Activated Frog Eggs and Cell Division

  • Centrifugation of Frog Eggs

    • Activated frog eggs are broken open through gentle centrifugation.

    • This process also separates the cytoplasm from other cellular components.

    • Undiluted cytoplasm is collected for further experiments.

Mammalian Cell-Cycle Control System

  • Challenges in Observing Individual Cells

    • Observing cells in intact mammals is difficult.

    • Most mammalian cell-cycle control studies utilize cells isolated from tissues or tumors.

    • These cells are grown in plastic culture dishes with essential nutrients.

  • Replicative Cell Senescence

    • When normal mammalian tissue cells are cultured, they often stop dividing after a limited number of cycles.

    • Example: Human fibroblasts cease to divide after 25-40 divisions due to replicative cell senescence.

  • Immortalized Cell Lines

    • Mutations in mammalian cells can lead to immortalized cell lines that proliferate indefinitely in culture.

    • These lines are not normal but are widely used due to:

    • Unlimited source of genetically homogeneous cells.

    • Sufficient size for detailed cytological observations.

    • Facilitation of biochemical analysis of proteins involved in cell-cycle control.

  • Cancer Studies

    • Studies of cultured mammalian cells are crucial for understanding the mechanisms of cell proliferation control.

    • Understanding these controls is also important for addressing the loss of control in cancer cells, as discussed in Chapter 23.

Studying Cell-Cycle Progression

  • Methods to Identify Cell Stage

    • Observation via a microscope allows visualization of cells in mitosis.

    • S-phase cells cannot be directly observed but can be identified.

    • Labeling Techniques:

      • Use of visualizable molecules incorporated into new DNA, such as ³H-thymidine or BrdU (bromo-deoxyuridine).

      • Cells incorporating ³H-thymidine are visualized through autoradiography.

      • Cells incorporating BrdU are identified using anti-BrdU antibodies.

  • Labeling Index

    • In rapidly proliferating but asynchronously dividing cells, about 30-40% are typically in S phase at any given time.

    • From the labeled proportion, the S phase duration can be estimated relative to the total cell cycle duration.

  • Mitotic Index

    • The proportion of cells undergoing mitosis helps estimate the M phase duration.

  • DNA Content Measurement

    • DNA content doubles during S phase, allowing the use of DNA-binding fluorescent dyes analyzed by flow cytometry.

    • Flow cytometry enables the analysis of large cell populations rapidly and extracts data on G1, S, and G2+M phase durations.

Cell Reproduction Process

  • Basic Steps

    • Cell reproduction starts with content duplication, followed by distribution into two genetically identical daughter cells.

    • The cycle consists of two major phases:

    • S phase (Synthesis): DNA duplication takes 10-12 hours. Occupies about half the cycle time in a typical mammalian cell.

    • M phase (Mitosis): Shorter than S phase (less than an hour), involves dramatic events including nuclear division.

  • Mitosis Process

    • Starts with chromosome condensation.

    • Chromosomes condense into compact forms required for segregation.

    • The nuclear envelope degrades followed by microtubule attachment to chromatids.

    • Metaphase: Chromosomes aligned at the mitotic spindle's equator.

    • Anaphase: Sister chromatids separate and move to opposite poles.

    • Cytokinesis: The cell divides cytoplasm, completing the process.

The Importance of G1 and G2 Phases

  • Interphase Structure

    • Traditional cell cycle divided into four phases: G1, S, G2, and M.

    • Interphase: Comprises G1, S, and G2.

    • G1 may take 23 hours of a 24-hour cycle while M phase takes just 1 hour.

  • Role of Gap Phases (G1 and G2)

    • Provide time not only for growth but also for environmental monitoring to ensure readiness for DNA replication and mitosis.

    • G1 Phase Importance: Its duration can change based on external conditions.

    • Cells can enter a resting state (Go) if unfavorable conditions prevail, remaining there for extended periods.

    • When conditions are favorable and growth signals are present, cells progress past the commitment point (Start or restriction point) in G1, committing to DNA replication.

Cell-Cycle Control System Conservation

  • Eukaryotic Similarities

    • The underlying organization of the cell cycle is largely conserved across eukaryotes, dating back over a billion years.

    • Many control system proteins function across species, allowing research findings in one organism to inform studies in others.

Genetic Dissection of the Cell Cycle in Yeasts

Comparison of Yeast Species

  • Fission yeast (Schizosaccharomyces pombe) grows via elongation and divides by septum formation.

  • Budding yeast (Saccharomyces cerevisiae) forms a bud that separates after mitosis.