Eukaryotic cell cycle control

Phases of the Cell Cycle

1. G0 (Quiescent phase)

2. G1 (Gap 1)

3. S (Synthesis phase)

4. G2 (Gap 2)

5. M Phase (Mitosis)

G0 (Quiescent phase)

• Non-dividing state

• ~95% of cells remain here

• Cells can re-enter the cycle if stimulated

G1 (Gap 1)

• Cell growth

• Protein synthesis

• Decision point: divide or stay in G0

S (Synthesis phase)

• DNA replication occurs

• Each chromosome duplicated

G2 (Gap 2)

• Preparation for mitosis

• Error checking (DNA integrity)

M Phase (Mitosis)

• Division of nucleus and cytoplasm

Key mitotic events:

• Chromosome condensation

• Nuclear envelope breakdown

• Spindle formation

• Chromosomes align at metaphase plate

• Sister chromatids separate

• Cytokinesis → two daughter cells

Cyclin-CDK System

• Cyclins: regulatory proteins (levels fluctuate)

• CDKs: enzymes (constant levels, activated by cyclins)

Function:

• Drive transitions between phases

• Ensure:

  • DNA replicates once only

  • Chromosomes segregate correctly

Temporal Coordination (Timing)

The cell cycle must occur in the correct order:

1. Growth (G1)

2. DNA replication (S)

3. Preparation (G2)

4. Division (M)

Each step depends on completion of the previous step

Spatial Coordination (Location)

Processes must occur in the correct cellular locations:

• DNA replication → nucleus

• Spindle formation → cytoplasm

• Chromosome segregation → mitotic spindle

Coordinated Cellular Events

• Centrosome duplication

• Spindle assembly

• Chromosome condensation

• Nuclear envelope breakdown

These must be synchronized for accurate division

Why Coordination Matters

• Prevents:

  • DNA re-replication

  • Chromosome mis-segregation

• Ensures genomic stability

Xenopus Oocytes

• Large cells → easy to manipulate

• Naturally synchronized

• Arrested at specific stages

Oocyte Maturation

Initial state:

• Oocytes arrested in G2-like state

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Trigger:

• Progesterone hormone

➡ Induces:

• Entry into meiosis I

• Progression toward meiosis II

Key Experimental Insight

• Cytoplasm from mature oocytes can:

  • Trigger maturation in immature oocytes

  • WITHOUT progesterone

Conclusion

➡ A soluble cytoplasmic factor controls maturation
➡ This factor = Maturation Promoting Factor (MPF)

experimental evidence that led to the definition and characterization of MPF

• Cell Fusion Experiments

  • Discovery of MPF (M-phase factor)

  • S-phase Promoting Factor

• Oocyte Cytoplasm Transfer

• MPF Activity Correlation

• MPF is a Kinase

Discovery of MPF (M-phase factor)

Experiment:

• Fuse:

  • M phase cell + G1/G2 cell

Result:

• Partner cell enters mitosis

Conclusion:

M-phase cells contain a diffusible mitotic factor (MPF)

S-phase Promoting Factor

Experiment:

• Fuse:

  • S phase cell + G1 cell → DNA replication occurs

  • S phase cell + G2 cell → NO replication

Conclusion:

• S-phase factor exists

• Only G1 cells respond

Oocyte Cytoplasm Transfer

• Cytoplasm from mature oocytes:

  • Induces mitosis in immature oocytes

Confirms:

• MPF is soluble and transferable

MPF Activity Correlation

• MPF activity:

  • Peaks during mitosis

  • Declines after mitosis

Direct link between MPF and mitotic entry

MPF is a Kinase

• Phosphorylates proteins (radioactive phosphate experiments)

Drives mitotic processes

Cyclin Discovery Experiment

Method:

• Fertilized sea urchin eggs

• Added radioactive methionine

• Tracked newly synthesized proteins

Observation:

• Protein levels:

  • Increase before mitosis

  • Rapidly decrease after

Named Cyclin

Cyclin and MPF

• MPF = Cyclin B + CDK

• Cyclin controls CDK activity

Cyclin Oscillation

• Cyclin B accumulates → mitosis begins

• Cyclin B degraded → mitosis ends

Key experiment: Cyclin required for mitosis

Blocking protein synthesis → no mitosis

Key experiment: Cyclin B mRNA addition

Restores mitotic activity

Key experiment: Non-degradable Cyclin B

• Cells enter mitosis

• Cannot exit

Conclusion:

Cyclin degradation is required for mitotic exit

Composition of MPF

• Cyclin B (regulatory)

• CDK (kinase)

Function of MPF

MPF triggers:

• Chromosome condensation

• Nuclear envelope breakdown

• Spindle formation

• Chromosome alignment

MPF Regulation of mitosis Mechanism

1. Cyclin B accumulates

2. CDK activated → MPF formed

3. Cell enters mitosis

4. Cyclin B degraded

5. MPF inactivated → mitotic exit

➡ MPF activity must rise and fall cyclically