20250207_Cytoskeleton Lecture 1_actin (1)

Cytoskeleton Overview

• Definition: Complex networks of protein filaments that perform multiple functions.

• Functions:

  • Structural Support: Maintains cell shape, migration, and division.

  • Spatial Organization & Intracellular Transport.

• Characteristics:

  • Highly dynamic & adaptable.

  • Composed of polymers held together by weak noncovalent interactions, allowing for easy assembly and disassembly.

  • Stable overall structure with constantly changing individual subunits.

Types of Cytoskeletal Filaments

1. Actin Filaments

• Function: Provide strength & shape at the cell cortex beneath the plasma membrane.

• Structural Features:

  • Form dynamic projections (e.g. filopodia) or stable structures (e.g. microvilli).

2. Microtubules

• Function: Form arrays from nucleus to plasma membrane for intracellular transport.

• Key Role in Cell Division: Generate the mitotic spindle that segregates chromosomes.

3. Intermediate Filaments

• Function: Line the nuclear envelope to protect DNA and provide mechanical strength to specialized cell types (e.g. skin, hair).

Actin Filaments Structure

• Composition: Comprised of individual actin subunits (G-actin) that bind ATP/ADP tightly.

• Polarity: Each subunit has a plus (adding site) and minus end (losing site).

• Filament Formation: Monomers assemble in a head-to-tail manner forming helical filaments (F-actin).

• Dynamics: Plus end is more dynamic; monomers add and lose more quickly at this end.

Actin Nucleation

• Initial Binding: Two actin subunits can bind spontaneously, but the association is unstable.

• Role of Trimer Formation: Addition of a third subunit creates a stable trimer (nucleus) that allows for faster polymerization.

• Critical Concentration: The point at which the rate of addition of subunits equals the rate of loss.

Kinetics of Polymerization

• Lag Phase: Initial monomer assembly and disassembly, forming small oligomers for nucleation.

• Growth Phase: Rapid addition of subunits to filament ends; decrease in G-actin concentration.

• Equilibrium Phase: Rate of addition matches dissociation rate; G-actin reaches critical concentration.

ATP and ADP Binding to Actin

• Hydrolysis: Actin catalyzes ATP to ADP, which is slow for free subunits; occurs faster when part of a filament.

• Dissociation: ADP-bound actin has reduced affinity for other subunits, leading to an increased likelihood of dissociation.

• ATP Cap Formation: Rapid addition of ATP-bound subunits can lead to the establishment of a stable ATP cap at filament ends.

Actin Treadmilling

• Dynamics: In intermediate concentrations, actin subunits are added rapidly to the plus end and lost from the minus end, maintaining filament integrity.

• Preservation of Structure: Allows flexibility during cellular processes through a net assembly at the plus end and disassembly at the minus end.

Actin Binding Proteins

1. Monomer Binding Proteins

• Profilin: Promotes binding of actin to the plus end and blocks minus end association.

• Thymosin: Prevents actin interactions at both ends, lowering free monomer concentration.

2. Actin Nucleating Proteins

• Arp2/3 Complex: Forms branched structures and is activated by Nucleation Promoting Factors (NPF).

• Formins: Ring-like complexes that stimulate filament growth at the plus end.

3. Filament Binding Proteins

• Tropomyosin: Side-binding stabilizer, reduces dynamics at the sides of the filament.

• CapZ: End-binding protein that prevents addition and loss of subunits at plus ends.

4. Filament Severing Proteins

• Gelsolin: Inserts into gaps in filaments, severing them and preventing further growth.

• Cofilin: Twists filaments, weakening binding and promoting disassembly, helping with filament turnover during cell migration.

Myosin Motor Protein

• Function: Generates force and movement along actin filaments.

• Structure: Composed of heavy chains forming coiled-coil dimers with globular heads that bind and hydrolyze ATP.

Myosin Movement Cycle

1. Steps of Movement

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

• Released: ATP binding to the head reduces affinity for actin.

• Cocked: ATP binding causes conformational change; ATP hydrolysis occurs.

• Re-binding: Head re-binds actin, and phosphate release initiates power stroke.

Muscle Contraction Mechanism

• Components: Interaction between thick (myosin) and thin (actin) filaments.

• Process: Myosin movement along actin drives muscle contraction, facilitating movement across the cellular structures.

Non-Muscle Myosin Activity

• Can assemble transiently in non-muscle cells; assembly regulated through phosphorylation by Myosin Light Chain Kinase.

• Essential for cell movement and shape changes, particularly in processes like cytokinesis.