20250210_Cytoskeleton Lecture 2

Cytoskeleton Overview

• Covered in Molecular Biology of the Cell (7th Edition): Chapter 16

• Importance of understanding the cytoskeleton in cellular structure and function.

Actin Filaments

Assembly of Actin

• G-actin monomers assemble head to tail to form actin filaments.

  • Plus end grows faster than the minus end.

  • Subunits hydrolyze ATP to ADP once part of a filament.

  • ADP-bound subunits dissociate from filament ends, leading to actin treadmilling.

Actin Binding Proteins

• Different classes include:

  • Monomer Binding Proteins

  • Filament Binding Proteins

  • Actin Nucleating Proteins

  • Filament Severing Proteins

  • Filament Crosslinking Proteins

Filament Severing Protein: Cofilin

• Cofilin binds side of actin filaments causing them to twist tighter.

  • This increased tension weakens subunit binding, leading to easier disassembly.

  • Binds preferentially to ADP-bound actin which is usually older.

  • Vital for cell migration.

Myosin Motor Proteins

Myosin Structure and Function

• Myosin is an actin-associated motor protein driving cellular movement.

  • Myosin II Dimer:

    • Walks along actin filaments using ATP.

    • Heavy chains composed of long α-helical sequences form a coiled-coil structure.

    • Contains a globular head that binds actin and hydrolyzes ATP.

    • Light chains modify head movement via phosphorylation.

Myosin Movement Cycle

Stages of Myosin Movement

• Attached: Myosin head tightly bound to actin without ATP.

• Released: ATP binds and causes conformational change, reducing affinity for actin.

• Cocked: ATP hydrolyzed; ADP and phosphate retained, causing the head to swing.

• Re-binding: Head re-binds actin; phosphate release generates force (power stroke), moving head closer to actin filament's plus end.

• Each head operates independently in this cycle.

Muscle Contraction Mechanics

• Thick filaments (myosin) and thin filaments (actin).

• During contraction:

  1. Action potential triggers Ca2+ influx from the ER.

  2. Troponin binds Ca2+ and moves tropomyosin away from myosin binding sites.

  3. Myosin can bind actin, leading to contraction.

Non-Muscle Myosin Functions

• Non-muscle isoforms of myosin temporarily associate with actin in non-muscle cells.

  • Phosphorylation regulated by Myosin Light Chain Kinase enables actin binding and filament assembly.

  • Essential for functions like cell movement and cytokinesis.

Cytoskeletal Filaments

Types and Functions

1. Actin Filaments:

   • Provide structural strength and shape beneath the plasma membrane; allow dynamic cell projections (e.g., filopodia).

2. Microtubules:

   • Facilitate intracellular transport, form the mitotic spindle for chromosome segregation during division.

3. Intermediate Filaments:

   • Line the nuclear envelope, providing mechanical strength to cells, especially in specialized cells like skin and hair.

Microtubule Structure and Assembly

Tubulin Polymers

• Made of heterodimers (α-tubulin and β-tubulin).

• α-tubulin binds GTP; β-tubulin can bind GTP or GDP.

• Protofilaments formed head-to-tail from subunits − plus end oriented towards open β-tubulin end.

Microtubule Characteristics

• Composed of 13 protofilaments forming a hollow structure.

• Microtubules are stiff due to multiple contacts between tubulin subunits.

• Dynamic ends: Plus ends grow/shrink faster than minus ends.

• Nucleation required for assembly.

GTP Dynamics in Microtubules

• α-tubulin is always GTP-bound; β-tubulin can hydrolyze GTP to GDP.

• GTP cap formation occurs when there’s a high concentration of free tubulin, stabilizing microtubule.

Dynamic Instability of Microtubules

• Loss of GTP cap leads to rapid disassembly of GDP-bound tubulin.

• Microtubules can oscillate between growth (polymerization) and shrinkage (depolymerization).

Microtubule Accessory Proteins

Nucleating and Sequestering Proteins

1. Nucleating Proteins:

   • γ-tubulin: assembles into the γ-Tubulin Ring Complex (γ-TuRC) facilitating nucleation.

   • Augmin: binds to existing microtubules, recruiting γ-TuRCs for new microtubule formation at angles.

2. Sequestering Proteins:

   • Stathmin: binds tubulin dimers, preventing assembly and promoting microtubule shrinkage. Phosphorylation reduces this affinity, enhancing growth.

Microtubule End-Binding and Severing Proteins

1. End-Binding Proteins:

   • Kinesin-13: induces microtubule catastrophe.

   • XMAP215: stabilizes plus ends and promotes rapid growth.

2. Severing Proteins:

   • Katanin: removes subunits from microtubule wall, enhancing growth by introducing new GTP-bound subunits after severing.