14. Cell Cycle Checkpoints and Mitotic Regulation

Foundations of Cell Cycle Regulation and the Logic of Checkpoints

The cell cycle is a complex, highly regulated process essential for the division of approximately 10 trillion10 \text{ trillion} cells in the human body. The fundamental question addressed is why the process must be so complicated. The answer lies in the necessity of redundancy and regulation to ensure genomic integrity. A "checkpoint" is a regulatory process that functions to verify if specific biological criteria are met before the cell proceeds to subsequent phases. These checkpoints act as gatekeepers to prevent errors like DNA damage or improper chromosome segregation from being propagated.

Core Regulatory Questions at Checkpoints

  1. Check if everything is correct or not!

  2. DNA Integrity: Is all DNA replicated accurately?

  3. Environmental Status: Is the surrounding environment favorable for cell division (e.g., presence of nutrients and growth factors)?

  4. Structural Readiness: Are all chromosomes correctly attached to the spindle?

Major Transition Points in the Cell Cycle

  • Start Transition (G1 to S): The cell enters the cycle and proceeds to S phase. This is the biggest commitment in terms of energy. The primary check here is whether the environment is favorable.

  • G2/M Transition: The cell enters mitosis. The checks include DNA replication completion and environmental favorability.

  • Metaphase-to-Anaphase Transition: This triggers anaphase and proceeds to cytokinesis. The primary requirement is that all chromosomes are properly attached to the spindle.

The G1 Phase and the Restriction Point (Checkpoint 1)

The G1 checkpoint is the point of entry into the cell cycle. Cells that are starved of serum (growth factors) will arrest in G1. If conditions are unfavorable, the cell may enter a quiescent state known as G0.

Growth Factor-Mediated Signaling

External signals such as Growth Factors act on receptors in the cell membrane, activating a signaling pathway that leads into the nucleus. Downstream of the Mitogen-Activated Protein Kinase (MAPK) pathway, several events occur:

  1. Early Genes: Activation of genes like Myc.

  2. Cyclin/CDK Production: Production of CDK4/6 and Cyclin D.

  3. G1-CDK Activity: The G1-Cdk complex (Cyclin D + CDK4/6) triggers the transcription of S-phase genes.

The Role of Retinoblastoma (Rb) Protein

Rb is a tumor suppressor gene originally discovered in association with childhood retinoblastoma tumors. It acts as a negative regulator of the cell cycle.

  • Without Growth Factors: Active Rb protein binds to the E2F protein (a transcription factor), keeping it inactivated. This prevents the transcription of S-phase genes like Cyclin E or Cyclin A.

  • With Growth Factors: G1-Cdk (Cyclin D/CDK4/6) phosphorylates the Rb protein. Once phosphorylated, Rb changes conformation and releases the E2F protein.

  • Positive Feedback: Active E2F promotes the transcription of its own gene and the genes for G1/S-cyclin (Cyclin E) and S-cyclin (Cyclin A). The resulting G1/S-Cdk further phosphorylates Rb, ensuring a robust, irreversible commitment to entering the S phase and initiating DNA synthesis.

DNA Damage Response (G1 Checkpoint)

If DNA damage is detected, a specific checkpoint is induced:

  • The ATM kinase is activated.

  • This leads to the activation of p21, a Cdk inhibitor protein.

  • The cell cycle stops at G1, preventing the cell from entering S phase until the damage is repaired, or it may enter G0.

The G2/M Transition and Mitosis Promoting Factor (MPF)

At the end of G2, the cell must decide whether to enter mitosis. Cell cultures subjected to DNA damaging reagents or those with incomplete replication will arrest here.

Replication Stress-Induced Checkpoint

Problems in replication, such as the presence of single-stranded DNA (ssDNA), activate a signaling cascade:

  1. ATR kinase is activated by ssDNA.

  2. ATR activates Chk1 kinase.

  3. Chk1 inhibits the phosphatase Cdc25 by adding an inhibitory phosphate.

  4. Cdc25 is normally responsible for removing the inhibitory phosphate from M-Cdk (Cdk1 + Cyclin B).

  5. If Cdc25 is inactive, Wee1 (a kinase) keeps an inhibitory phosphate on M-Cdk, halting the cell cycle at G2.

Activation of M-Cdk (MPF)

When conditions are favorable and DNA is fully replicated:

  • Cdk-activating kinase (CAK) adds an activating phosphate.

  • Cdc25 removes the inhibitory phosphate placed by Wee1.

  • This creates active M-Cdk (also known as Maturation/Mitosis Promoting Factor, or MPF).

