The Cell Cycle and Mitosis

Cell Cycle Architecture

• The eukaryotic cell cycle is divided into two broad parts:
  • Interphase – all of the cycle outside of M phase
  • M phase (Mitosis + Cytokinesis)

Standard ordering: G1 \rightarrow S \rightarrow G2 \rightarrow M

Complete example timing from lecture (typical mammalian somatic cell):
• G1 \approx 10\,\text{h} • S \approx 8\,\text{h} • G2 \approx 5\,\text{h}
• M \approx 1\,\text{h}
• Total \approx 24\,\text{h}

Interphase Details

• G1 (Gap-1)
  • Cell growth, RNA & protein synthesis, preparation for DNA replication.
  • Environmental sensing & major checkpoint (restriction point/R-point).
  • Length is the most variable of all phases; highly influenced by nutrients & mitogens.

• S (Synthesis)
  • Entire nuclear DNA is duplicated.
  • Histone production up-regulated; centrosome replication begins.
  • BrdU or ^3\text{H}-Thymidine incorporation are classical markers.

• G2 (Gap-2)
  • Proof-reading of replicated DNA, repair, synthesis of M-phase proteins.
  • Contains G2/M checkpoint (DNA damage & unreplicated DNA response).
  • Cells usually display doubled centrosomes positioned on opposite sides of nucleus.

Mitosis – Sub-phases & Key Events

Prophase

• Chromatin condenses ⇒ discrete chromosomes become visible.

• Each chromosome already consists of 2 sister chromatids joined at a centromere.

• Nucleolus disappears; nuclear envelope begins to fragment.

• Mitotic spindle (microtubule array) starts assembling between centrosomes.

• Chromosomes have no organised orientation yet.

Prometaphase (early metaphase – shown on detailed figure)

• Nuclear envelope completely breaks down → spindle microtubules enter nuclear space.

• Kinetochore complexes assemble on centromeres; microtubules attach.

• Chromosomes exhibit rapid, jerky movements driven by microtubule dynamics.

Metaphase

• Chromosomes align on the metaphase (equatorial) plate.

• Mitotic spindle fully organised; microtubules span from each spindle pole to kinetochores.

• Disappearance of the nuclear membrane is complete, allowing clear microscopy visualisation.

Anaphase

• Cohesin complexes are cleaved at centromeres ⇒ sister chromatids separate.

• Resulting daughter chromosomes move toward opposite poles along shortening kinetochore microtubules.

• Polar microtubules lengthen, further separating poles.

Telophase

• Spindle apparatus disassembles.

• Chromosomes de-condense back to diffuse chromatin.

• Nuclear envelopes re-form around each chromosome set; nucleoli reappear.

Cytokinesis

• Actomyosin contractile ring constricts the cell membrane → cleavage furrow.

• Cytoplasm is partitioned, yielding two genetically identical daughter cells.

Structural & Molecular Components

• Centrosome/centriole pair: microtubule organising centres at spindle poles.

• Spindle microtubule classes:
  • Kinetochore (attach to centromere)
  • Polar (interdigitate at mid-zone, push poles apart)
  • Astral (anchor spindle to cortex).

• Kinetochore: multi-protein complex providing motor activity & SAC (spindle-assembly checkpoint) signalling.

Plant vs. Animal Mitosis (diagram reference)

• Major difference: plant cells lack centrioles; spindle originates from cortical MTOCs.

• Cytokinesis:
  • Animals – contractile ring → cleavage furrow.
  • Plants – vesicle-derived cell plate forms new cell wall.

Techniques to Measure Phase Durations

1. Mitotic Arrest with Colcemid (microtubule antagonist)

• Colcemid prevents spindle fibre assembly ⇒ cells accumulate in metaphase.

• Plot % of metaphase cells vs. time after drug addition.
  • When curve plateaus (≈100 % at \sim 24\,\text{h} in example) → estimate complete cycle length.

2. Mitotic Index Method

• Visually count cells in M phase (e.g., 3 of 75).

Apply basic proportionality: tM = \dfrac{NM}{N{total}} \times T{cycle}
• Example: \dfrac{3}{75} \times 24\,\text{h} = 0.96\,\text{h} (mitosis duration).

3. BrdU Labelling for S-phase

• Supply cells with BrdU (thymidine analogue) briefly.

• Detect incorporation via anti-BrdU antibody.

Fraction labelled gives S length: tS = f{BrdU} \times T_{cycle}
• Example: \dfrac{33}{100} \times 24\,\text{h} = 7.92\,\text{h}.

4. Synchronous Release & ^3\text{H}-Thymidine Pulse

Arrest population in metaphase with Colcemid (>24/h) then wash out → cells start G1 synchronously.

• Every hour add ^3\text{H}-Thymidine for 1 h; measure DNA radioactivity.

• Peak in incorporation marks entry into S.

• Example data: G_1 \approx 10\,\text{h},\; S \approx 8\,\text{h}.

5. Hydroxyurea Block-Chase for G2 Length

• Label cells with thymidine analogue, then add hydroxyurea (inhibits dNTP synthesis).
  • Cells already in mid-S arrest; others finish current cycle and halt at next S start.

• Wash out & add Colcemid; measure time when labelled cells appear in M → gives G_2.

Conceptual & Practical Significance

• Precise knowledge of cell-cycle kinetics underpins:
  • Cancer biology (hyper-proliferation, checkpoint defects, chemotherapeutic targeting).
  • Developmental biology (timed divisions during morphogenesis).
  • Regenerative medicine & tissue engineering (stem-cell expansion control).

• Drugs like Colcemid, hydroxyurea, taxol serve both as research tools and clinical agents; ethical use requires minimising off-target toxicity and environmental release.

Key Formulae & Quantitative Relationships

Phase time from fraction labelled/indexed: t{phase} = f{phase} \times T_{cycle}.

Example full breakdown:
T{cycle} = t{G1}+tS+t{G2}+t_M
24\,\text{h} = 10\,\text{h} + 8\,\text{h} + 5\,\text{h} + 1\,\text{h}.