Prokaryotic Cytoskeleton and Its Functions

• Proteins in prokaryotic cells play roles similar to those of the cytoskeleton in eukaryotic cells.
• Key examples include:
  1. Actin-like MreB protein: Involved in DNA segregation during cell division.
  2. Tubulin-like FtsZ protein: Determines when bacterial cells will divide.
  3. Intermediate filament-like crescentin protein: Regulates bacterial cell shape.
• The FtsZ protein is also produced by organelles in eukaryotes like chloroplasts and mitochondria, and it localizes to the division sites of these organelles.

Dynamic Nature of the Cytoskeleton

• The cytoskeleton is a key focus of research, especially for its roles in cell motility and structure.

• Examples of cytoskeletal components:

  • Microfilaments: Essential parts of muscle fibers and contribute to structural integrity.
  • Microtubules: Serve as the framework for cilia and flagella, which help cells move through fluid environments.

• Historically, the cytoskeleton's large structures were studied using light microscopy before advanced techniques revealed their dynamic assembly and disassembly.

Techniques for Visualizing the Cytoskeleton

1. Fluorescence Microscopy on Fixed Specimens:

   • Fluorescent compounds or antibodies bind to cytoskeletal proteins, labeling them in preserved cells.
   • Example: Staining actin filaments in fibroblasts shows actin bundles under a fluorescence microscope.

2. Live Cell Fluorescence Microscopy:

   • Fluorescently tagged cytoskeletal proteins are introduced into living cells.
   • Example: Fluorescent tubulin molecules are incorporated into microtubules, which are easily visualized.

3. Computer-Enhanced Digital Video Microscopy:

   • High-resolution digital images from microscopes are processed to remove contrast-obscuring artifacts, improving visibility.

4. Electron Microscopy:

   • Provides detailed resolution of individual filaments using techniques like freeze-etching or deep-etching.
   • Can visualize the fine structure of the cytoskeleton.

Drugs Affecting Microtubule Assembly

1. Colchicine:

   • Derived from autumn crocus (Colchicum autumnale), it binds to tubulin and prevents it from forming microtubules.
   • Leads to the disassembly of existing microtubules.

2. Vinblastine and Vincristine:

   • Compounds from periwinkle plant (Catharanthus roseus), causing tubulin to aggregate inside the cell.
   • Used in anticancer treatments to stop cell division, as cancer cells divide rapidly.

3. Nocodazole:

   • A synthetic drug that inhibits microtubule assembly but is reversible after removal.

4. Taxol:

   • Derived from Taxus brevifolia (Pacific yew tree), it stabilizes microtubules, preventing their disassembly.
   • Promotes free tubulin’s incorporation into microtubules, blocking mitosis.
   • Commonly used in cancer treatments, especially for breast cancer.

Effects on Mitosis

• Drugs like taxol and colchicine disrupt the mitotic spindle, halting cell division.
• Their mechanisms differ:
  • Taxol stabilizes microtubules, preventing them from breaking down.
  • Colchicine prevents microtubules from forming.
• These effects make such drugs effective against rapidly dividing cancer cells, which rely on mitosis for proliferation.



Drugs Affecting Microfilaments

• Drugs can interfere with the assembly or stability of microfilaments, processes that are critical to cellular function.
• These drugs often disrupt the polymerization of actin filaments, affecting cellular structure and movement.

Key Drugs Affecting Microfilaments:

1. Cytochalasin D:

   • A fungal toxin that prevents new actin monomers from adding to the minus ends of microfilaments.
   • This inhibition leads to the depolymerization of microfilaments in treated cells, as existing filaments break down.

2. Latrunculin A:

   • A marine toxin isolated from the Red Sea sponge (Latrunculia magnifica).
   • It binds to actin monomers, sequestering them and preventing them from assembling into filaments.
   • The result is a reduction in microfilament content in treated cells.

3. Phalloidin:

   • A cyclic peptide toxin from the death cap mushroom (Amanita phalloides).
   • It stabilizes microfilaments, preventing their breakdown.
   • Fluorescently labeled phalloidin is also a tool for visualizing F-actin under a fluorescence microscope.

Microtubules: Structure and Functions

Overview of Microtubules

• Microtubules are the largest components of the cytoskeleton, playing vital roles in cellular shape, movement, and organization.
• In eukaryotic cells, they are categorized as:
  1. Cytoplasmic microtubules: Loosely organized, dynamic structures.
  2. Axonemal microtubules: Stable structures found in cilia, flagella, and basal bodies.

Functions of Cytoplasmic Microtubules

1. Structural Support:

   • Maintain axons and nerve cell extensions in animal cells.

2. Cell Shape and Division:

   • Help migrating animal cells retain their polarized shape.
   • In plant cells, they regulate the orientation of cellulose microfibrils during cell wall formation.

3. Chromosome Movement:

   • Facilitate the segregation of chromosomes during mitosis and meiosis.

4. Vesicle Transport:

   • Provide an organized system of tracks for vesicle movement within the cell.

Basic Structure of Microtubules

• Microtubules are hollow cylinders with a diameter of ~25 nm and an inner diameter of ~15 nm.
• They are composed of protofilaments, arranged longitudinally.
  • Each protofilament consists of α-tubulin and β-tubulin subunits forming a heterodimer.
• Typically, 13 protofilaments assemble side by side around a central hollow lumen.

Properties of Tubulin Heterodimers:

1. Each heterodimer contains:
   • One molecule of α-tubulin.
   • One molecule of β-tubulin.
2. The individual subunits bind to form a stable αβ-heterodimer.
3. Structural studies reveal that both tubulins share:
   • Nearly identical 3D structures.
   • ~40% amino acid sequence similarity.

Domains of Tubulin Subunits

1. N-terminal GTP-binding domain: Important for energy regulation and microtubule assembly.
2. Middle domain: Site for colchicine binding (a drug that blocks microtubule assembly).
3. C-terminal domain: Interacts with microtubule-associated proteins (MAPs).

Axonemal Microtubules

• Axonemal microtubules are highly organized and stable.
• Found in structures such as cilia and flagella, they are essential for cellular motility.