9. Molecular Biology: Detailed Notes on Actin Cytoskeleton Dynamics and Regulation

Course Overview and Introduction to the Cytoskeleton

This course covers several foundational topics in molecular and cellular biology, including:

Chemical Foundations
Protein Structure, Function, and Regulations
The pathway from Gene to Protein
Protein Targeting and Sorting
Vesicular Transport
Cell Signaling
Cytoskeleton: Actin and Molecular Motors
Microtubules
Cell Cycle, Mitosis, and Meiosis
Stem Cells
Cell Death (Apoptosis)
Cancer
Membrane Structure and Membrane Transport
ATP Synthase

The Cytoskeleton and Movement

The cytoskeleton is responsible for both the internal architecture of the cell and various forms of movement. It acts as both the skeleton and the muscle system of the cell. Key components discussed include:

Filaments
Nucleus and DNA
Actin
Tubulin

Comparison of the Three Major Cytoskeletal Filaments

There are three distinct types of cytoplasmic filaments that differ in their physical properties, polarity, and dynamics.

1. Actin Filaments (Microfilaments)

Subunit: Actin-ATP.
Structure: Thin and flexible filaments.
Polarity: Polar.
Dynamics: Highly dynamic.
Functions: - Whole cell locomotion. - Formation of the contractile ring during cytokinesis. - Interaction with Myosin motors.

2. Intermediate Filaments

Subunit: Molecularly diverse; do not use NTPs (no ATP or GTP requirement).
Structure: Thin-ish and flexible; act as internal cables for mechanical strength. Stable structures.
Polarity: Non-polar.
Dynamics: Less dynamic than actin or microtubules.
Functions: - Providing mechanical strength to cells and tissues. - Note: There are no known associated motor proteins for intermediate filaments.

3. Microtubules

Subunit: Heterodimer-GTP.
Structure: Rigid, hollow tubes.
Polarity: Polar.
Dynamics: Highly dynamic.
Functions: - Positioning organelles within the cell. - Forming the mitotic spindle for chromosome segregation. - Intracellular transport. - Components of cilia and flagella. - Interaction with Kinesin and Dynein motors.

Detailed Focus: Actin Structure and Formation

Evolution and Abundance

Actin is evolutionarily conserved and highly abundant within the cell.
It contains a central cleft that binds to ATP.

Monomers vs. Polymers

G-actin (Globular actin): The monomeric form of actin. By convention, it consists of a $4$ -domain structure.
F-actin (Filamentous actin): The polymeric form, organized into a filament.
Polarity: The filament has two distinct ends: - The Plus end ( $+$ ), also known as the Barbed end. - The Minus end ( $-$ ), also known as the Pointed end.

Mechanism of Polymerization

Nucleation: The rate-limiting step where G-actin monomers must find partners to form a stable nucleus (typically a triplet). Once a nucleus of $3$ actin monomers is formed, it becomes much easier to recruit more monomers.
Elongation: The rapid addition of monomers to the growing filament.
Steady State: A phase where the mass of the filaments remains constant over time because the rate of assembly equals the rate of disassembly.

The Role of ATP in Polymerization

ATP-bound G-actin monomers have a high affinity for filaments and prefer to integrate and stay within them.
ATP-actin monomers primarily add to the plus end (barbed end).
ATP hydrolysis occurs shortly after the monomer binds to the filament, converting it to ADP-actin.
ADP-actin monomers have a lower affinity for the filament and tend to fall off.

Actin Filament Treadmilling

The Treadmilling Process

Filament treadmilling refers to the process where growth occurs at the plus end while disassembly occurs simultaneously at the minus end.
A student metaphor: "As much as you run, you are in the same place!"
Directional Movement: Treadmilling is the driving force for directional cell migration. The addition of ATP-G-actin at the ( $+$ ) end and the removal of ADP-G-actin at the ( $-$ ) end shifts the filament position in the direction of migration.

