Cell Cycle and Growth Control

Cell Cycle Overview

• The cell cycle and cell growth control are crucial for development and disease, particularly cancer.
• Focus on how cells decide when to divide and how they communicate with neighbors.

Phases of the Cell Cycle

• The cell cycle is often visualized as a clock, including:
  • M phase (mitosis): Cell division occurs.
  • G1 phase: Cell growth and preparation for DNA synthesis.
  • S phase: DNA synthesis occurs.
  • G2 phase: Further growth and preparation for mitosis.

Detailed Phase Activities

• Mitosis (M Phase):
  • The cell splits into two daughter cells.
  • Requires a mitotic spindle made of microtubules to pull apart chromosomes.
• G1 and G2 Phases:
  • Cell grows by making more of its components:
    • Increased plasma membrane.
    • More organelles.
• S Phase (DNA Synthesis):
  • DNA is copied accurately to maintain the integrity of the genome.
  • The genome contains all the genes needed to encode an entire organism.
• Post-DNA Synthesis:
  • Additional steps to prepare for mitosis, including organization of cell parts.
  • Critical checkpoints ensure all DNA is copied correctly before division.

Studying the Cell Cycle

• Cell cultures contain millions of cells at different stages of the cell cycle.
• Cells are typically distributed randomly across all phases.
• To study the cell cycle effectively, cells need to be synchronized.

Cell Synchronization

• Synchronization involves disrupting the cell population so that cells cannot progress past a certain point in the cycle.
• Example: Blocking microtubule growth using drugs like Colcemide.

Using Colcemide

• Colcemide prevents assembly of microtubules, thus inhibiting mitotic spindle formation.
• Cells get stuck early in M phase.
  • Colcemide \rightarrow No \, microtubule \, assembly \rightarrow Blocked \, M \, phase
• Application to an asynchronous cell population:
  • Cells in later stages of M phase complete mitosis and enter G1.
  • New cells cannot enter G1, creating a gap in the cell cycle.
  • Asynchronous \, cells + Colcemide \rightarrow Bunch \, up \, at \, start \, of \, M \, phase

Visualizing Synchronization

• Plotting the number of cells in M phase over time shows:
  • Initially, a normal number of cells in M phase.
  • After adding Colcemide, more cells accumulate in M phase.
  • After approximately 24 hours (for human cells), nearly all cells are in M phase.

Creating a Synchronous Cell Population

• Add Colcemide to the culture for about 24 hours to synchronize cells in M phase.
• Removing Colcemide allows cells to proceed through the cycle together.
• Cells move through G1, S, G2, and back to M phase synchronously.

Measuring S Phase

• Synchronized cell populations allow for precise measurements of cell cycle phases, such as S phase.
• S phase: DNA synthesis
  • Old strand + DNA Polymerase + ATP + Building Blocks(A,G,T,C) -> New complementary strand.
  • Labeled versions of building blocks used to mark DNA synthesis

Using Tritiated Thymidine

• Tritiated thymidine (radioactive T) is used to label newly synthesized DNA.
• Cells are fed tritiated thymidine, which is incorporated into the new DNA strands during S phase.

Measuring Incorporated Tritiated Thymidine

• After washing away excess tritiated thymidine, measure the radioactivity of the cells.
• The amount of incorporated tritiated thymidine indicates the level of DNA synthesis.

Data Interpretation

• Plotting incorporated tritiated thymidine over time:
  • An initial flat line indicates no DNA synthesis.
  • A rising phase indicates active DNA synthesis (S phase).
  • A plateau indicates the end of S phase.
• The length of S phase can be determined from the duration of the rising phase.
  G1 \rightarrow S \rightarrow G2

Measuring the Entire Cell Cycle

• The entire cell cycle length can be measured from the beginning of one S phase to the beginning of the next.
• Cell \, Cycle = G2 + M + G1 + S

Alternative Measurement: DNA Content per Cell

• Measure the total amount of DNA in a cell, using fluorescent labels that bind to DNA.

Flow Cytometry

• Flow cytometry is used to measure the fluorescence of individual cells.
• A flow cytometer sucks up cells and sends them through a narrow channel one by one.
• A camera measures the brightness of each cell in different fluorescent channels.
• The amount of fluorescence is proportional to the amount of DNA in the cell
• Data is plotted as a histogram, showing the number of cells versus DNA content.

Interpreting Flow Cytometry Data

• In an asynchronous population, the histogram shows two peaks:
  • A peak at a lower DNA content (cells in G1).
  • A peak at twice the DNA content (cells in G2 and M).
  • Cells in S phase are distributed between the two peaks.
• The height of the peaks indicates the relative number of cells in each phase.

Phase Length Interpretation

• A larger G1 peak suggests that G1 is longer than G2 plus M.

Cell Cycle Regulation

• To study cell cycle regulation, experiments were conducted using cow eggs.

Experiment

• Inject cytoplasm from a cell in M phase into an unfertilized oocyte.
• The oocyte suddenly enters M phase, forming a mitotic spindle.
• This indicates that certain chemicals or proteins in the cytoplasm control the start of M phase.

Key Proteins: M-Cdk and Cyclin

• M-Cdk (M phase-promoting factor):
  • Cell division Kinase with activity that rises and falls during the cell cycle, peaking in M phase.
• Cyclin:
  • Concentrations rise gradually throughout the cell cycle, reaching a maximum in M phase.
  • Cyclin activates M-Cdk.
  • At the end of M phase, cyclin is destroyed, causing M-Cdk activity to drop.
    \text{Cyclin Concentration} \uparrow \rightarrow \text{Active M-Cdk} \uparrow \rightarrow \text{Enter M Phase}

Cyclin as a Clock

• Cyclin acts as a clock, controlling the timing of M phase.
• The rate of cyclin synthesis determines the slope of its concentration increase.
• When cyclin reaches a threshold, it activates M-Cdk, triggering M phase.
• At the end of metaphase, cyclin is destroyed.

Discussion Section

• Analyze data to determine the length of different cell cycle phases.
• Learn about various experiments used to measure the cell cycle.