Microfilaments, Muscle and Myosins Notes

Microfilaments, Muscle, and Myosins

Roles of Actin Networks at the Cell Cortex

  • Actin networks play crucial roles at the cell cortex.
  • Microfilaments (actin) are 7-9 nm in diameter.
  • Microtubules (alpha/beta-tubulin dimers) are approximately 25 nm in diameter.
  • Intermediate filaments (various subunits) are around 10 nm in diameter.

Comparison of Microfilaments, Microtubules, and Intermediate Filaments

Microfilaments (Actin)
  • Subunit Binding: Actin binds ATP.
  • Structure: Forms rigid gels, networks, and linear bundles.
  • Assembly: Regulated assembly from many locations; highly dynamic and polarized.
  • Function: Tracks for myosins; contractile machinery and network at the cell cortex.
Microtubules (αβ-tubulin)
  • Subunit Binding: αβ-tubulin binds GTP.
  • Structure: Rigid and not easily bent.
  • Assembly: Regulated assembly from few locations; highly dynamic and polarized.
  • Function: Tracks for kinesins and dyneins; organization and long-range transport of organelles.
Intermediate Filaments
  • Subunit Binding: IF subunits don't bind a nucleotide.
  • Structure: Great tensile strength.
  • Assembly: Assembled onto pre-existing filaments; less dynamic and unpolarized.
  • Function: Cell and tissue integrity; no motors.

Cellular Structures Involving Microfilaments

  • Microvilli
  • Cell junctions
  • Adherens belt
  • Filopodia
  • Lamellipodium/leading edge
  • Stress fibers
  • Contractile ring

Actin Structure and Assembly

  • G-actin: Globular protein that binds ATP within a central cleft.
  • F-actin: Filamentous actin, formed by two strands of subunits.
  • Repeating Unit: 28 subunits (14 per strand) over 72 nm.
  • Helical Twist: Clockwise; symmetry every 36 nm.
  • Polarity: ATP-binding cleft exposed at the minus end.

Actin Filament Dynamics and Treadmilling

  • ATP-G-actin Addition: 10x faster at the plus (+) end than the minus (-) end.
  • Dissociation Rate: Similar at both ends.
  • ATP Hydrolysis: ATP hydrolyzes to ADP-Pi in the filament, followed by slow Pi release, resulting in ATP-actin, ADP-Pi-actin, and ADP-actin.
  • Treadmilling: ATP-actin added preferentially at the plus end, ADP-actin disassembles at the minus end.
  • Critical Concentration: Cc=koff/konCc = koff/kon (ratio of dissociation rate to association rate).

Compounds Affecting Actin Filament Stability

  • Cytochalasin D: Binds actin monomers and prevents polymerization.
  • Latrunculin A: Binds actin monomers and prevents polymerization.
  • Phalloidin: Binds and stabilizes actin filaments; can be fluorescently labeled for staining.

Regulation of Actin Polymerization

  • G-actin Concentration: In cells, G-actin is 1000 times more concentrated than the critical concentration.
  • Thymosin-β4: Binds most G-actin, preventing its incorporation into filaments. Microinjection of Thymosin-β4 inhibits actin filament assembly.
  • Profilin: Binds G-actin (weaker than thymosin-β4 but stronger than actin + ends) and promotes ATP replacement, facilitating actin incorporation into filaments. It is mostly at the plasma membrane bound to PIP2.

Cofilin and Actin Filament Destabilization

  • Cofilin (Actin Depolymerizing Factor/ADF): Binds to the sides of ADP-actin in the filament, inducing fragmentation.
  • Function: Replenishes the pool of free ADP-actin, which can be recharged by profilin.
  • Mechanism: Cofilin chops off 18-20mers, then Aip1 (actin-interacting protein 1) chops these into monomers.

Actin Binding Proteins: The Toolbox

  1. Monomer Binding Proteins:
    • Profilin
    • Thymosin β4
  2. Filament Severing Proteins:
    • Cofilin
  3. Filament Capping Proteins:
    • Plus end: CapZ
    • Minus end: Tropomodulin
  4. Filament Nucleators:
    • Straight: Formin
    • Branched: Arp2/Arp3 complex
  5. Bundling Proteins:
    • Fimbrin
    • α-Actinin
  6. Filament Side Binders:
    • Tropomyosin
  7. Molecular Motors:
    • Myosins (14 classes)

Spatial Regulation of Actin Filament Formation

  • Formin: Multi-domain protein for straight actin filament formation.
    • Rho GTPase binding domain (RBD)
    • Profilin-ATP-actin binding domain (FH1)
    • Filament nucleating domain (FH2)
  • Inactive State: RBD binds and inhibits the FH2 domain.
  • Activation: When Rho GTPase is activated, formin is recruited to the plasma membrane, causing a conformational shift that releases the FH1 and FH2 domains.

Arp2/Arp3 Complex and Branched Actin Filaments

  • Function: Induces branching of actin filaments at a fixed angle of 70 degrees.
  • Location: Frequently located near the cell membrane.
  • Activation: Activated by proteins like WASp and WAVE.