Regulation of Treadmilling In Vivo

In the cell, treadmilling is tightly regulated by specific proteins to ensure movement is controlled and occurs on the correct side of the cell.

1. Profilin

Binds to ADP-actin and promotes the exchange of ADP for ATP.
It binds at the barbed end region of the actin monomer.
Function: It essentially "recharges" the monomer and serves as a gatekeeper, ensuring new monomers can only be added to the front ( $+$ end) and cannot be added to the back.

2. Cofilin

Binds to the end of the filament where ADP-bound actin is located (the minus end).
Function: It breaks the filament down (depolymerization) into "junk" fragments, allowing the monomers to exit and enter the cycle again.

3. Thymosin- $\beta 4$

Binds to ATP-actin monomers to prevent them from polymerizing.
Function: It acts as a storage reservoir for actin monomers (e.g., in platelets), keeping them available but inactive until needed.

Structural Regulation and Capping

Capping Proteins

Capping proteins are used to regulate the length and stability of actin polymers by blocking further assembly or disassembly.

CapZ: Binds to and "locks" the plus end ( $+$ ). This prevents new actin from being added, effectively stopping growth at that end.
Tropomodulin: Binds to and caps the minus end ( $-$ ).
Result: Capping makes the structure rigid and prevents any further change in filament length.

Nucleation and Specialized Proteins

Nucleation is the rate-limiting step of actin formation. In cells, spontaneous nucleation is too slow, so specialized proteins are used to regulate where and when filaments form.

Formins

Structure: Contains FH2 domains that exist as dimers.
Function: Formins promote the formation of long, linear filaments. The FH2 dimer holds the actin and helps build the chain longer.
Regulation: Formins help regulate nucleation and make treadmilling occur faster.

Formin Activation at the Plasma Membrane

Actin filaments are often concentrated at the cell surface to drive membrane-associated processes.

Inactive State: The Formin protein is folded and inactive in the cytosol.
Activation: A Rho GTP protein (activated by GTP) binds to the Rho-binding domain (RBD) of the Formin.
Recruitment: This activates the FH1 and FH2 domains. Profilin-ATP-actin is then recruited to the FH1 domain and transferred to the FH2 domain to begin polymerizing the ( $+$ ) end at the plasma membrane.

Arp2/3 Complex (Branching)

Function: Unlike Formins which create linear chains, the Arp2/3 complex binds to the sides of pre-existing actin filaments to nucleate new branches.
Structure: Branches form at a characteristic $70^{\circ}$ angle.
Role: This branching is essential for creating the dense networks found in the lamellipodia/leading edge of moving cells.

Functional Roles of Actin Structures

Actin-based structures perform diverse cellular functions:

Microvilli: Found in epithelial cells of the small intestine for surface area expansion.
Cell Cortex: Provides structural support under the plasma membrane.
Adherens Belt: Helps in cell-to-cell adhesion and movement.
Filopodia and Lamellipodia: Projections at the leading edge that help the cell move.
Stress Fibers: Contractile bundles in the cell.
Phagocytosis: Movement required to engulf particles.
Endocytic Vesicles: Moving vesicles into the cell.
Contractile Ring: Responsible for separating the cell into two during cytokinesis.

Case Study: Phagocytosis

Branching directed by Arp2/3 is crucial for phagocytosis:

Antibodies ( $Ab$ ) coat a bacterium (Opsonized bacterium).
Fc receptors on a leukocyte (neutrophil) recognize the antibodies.
This triggers the Arp2/3 complex and Formin proteins to assemble F-actin.
The actin assembly drives the membrane to surround the bacterium, forming a Phagosome (vesicle).
The phagosome eventually fuses with Lysosomes for degradation.

Summary Recap

The Three Filaments: Actin and microtubules are polar and dynamic; intermediate filaments are non-polar and stable.
Actin Treadmilling: Regulates directional cell movement.
Regulation: Dynamics are driven by nucleation and branching proteins (Formin and Arp2/3), which define cellular movement and shape.