Lecture 12: Microfilament Composition, Dynamics, and Regulation

Overview of the Eukaryotic Cytoskeleton

The eukaryotic cell cytoskeleton consists of three distinct components that provide structure, organization, and motility. This lecture (Lecture 12) initiates the material for Exam 3, specifically focusing on microfilaments.

• Microfilaments (Actin Filaments): Consist of actin subunits and bind ATP.

• Microtubules: Consist of $\alpha/\beta$ tubulin dimers and bind GTP.

• Intermediate Filaments: Composed of various proteins and do not bind a nucleotide.

Comparative Characteristics of Cytoskeletal Elements

• Structure and Rigidity:

  • Microfilaments: Form rigid gels, networks, and linear bundles.

  • Microtubules: Highly rigid and not easily bent.

  • Intermediate Filaments: Provide great tensile strength.

• Assembly and Dynamics:

  • Microfilaments: Assembly is regulated and occurs from a large number of locations. They are highly dynamic (assembled/disassembled).

  • Microtubules: Assembly is regulated but limited to a small number of specific locations. They are highly dynamic.

  • Intermediate Filaments: Assembled onto pre-existing filaments. They are significantly less dynamic than the other two types.

• Polarity:

  • Microfilaments and Microtubules: Polarized (possess distinct plus and minus ends).

  • Intermediate Filaments: Not polarized; they lack directional orientation.

• Motor Proteins:

  • Microfilaments: Act as a track for myosin.

  • Microtubules: Act as a track for kinesins and dyneins.

  • Intermediate Filaments: No associated motor proteins.

• Primary Functions:

  • Microfilaments: Contractile machinery, cell cortex (inner plasma membrane network), cell shape, and motility.

  • Microtubules: Organization, chromosome segregation (mitosis/meiosis), and long-range transport of organelles.

  • Intermediate Filaments: Providing cell and tissue integrity.

Microfilament Structures and Locations in Cells

Microfilaments are central to the architecture of both stationary and migrating cells.

In Polarized Epithelial Cells

• Apical Domain: Contains microvilli, which are extensions based on microfilaments.

• Basolateral Domain: Separated from the apical side by tight junctions.

• Cell Cortex: A red-appearing "crisscross" network surrounding the inside of the cell, composed of microfilaments.

• Nuclear Envelope: Surrounded by intermediate filaments called lamins.

In Migrating Cells

• Leading Edge: The front of the cell that develops extensions for migration.

• Filopodia: Long, thin extensions used for sensory and exploratory functions.

• Lamellipodia: Broad, sheet-like protrusions designed for persistent protrusion.

• Stress Fibers: Contractile bundles of actin and non-muscle myosin used for cell body translocation.

Other Microfilament-Based Structures

• Adherens Junctions: Important for cell-cell interactions to form tissues.

• Phagocytosis: Actin machinery is required for fibroblasts/macrophages to engulf material.

• Endocytic Vesicles: Move along microfilament tracks from the plasma membrane for transport to lysosomes.

• Contractile Ring: Forms during cytokinesis to divide the cytoplasm during mitosis/meiosis.

Composition and Structure of Actin

Actin exists in two primary forms within the cell:

G-Actin (Globular Actin)

• This is the monomeric form of actin.

• Structure: Divided into two approximately equal lobes with a central ATP-binding cleft.

• Subdomains: Contains four subdomains ($1, 2, 3,$ and $4$). The N and C termini both lie in domain $1$.

• Properties: Functions as an ATPase; it binds ATP and can hydrolyze it.

F-Actin (Fibrous/Filament Actin)

• This is the polymeric form of actin.

• Structure: Consists of two strands of monomers wrapped around each other.

• Repeat Unit: A repeating unit consists of $28$ actin subunits ($14$ monomers on each strand).

• Dimensions: Covers a distance of $36$ to $72\,nm$ per repeat. Because of the twist, thickness alternates between $7\,nm$ and $9\,nm$.

• Polarity: All ATP-binding clefts point toward the same end of the filament. This establishes the orientation:

  • Minus ($-$) End: The pointed end.

  • Plus ($+$) End: The barbed end.

• Decoration Experiment: When decorated with myosin heads, the myosin forms "arrowheads" pointing toward the negative end, while the "barbed" part points toward the plus end.

Microfilament Dynamics and Assembly

Actin assembly is a concentration-dependent process characterized by three phases: Nucleation, Elongation, and Steady State.

The Critical Concentration ($C_C$)

• Below the $C_C$, actin exists only in its monomeric (G-actin) form.

• At the $C_C$, filaments start to assemble. The mass of the monomeric pool remains constant while additional actin added to the system increases the mass of the filaments.

Structural Assembly Steps

1. Nucleation: The slow, initial step where multiple actin monomers assemble into a "nucleus" or seed. Adding pre-formed nuclei (seeds) bypasses this slow lag phase.

2. Elongation: Once a nucleus is formed, it is extended in both directions. The plus end grows substantially faster than the minus end.

3. Steady State: A point where the rate of subunit addition equals the rate of subunit loss, and the total filament length remains constant.

Asymmetric Growth and Treadmilling

• Plus End Properties: High on-rate ($12$ units/sec). $C_C = 0.12\,\mu M$. Growth consists largely of ATP-actin.

• Minus End Properties: Low on-rate ($1.3$ units/sec). $C_C = 0.6\,\mu M$. Loss of subunits is more frequent here, typically as ADP-actin.