  • Positive Feedback: Active M-Cdk further activates Cdc25 and inhibits Wee1 to ensure a rapid transition into mitosis.

Molecular Functions of MPF and Mitosis Structures

MPF functions in concert with other kinases, such as Aurora Kinase and Polo-like kinases (PLK), to phosphorylate specific substrates that drive the structural changes of mitosis.

Structural Changes Driven by MPF

  • Nuclear Envelope Breakdown (NEBD): MPF and PKC phosphorylate nuclear lamins (specifically lamin tetramers) and nuclear pore proteins. This causes the laminate polymers to disassemble into phosphorylated lamin dimers, causing the nuclear envelope to breakdown. This process is reversible; the removal of phosphates allows the envelope to reform later.

  • Chromosome Condensation: MPF and Aurora B promote the compaction of chromatin into mitotic chromosomes.

  • Kinetochore Assembly: Attachment sites for spindle microtubules are formed.

  • Spindle Formation: Interphase microtubule arrays are replaced by mitotic asters.

Dimensions of Condensation

  • Interphase structures: ≈50 nm\approx 50\,nm (Nuclear pore) and ≈300 nm\approx 300\,nm fibers.

  • Condensing sections: ≈700 nm\approx 700\,nm.

  • Full mitotic chromosome: ≈1400 nm\approx 1400\,nm.

Chromosome Architecture: Cohesins and Condensins

Cohesins

Cohesin complexes hold sister chromatids together from S phase through metaphase. They consist of proteins including Smc1, Smc3, Scc1, Scc3, Pds5, and Wapl.

Condensins

Condensins regulate loop extrusion and supercoiling to compact individual sister chromatids.

  • Condensin II: Functions in G2/prophase for initial condensation.

  • Condensin I: Functions later for secondary loop formation.

  • Subunits include Smc2, Smc4, and Cap-H/H2 proteins. Aurora B and MPF are required for their regulation.

The Metaphase-to-Anaphase Transition (Checkpoint 3)

This checkpoint ensures that chromosomes are accurately segregated. Cells treated with microtubule inhibitors will arrest here.

The Bipolar Attachment and Tension Sensing

The most stable structure is bipolar chromosome attachment with tension. Tension is sensed via the Ndc80 complex and Aurora B kinase.

  • Low Tension: If a chromosome is not properly attached, Aurora B (located at the inner kinetochore) phosphorylates Ndc80 (at the outer kinetochore). Phosphorylated Ndc80 has a reduced affinity for microtubules, causing them to detach so a correct attachment can be attempted.

  • High Tension: When bipolar attachment is achieved, the kinetochores are pulled apart. Ndc80 moves out of the range of Aurora B, phosphorylation ceases, and the microtubule attachment becomes stable.

Triggering Anaphase: The APC/C Complex

For chromosomes to separate, the cohesive forces holding sister chromatids together must be destroyed.

  1. APC/C (Anaphase Promoting Complex/Cyclosome): An ubiquitin ligase that identifies targets for destruction.

  2. Securin: An inhibitory protein that keeps the enzyme Separase inactive.

  3. Cdc20: Activates APC/C.

  4. APC/C-Cdc20 ubiquitylates Securin, leading to its degradation.

  5. Released Separase (a protease) cleaves the Scc1 subunit of the cohesin complex.

  6. The "Sister Chromatin" is cut open, allowing segregation.

Spindle Assembly Checkpoint (SAC)

If a kinetochore is unattached, the SAC is activated:

  • The Mitotic Checkpoint Complex forms.

  • Cdc20 is inhibited.

  • APC/C remains OFF.

  • Chromosomes stop at metaphase and do not proceed to anaphase.

Questions & Discussion

Q: Which step is the biggest commitment? (Energy)A: The G1 phase/Start transition to begin the process, which involves entering the cell cycle and proceeding to the S phase.

Q: What is downstream of MAPK?A: Early genes like Myc, then CDK4/6 and Cyclin D, leading to G1-Cdk activity.

Q: What is the role of G1-CDK?A: It triggers transcription of S-phase genes (e.g., Cyclin E) by phosphorylating and inactivating the Rb protein.

Q: Where do spindle microtubules attach to chromosomes?A: They attach to the kinetochore, a protein complex built on the CENP-A (a histone H3 variant) at the centromere.

Q: How do cells sense chromosome attachment?A: Through tension sensing and the Spindle Assembly Checkpoint (SAC), where Aurora B phosphorylates Ndc80 in the absence of tension to prevent premature anaphase.**