Cytoskeleton Chapter: Actin Dynamics

Exam 3 Information

• Exam 3 is next Thursday, November 14th

Cytoskeleton 3: Actin

• This lecture investigates actin, its structure, dynamics, regulators, the biological structures it forms, and how cells use the actin cytoskeleton.
• Objectives:
  • Draw a cartoon of actin filament, highlighting polarity, dynamics, and the role of ATP.
  • Discuss G-actin, F-actin, and how factors regulate F-actin nucleation, structure, and dynamics.
  • Discuss the effects of various drugs that affect the actin cytoskeleton.
  • Describe various cellular processes that require various actin structures.
  • Compare and contrast the structure and function of various F-actin network structures.

Actin Cytoskeleton

• Actin filaments allow cells to adopt a variety of shapes and perform specific functions.
  • Examples of structures include villi, contractile bundles, sheet-like and finger-like protrusions, and contractile rings.

Actin Filaments

• Thin, flexible protein threads

• 7 nm in diameter

• Less rigid than microtubules

• Polarized: fast-growing plus (barbed) end and slow-growing minus (pointed) end

• Structure:

  • Actin molecule
  • Pointed end
  • Barbed end
  • 37 nm
  • Diameter 7 nm
  • Two protofilaments
  • Short pitch
  • Long pitch
  • Right-handed twist

• Basic subunit is a single actin monomer that polymerizes into a helical chain.

• Unlike microtubules, actin binds to ATP.

• Interactions within the filament and between other protofilaments (short and long pitch interactions).

Actin Structure (ATPase)

• Actin has four subdomains.
• Subdomains 1 and 3 are similar.
• ATP binds at the center of the molecule.
• This contrasts with the GTP binding site on Beta-tubulin, located between the beta and alpha subunits.
• The ATP catalytic machinery is entirely within the actin protein.
• Hydrolysis is activated through allostery when actin polymerizes.
• Subdomains 1 and 2 face outward, while subdomains 3 and 4 face inward.
• G-actin polymerizes in the ATP-bound state.
• Actin hydrolyzes ATP to ADP; this results in a conformational change, weakening actin-actin contacts, and can lead to minus end depolymerization.

Actin Polymerization/Depolymerization

• Actin exhibits a similar polymerization and depolymerization cycle as microtubules, but with differences:
  • Monomeric actin binds to ATP and polymerizes at the plus end.
  • Upon polymerization, actin's endogenous ATPase activity cleaves ATP to ADP.
  • ATP hydrolysis can be thought of as a molecular "clock"—older actin filaments with ADP are unstable and disassemble from the minus end.

Tubulin Versus Actin (Key Differences)

• Tubulin is a heterodimer
• Requires GTP for polymerization
• Residues that contribute to hydrolysis are supplied by longitudinal lattice mates
• GTP hydrolysis causes minor conformational changes
• 13 protofilaments (straight)
• Dynamics occur at the MT plus end
• Actin is a monomer
• Requires ATP for polymerization
• Residues that contribute to hydrolysis are supplied by the monomer
• ATP hydrolysis causes conformational changes
• 2 protofilaments (right-handed twist)
• Grows at the barbed end (plus end), and depolymerizes at the pointed end (minus end)

Actin Network Structures

• Actin can form filopodia and lamellipodia.
• Formation, regulation, and dynamics of these networks are regulated by distinct sets of actin-binding proteins.
  • Example proteins include Ena/VASP, Arp2/3 and Formin.

Actin Binding Proteins (Illustrated in Green)

• Actin is induced to form various structures based on its interaction with actin-binding proteins and its own set of motor proteins.
  • Example proteins include bundling proteins, severing proteins, cross-linking proteins, and capping proteins.
  • Proteins regulate the type of actin network locally formed in regions of the cell, as well as the dynamics of the network.

Actin Nucleation (Arp2/3)

• Arp2/3 complex nucleates actin filaments at an angle from a mother filament.
  • Daughter actin filaments grow at a roughly 70-degree angle from the mother filament.
  • The Arp2/3 complex recruits Wasp-Profilin-Actin complexes.
  • This recruitment initiates the nucleation and polymerization into daughter filaments.
• The Arp2/3 network is mainly located in lamellipodia.

