Cytoskeleton Chapter: Actin Dynamics
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Questions and Answers

What is the diameter of actin filaments?

  • 15 nm
  • 5 nm
  • 10 nm
  • 7 nm (correct)

    • Which end of the actin filament is referred to as the plus end?

  • The stubby end
  • The tapered end
  • The pointed end
  • The barbed end (correct)

    • What happens to ATP after actin polymerizes?

  • It binds to other proteins for stability.
  • It is converted to ADP and releases energy. (correct)
  • It triggers actin filament contraction.
  • It remains unchanged in the filament.

    • How does the hydrolysis of ATP affect actin polymerization?

    <p>It induces conformational changes that weaken interactions.</p> Signup and view all the answers

    Which of the following statements is true about actin's catalytic machinery?

    <p>It is entirely within the actin protein itself.</p> Signup and view all the answers

    What role does ATP hydrolysis play in the actin polymerization cycle?

    <p>It acts as a molecular clock for regulating disassembly.</p> Signup and view all the answers

    Which of the following structures do actin filaments form?

    <p>Filopodia and lamellipodia.</p> Signup and view all the answers

    Which protein complex is essential for the nucleation of new actin filaments?

    <p>Arp2/3 complex.</p> Signup and view all the answers

    What is required for the activation of the Arp2/3 complex?

    <p>A mother filament and G-actin bound to WASP.</p> Signup and view all the answers

    How do actin-binding proteins influence actin polymerization?

    <p>They regulate the dynamics and formation of actin networks.</p> Signup and view all the answers

    After ATP is hydrolyzed, what happens to older actin filaments?

    <p>They are more likely to disassemble from the minus end.</p> Signup and view all the answers

    Which protein is involved in the recruitment of free actin to the Arp2/3 complex?

    <p>WASP.</p> Signup and view all the answers

    What initiates the polymerization of daughter filaments from the mother F-actin filament?

    <p>Activation of the Arp2/3 complex.</p> Signup and view all the answers

    What effect does the strain from cofilin binding have on actin filaments?

    <p>It leads to their severing.</p> Signup and view all the answers

    What is the function of the cell cortex made of actin filaments?

    <p>To provide mechanical strength to the cell membrane.</p> Signup and view all the answers

    How does the mutation of filamin in melanoma cells affect their morphology?

    <p>It results in an increase in membrane blebs.</p> Signup and view all the answers

    What structural feature is characteristic of the leading edge of a migrating cell?

    <p>High concentration of actin resulting in lamellipodia.</p> Signup and view all the answers

    What role does filamin serve in the context of actin filaments?

    <p>It cross-links actin filaments into a meshwork.</p> Signup and view all the answers

    What is the primary feature observed in cancer cells with disrupted filamin function?

    <p>Robust motility.</p> Signup and view all the answers

    What type of cellular structure is seen at the leading edge of a crawling cell?

    <p>Lamellipodium.</p> Signup and view all the answers

    In the actin filaments of lamellipodia, how are the filaments arranged?

    <p>Oriented with plus ends at the cell margin.</p> Signup and view all the answers

    What angle does the daughter actin filament branch off from the mother filament?

    <p>70 degrees</p> Signup and view all the answers

    What effect does profilin have on G-actin?

    <p>Facilitates nucleotide exchange</p> Signup and view all the answers

    What mechanism is primarily responsible for actin dynamics exhibiting treadmilling behavior?

    <p>Hydrolysis of ATP to ADP</p> Signup and view all the answers

    How do formins contribute to actin polymerization?

    <p>By delivering actin to the filament plus end</p> Signup and view all the answers

    What role do capping proteins play in filament length regulation?

    <p>They bind to the plus end of actin filaments</p> Signup and view all the answers

    What characteristic of profilin allows it to induce a more open conformation in actin?

    <p>Its binding to the hinge region of actin</p> Signup and view all the answers

    How does cofilin affect the actin lattice in the ADP state?

    <p>It destabilizes the lattice structure</p> Signup and view all the answers

    What distinguishes filopodia from lamellipodia in terms of actin structure?

    <p>Formation of long unbranched bundles</p> Signup and view all the answers

    What is the primary role of actin filament polymerization at the plus ends?

    <p>To produce force that pushes the leading edge forward</p> Signup and view all the answers

    Which protein functions as an actin nucleator in filament formation?

    <p>ARP complex</p> Signup and view all the answers

    How does cofilin contribute to actin filament dynamics?

    <p>It binds to older filaments and promotes their disassembly</p> Signup and view all the answers

    What is the significance of the angle at which the daughter filament grows from the mother filament?

    <p>It determines the force exerted on the membrane</p> Signup and view all the answers

    What triggers disassembly of aged mother actin filaments?

    <p>Cofilin attachment</p> Signup and view all the answers

    What determines the formation of different actin structures based on cell type?

    <p>Signaling pathways involving small GTPases</p> Signup and view all the answers

    Which small GTPase is NOT mentioned in connection with actin structure formation?

    <p>Rab</p> Signup and view all the answers

    Which process is coordinated with F-actin dynamics during neuronal cell migration?

    <p>Microtubule-motor based delivery of factors to growth cones</p> Signup and view all the answers

    Study Notes

    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.

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