Cell Cycle and Regulation

Flow Cytometry and Cell Sorting

  • Flow cytometry allows for the analysis of individual cells as they pass through a laser.

    • Each cell is examined and data is collected for various properties.

    • Output produces a graph representing the distribution of cell properties (e.g., size, granularity).

  • Unlike flow cytometry, cell sorting separates cells into different tubes based on specific criteria.

    • For instance, cells can be sorted based on the number of DNA copies:

    • One copy of DNA → Tube 1

    • Two copies of DNA → Tube 2

    • Cells in between → Tube 3

  • Applications of sorting include studying specific cell populations such as G1 and G2/M phase cells for future experiments.

  • Cost Consideration: Sorting is typically more expensive than basic flow cytometry due to the complexity of separating and collecting cells into multiple tubes.

Cell Cycle Checkpoints

  • The cell cycle has three major checkpoints that ensure proper progression through each phase:

    1. G1/S Checkpoint:

    • Assess if the environment is favorable for cell division.

    • Key questions considered:

      • Is the cell size stable?

      • Does it have sufficient resources (enzymes, space)?

    1. G2/M Checkpoint:

    • Confirm that DNA replication is complete before mitosis begins.

    • Questions include:

      • Is there adequate ATP?

      • Has all DNA been replicated correctly?

    1. Metaphase/Anaphase Transition (APC Checkpoint):

    • Checks if all chromosomes are correctly attached to the spindle apparatus.

    • If not attached properly, the cell will not move to anaphase, preventing division.

Cyclins and Cyclin-Dependent Kinases (CDKs)

  • Cyclins are proteins whose levels fluctuate throughout the cell cycle and play a critical role in regulating checkpoints.

    • Types of cyclins include:

    • Cyclin D (G1 phase)

    • Cyclin E (G1/S transition)

    • Cyclin A (S phase)

    • Cyclin B (M phase)

  • Function of Cyclins:

    • Cyclins activate CDKs, which are enzymes that phosphorylate target proteins, driving the cell cycle forward by activating signaling pathways.

  • Process of Activation:

    • Cyclin binds to a CDK, leading to partial activation.

    • Further phosphorylation by the CDK activating kinase (CAK) is required for full activation.

    • Inhibition of CDKs:

    • Inhibitory phosphorylation can occur, which prevents kinase activity when a second phosphate is added.

    • CDK inhibitor proteins (CKIs), such as p27, can also inhibit active cyclin-CDK complexes.

    • Key regulators: CDK1, CDK2, CDK4, and CDK6 are commonly involved.

  • Role of APC (Anaphase Promoting Complex):

    • Essential for regulating the transition from metaphase to anaphase, by tagging cyclins for degradation to move forward in the cycle.

    • APC is part of the ubiquitin ligase family, promoting the degradation of cyclins like Cyclin B when mitosis concludes.

The Cell Cycle and Phases

  • Overview of Checkpoints:

    • G1/S promotes transition into S phase. G2/M confirms readiness for mitosis.

    • Each transition is crucial to ensure cellular integrity before reproduction.

S Phase: DNA Replication

  • During S phase, DNA replication must be conducted carefully to produce two identical copies, referred to as sister chromatids.

    • The formation of the pre replicative complex (pre-RC) is critical for initiating DNA replication at origins that are rich in adenine and thymine (A&T).

    • Components of the pre-RC include:

    • ORC (Origin Recognition Complex)

    • CDC6 and CDT1 as helper proteins

    • MCM, a helicase that unwinds DNA for replication

  • Replication Activation:

    • The activation of S phase-specific CDKs leads to the promotion of appropriate protein complexes to facilitate the start of DNA replication.

Proteins Involved in Cohesion and Condensation

  • Cohesin: Holds sister chromatids together post-replication.

    • Structure: Comprised of SMC (Structural Maintenance of Chromosomes) molecules.

    • Condensin: Tightly condenses chromosomes for efficient segregation during mitosis.

Overview of Mitosis

  • Mitosis can be divided into distinct phases:

    1. Prophase: Chromatin condenses, nuclear envelope begins to break down, spindle apparatus forms.

    2. Metaphase: Chromosomes align at the cell equator, checked for proper attachment.

    3. Anaphase: Sister chromatids separate and are pulled toward opposite poles of the cell.

    4. Telophase: Chromatids reach poles, nuclear envelope reform around each set.

    • A critical checkpoint before anaphase: Is regulated by APC ensuring all chromosomes are attached before proceeding.

Kinetochore Dynamics

  • The kinetochore is a crucial protein structure where spindle microtubules attach to chromosomes.

    • Chromatids must be bi-oriented, meaning each sister chromatid must attach to microtubules from opposite poles.

    • Microtubule dynamics play a role in the attachment and movement of chromosomes during division, ensuring correct segregation.

    • Forces involved:

    • Depolymerization of microtubules at their plus ends

    • Microtubule flux at the minus ends

    • Polar ejection forces from kinesins directing proper alignment.

Conclusion and Summary

  • The precise orchestration of cell cycle and division mechanisms is critical for cellular health and development. Defects in these processes can lead to significant consequences, including tumorigenesis. Understanding the molecular players and regulatory mechanisms is essential for insights into developmental biology and oncology.