Actin Nucleation (Continued)

• The Arp2/3 complex activation requires the full complex and the necessary factors (Actin, ATP and WASP)
• In the lamellipodium, Arp2/3 complex activates actin filaments that grow from mother filaments at an angle of approximately 70°.
• This process is regulated by GTPases such as Rho, Rac, or Cdc42.

Actin Sequestering (Profilin)

• Profilin binds to free ADP-bound G-actin and induces nucleotide exchange
  • Profilin has a higher affinity for ATP-bound G-actin
  • Actin hydrolyses ATP to ADP
  • Dissociation and depolymerization occurs at the minus end – overall creating the treadmilling behavior of actin dynamics
• Actin has conformational changes due to ATP hydrolysis
  • The ATP-bound state is slightly opened relative to the ADP state. These conformational changes affect lattice stability.
  • To be able to open and close, there is a hinge-like region at the base of actin.
• Profilin binds the hinge region
  • Inducing a more open conformation amenable for ADP release.

Actin Capping

• Filament length regulation is controlled by competing activities of capping proteins.
• Anti-capping proteins, such as Ena/VASP, and capping proteins, regulate filament length
• Capping proteins bind to and stabilize the F-actin plus end.
  • Localizations and activity of capping proteins can be used to limit filament length.

Actin Severing (Cofilin)

• Cofilin binds to the actin lattice that is in the ADP state.
  • Binding happens cooperatively.
  • Cofilin binds this lattice, elicits some conformational changes in the lattice locally, and this altered lattice structure has high affinity for more cofilin molecules to bind
  • Strain from these changes leads to severing of actin.
• Cofilin-bound actin lattices and severed actin filaments serves as seeds for more nucleation

Drugs that Affect Actin Dynamics

• Drugs can be used to understand actin dynamics and function in cells by:
  • Phallidin: Binds filaments; prevents depolymerization
  • Cytochalasin: Caps filament plus ends; prevents polymerization; depolymerizes filament at minus ends
  • Latrunculin: Binds actin monomers; prevents polymerization

General Actin Functions

• Actin filaments are assembled into a scaffolding that gives cells their shape.
• Actin filaments are used to generate forces during cellular movements (pushing and pulling), including motility.

Cell Cortex

• The cortex is a region of cytoplasm just beneath the plasma membrane.
• It’s composed of actin filaments and actin cross-linking proteins.
• The cortex provides mechanical strength to the cell membrane and helps cells maintain their shape.
• Filamin cross-links actin filaments into a meshwork.

Lamellipodium

• This is a structure that is rich in actin and is found in the leading edge of migrating cells.
• Migrating cells expressing GFP-actin show a brightly stained leading edge with a membrane that appears to be ruffling.

Actin Polymerization in Lamellipodia

• Polymerization of actin filaments at their plus ends produces the force that pushes the leading edge forward.
• The growth of thousands of individual actin filaments results in sufficient force to push the membrane.
  • Actin filaments are organized into a branched network by the actions of several proteins, mainly ARP (actin-related protein) complex and capping protein.

Regulation of Actin Structures

• Signaling pathways, involving small GTPases, regulate different actin structural formations.
  • Activation of Rho, Rac, or Cdc42 triggers different pathways that drive distinct F-actin network formations.
• Regional activation of GTPases leads to distinct actin structures in different regions of a cell.
• These GTPases are membrane-associated and drive the formations close to the membrane.

Growth Cones and Neuronal Migration

• Growth cones are critical for neuronal cell migration.
• Microtubules and F-actin dynamics are coordinated in the process.
• Microtubule-motor-based delivery of factors to the growth cone, along with co-regulation of actin and microtubule dynamics.
• Spectraplakins are critical for coordinating actin and microtubule dynamics.
• Spectraplakins play critical roles in neuronal migration, which is required for brain development.

This quiz delves into the intricacies of actin within the cytoskeleton, exploring its structure, dynamics, and regulatory mechanisms. You'll learn about G-actin and F-actin, their roles, associated drugs, and the diverse cellular processes that rely on actin structures. Prepare to illustrate actin filaments and analyze their functional significance.