Ch 9

Overview of the Cytoskeleton

• The cytoskeleton is the skeletal system of eukaryotic cells, composed of three filamentous structures:

  1. Microtubules - Long, hollow unbranched tubes made of tubulin.

  2. Microfilaments - Solid, thinner branching network made of actin.

  3. Intermediate Filaments - Tough, rope-like fibers composed of globular proteins.

• Functions of the Cytoskeleton:

  1. Provides structural support and maintains cell shape.

  2. Acts as a framework for positioning organelles.

  3. Functions as a network of tracks for intracellular transport.

  4. Generates forces needed for cellular locomotion.

  5. Plays a crucial role in cell division, particularly in chromosome segregation.

II. Microtubules

A. Structure and Composition

• Hollow, rigid tubular structures with an outer diameter of 25 nm and wall thickness of 4 nm.

• Composed of αβ-tubulin heterodimers, assembled into protofilaments.

• Polarity: Plus-end (β-tubulin) and minus-end (α-tubulin).

B. Microtubule Associated Proteins (MAPs)

• Stabilize and regulate microtubules.

• Example: MAP2, which has three tubulin-binding sites.

• Tau protein (MAP) is involved in Alzheimer’s disease when excessively phosphorylated.

C. Microtubule Functions

1. Structural support and organization

   • Found in epithelial and nerve cells for organelle transport.

   • Forms the mitotic spindle during cell division.

2. Intracellular transport

   • Microtubules act as tracks for motor proteins (Kinesin and Dynein).

3. Motility

   • Present in cilia and flagella.

D. Microtubule Elongation and Regulation

• Microtubule Organizing Centers (MTOCs): Structures where microtubules originate (e.g., centrosomes).

• Microtubule growth and shrinkage regulated by GTP hydrolysis.

III. Motor Proteins

A. Kinesin

• Plus-end directed motor protein involved in anterograde transport.

• Structure:

  • Two identical heavy chains forming a stalk.

  • Two globular heads that bind microtubules and hydrolyze ATP.

• Movement:

  • Hand-over-hand model with 8 nm steps.

  • Highly processive (travels long distances without detaching).

• Kinesin variants:

  • Kinesin-1: Moves cargo towards plus-end.

  • Kinesin-14: Moves towards the minus-end.

  • Kinesin-13: Depolymerizes microtubules.

B. Dynein

• Minus-end directed motor protein responsible for retrograde transport.

• Cytoplasmic dynein transports vesicles, organelles, and chromosomes.

• Axonemal dynein drives the movement of cilia and flagella.

IV. Microfilaments (Actin Filaments)

A. Structure and Composition

• Composed of actin monomers assembled into a helical structure.

• Thin and flexible compared to microtubules.

B. Functions

1. Supports microvilli in epithelial cells.

2. Aids in cell motility (e.g., amoeboid movement).

3. Essential for cytokinesis during cell division.

C. Actin-Associated Proteins

• Filamin: Cross-links actin filaments.

• Thymosin and Profilin: Regulate actin polymerization.

• Myosin: Actin-based motor protein for intracellular transport and contraction.

V. Intermediate Filaments

A. Structure and Composition

• Rope-like fibers composed of various proteins (keratins, vimentin, laminins).

• More stable and durable than microtubules and microfilaments.

B. Functions

1. Provides mechanical strength to cells.

2. Supports nuclear structure (lamins).

3. Anchors organelles within the cytoplasm.

VI. Techniques to Study the Cytoskeleton

1. Fluorescence Microscopy

   • Uses fluorescently labeled proteins (e.g., GFP-tubulin) to visualize cytoskeletal structures.

2. Fluorescence Recovery After Photobleaching (FRAP)

   • Studies protein dynamics by bleaching a region and observing recovery.

3. Optical Tweezers

   • Uses laser beams to manipulate motor proteins.

4. Total Internal Reflection Fluorescence (TIRF) Microscopy

   • Observes molecular motors (e.g., kinesin moving on microtubules).

VII. Cytoskeleton-Related Diseases

1. Alzheimer’s Disease

   • Caused by hyperphosphorylated Tau protein, leading to neurofibrillary tangles.

2. Kartagener’s Syndrome

   • Defective dynein arms in cilia lead to respiratory issues and infertility.

3. Cancer

   • Microtubule-targeting drugs (e.g., Taxol) used in chemotherapy disrupt mitotic spindles.

VIII. Cilia and Flagella

A. Structure

• Composed of microtubules arranged in a 9+2 pattern.

• Movement powered by axonemal dynein.

B. Functions

• Cilia: Moves mucus in the respiratory tract.

• Flagella: Propels sperm cells.

IX. Intra-Flagellar Transport (IFT)

• Essential for the assembly and maintenance of cilia and flagella.

• Mediated by Kinesin-2 (anterograde transport) and Dynein (retrograde transport).

X. Applications of Cytoskeletal Research

1. Nanotechnology

   • Development of nanomachines for medical applications.

2. Medical Therapies

   • Microtubule inhibitors (e.g., Taxol) used in cancer treatment.

3. Regenerative Medicine

   • Cytoskeleton-based therapies for neurological disorders.