• Actin Treadmilling: Subunits added at the plus end appear to move through the filament and are eventually lost at the minus end. This gives the appearance of the filament moving across the cytosol even if its total length stays the same.

Regulation of Actin Dynamics by Interacting Proteins

Several classes of proteins regulate the assembly, disassembly, and structure of microfilaments.

Dynamics Regulators

• Profilin: Promotes ATP-for-ADP exchange on actin monomers, converting inactive ADP-actin into active ATP-actin ready for plus-end addition.

• Cofilin: Binds to ADP-actin filaments and promotes their disassembly from the minus end, feeding monomers back to the profilin cycle.

• Thymosin $\beta$: Acts as a sequestration protein (storage); it binds ATP-actin to prevent it from being added to filaments until needed.

Capping Proteins

• CapZ: Binds at the plus end, blocking assembly and disassembly at that end. It limits dynamics to the minus end.

• Tropomodulin: Binds at the minus end, preventing subunit loss and stabilizing the filament.

Nucleating and Branching Proteins

• Formin: A dimeric nucleator with an FH2 domain that rocks back and forth to allow the insertion of subunits. It stays at the plus end as it grows, protecting it from capping proteins. It creates long, unbranched filaments.

• Arp2/3 Complex: Nucleates branching. It binds to pre-existing F-actin and initiates a new filament at a specific angle. It remains at the minus end of the new branch.

  • NPF (Nucleation Promoting Factor): Required to activate Arp2/3. One example is WASP.

Actin-Based Movement and Pathogens

Listeria monocytogenes

• Listeria is a foodborne pathogen that uses actin polymerization for movement.

• Mechanism: It possesses a protein called ActA that mimics cellular Nucleation Promoting Factors (NPFs). It hijacks the host's Arp2/3 complex to create a "comet tail" of branched actin.

• Result: The force of polymerization pushes the bacterium through the cytoplasm and into adjacent cells.

Endocytosis

• Vesicle formation at the plasma membrane involves NPFs (like WASP) and Arp2/3.

• A branched actin network forms around the invagination, providing the force to move the endocytic vesicle through the cytoplasm.

Cross-Linking Proteins

Cross-linking proteins organize filaments into bundles (parallel) or networks (crisscross).

• Fimbrin: Cross-links parallel filaments; found in microvilli and filopodia.

• $\alpha$-Actinin: An anti-parallel dimer found in stress fibers and the muscle Z-line.

• Spectrin: An anti-parallel protein found in the cell cortex.

• Filamin: Forms crisscrossing networks of actin; found at the leading edge.

• Dystrophin: Links the actin cortex to transmembrane proteins in the plasma membrane. Mutations in this protein cause muscular dystrophy.

Myosin Motor Proteins

There are approximately $40$ different myosin proteins, including three major classes found widely in eukaryotes.

• Class I (Myosin I): Involved in endocytosis and linking actin to the membrane.

• Class II (Myosin II): Primarily involved in muscle contraction and cytokinesis.

• Class V (Myosin V): Involved in the transport of organelles along actin tracks.

Myosin II Structure and Function

• Structure: Consists of heavy chains (head, neck, and tail) and light chains (regulatory and essential). The head contains the actin-binding site and the nucleotide (ATP) binding site.

• Neck Length: The rate of movement is directly proportional to the neck size; longer necks result in faster movement.

• Step Size: Myosin V moves in a "hand-over-hand" fashion with steps as large as $72\,nm$.

The ATPase Cycle (Myosin Movement)

1. ATP Binding: Head releases from actin.

2. ATP Hydrolysis: ATP breaks down into ADP and $P_i$. The head rotates into a "cocked" state.

3. Actin Binding: The head binds to a new site on the actin filament closer to the plus end.

4. Power Stroke: $P_i$ is released, causing the head to straighten and pull the actin filament.

5. ADP Release: Cycle resets.

Muscular Contraction: The Sliding Filament Model

• Sarcomere: The basic unit of muscle contraction in myofibrils, bounded by Z-discs.

• Structural Composition:

  • I Band: Contains actin filaments attached to the Z-disc.

  • A Band: The middle part where myosin filaments overlap with actin.

• Contraction Mechanism: Triggered by calcium and ATP. Myosin heads move along actin toward the plus end, pulling the Z-discs closer together. This shortens the sarcomere without shortening the actual filaments.

Regulation by Rho Family GTPases

The actin cytoskeleton is regulated by extracellular signals (growth factors, LPA) via three main Rho family proteins: Cdc42, Rac, and Rho.

Specific Regulatory Pathways

• Cdc42: Activates WASP and Arp2/3. This leads to actin polymerization and the formation of filopodia.

• Rac: Activates WAV and Arp2/3. This leads to the formation of lamellipodia.

• Rho: Activates Formin (for linear actin) and Rho Kinase (to regulate myosin II). This leads to stress fiber formation and contraction.

Signal Integration and Crosstalk

• Signals are often integrated from multiple receptors.

• There is complex crosstalk: Cdc42 can activate Rac, which can activate Rho. Conversely, Rho can negatively feed back to inhibit Rac.

Wound Healing Assay

• Procedure: A dish of confluent cells is scratched ("wounded"), and the time to closure is measured.

• Research Findings:

  • Dominant Negative Rac: Disrupts healing by stopping Arp2/3 activity.

  • Dominant Negative Cdc42: Leads to non-oriented lamellae; movement occurs but lacks direction.

  • Dominant Negative Rho: Results in the strongest negative effect, completely preventing wound closure and cell movement.