G0 Phase

  • 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

  • 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.

P53 Protein

  • P53 detects DNA damage before S phase.
  • If DNA damage is detected, P53 halts the cell cycle to allow for repair.
  • Once DNA is repaired, P53 allows the cell cycle to proceed.
  • If DNA cannot be repaired, P53 triggers apoptosis (cell suicide).
  • Apoptosis prevents replication of damaged DNA that could lead to cancer.

Discovery of Cdks

  • 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 Mutants

  • 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.

Observing Cell Cycle Stages

  • 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.

Experimental Setup

  • 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.