WASp and Activation of Arp2/Arp3 Complex

  • WASp: Multi-domain protein.
    • Rho GTPase binding domain (RBD)
    • Actin binding domain
    • Arp2/Arp3 complex binding domain
  • Inactive State: RBD prevents binding to the Arp2/Arp3 complex.
  • Activation: When Cdc42 GTPase is activated (e.g., in response to a chemotactic signal), WASp is recruited to the plasma membrane, causing a conformational shift that releases the Arp2/Arp3 binding domain.

Actin Filament Bundling

  • Actin filaments can be packed into tight or loose bundles.
  • α-Actinin: Forms dimers that bundle parallel or anti-parallel filaments loosely, allowing Myosin II access.
  • Fimbrin: Bundles filaments of the same polarity tightly, excluding Myosin II.

Myosin Motors

  • Required for:
    • Organization of organelles
    • Chromosome segregation
    • Protein and RNA transport
    • Cell division
    • Cell motility and chemotaxis
    • Maintaining cell integrity
    • Body mechanics (food mastication, digestion, blood circulation, communication, reproduction, body movement).

Myosin Motor Superfamily

  • Multiple members, each with specific functions.
  • Examples: Myosin I, Myosin II, Myosin V; mutations can lead to deafness, blindness, or seizures.

Myosin Tails and Their Functions

  • The tail determines what myosin binds to and, therefore, its function.
  • Myosin I: Binds membranes; involved in endocytosis.
  • Myosin II: Forms dimers that associate into bi-directionally symmetrical bouquets.
  • Myosin V: Dimerizes but does not form large complexes; binds vesicles via adaptor molecules for transport to the plus ends of actin filaments.

Myosin II Structure and Function

  • Forms filaments from bi-symmetrical bouquets of dimers.
  • Coiled-Coil Tail Region: Holds dimers together; can be cleaved by Chymotrypsin.
  • S1 Heads: Monomeric heads with motor activity and essential/regulatory light chain binding sites; obtained after Papain digestion (removes S2 region).

Actin Filament Polarity

  • Determined via electron microscopy using purified monomeric myosin S1 heads.
  • Arrowhead Appearance: Points to the minus end of the filament.
  • Tropomyosin binding site changes on muscle actin to inhibit myosin binding when muscle is inactive and is moved out of the way to allow myosin to bind when muscle is active

Myosin Motility Studies

  • Studied by binding myosin to a coverslip and observing fluorescent actin filaments moving over it.
  • Mechanism: Principles apply to all myosins, though Myosin II is the primary example.
  • Myosin II has two heads that act independently.

Molecular Mechanism of Myosin II Action

  • Pi Dissociation: Slow in the absence of actin; adding actin raises its rate 300-fold (approximately 10 ATP molecules per myosin per second).
  • Power Stroke: Pi dissociation induces conformational changes in the head domain, amplified by a converter region, rotating the lever-like neck and moving the actin filament by a few nanometers.

Sliding Filament Theory

  • Myofibrils consist of sarcomeres.
  • Sarcomeres are composed of myosin (thick) and actin (thin) filaments.
  • Muscle contraction occurs via the sliding of actin filaments along myosin filaments.
  • Z disk: plus ends of actin filaments are capped by CapZ.
  • Minus ends: of the actin filaments are capped by tropomodulin.
  • Nebulin: determines the length of the actin filaments.

Muscle Cell Structure

  • Syncytial (multi-nucleated) cells up to ~50cm long, formed by fusion of myoblasts.
  • Contain myofibrils with repeating sarcomeres (basic units of muscle action).
  • Sarcomere Definition: Region between Z lines, with one dark band (myosin) and two half light bands (actin).
  • Contraction: Light band disappears as the overlap between myosin and actin filaments increases.

Nerve Impulses and Muscle Contraction

  • Depolarization: Nerve impulses depolarize the sarcolemma (plasma membrane).
  • T-tubules: Invaginations of the plasma membrane adjacent to the sarcoplasmic reticulum (SR).
  • Calcium Release: Action potential opens voltage-gated Ca++ channels, releasing a small burst of Ca++ into the cytosol, triggering massive Ca++ release from the SR.
  • Myosin Binding: Increased calcium concentration causes myosin to bind to actin.

Role of Tropomyosin and Troponin

  • Tropomyosin (TM): Covers the myosin-binding site on actin in the absence of Ca++.
  • Troponin (TN) Complex:
    • TN-T
    • TN-I
    • TN-C (calcium-binding subunit)
  • Mechanism: Ca++ binding to TN-C causes a conformational change, exposing the myosin-binding site by moving tropomyosin.

Non-Muscle Myosin II

  • Functions in:
    • Stress fibers
    • Contractile ring

Myosin Motor Movement

  • Influenced by:
    • Step size: Determined by the length of the neck linker region.
    • Duty ratio: Percentage of time the motor is bound to actin, controlled by ATPase activity.
  • Processive motors need a high duty ratio and large step size.

Measuring Myosin Motor Properties

  • Optical Tweezers (Optical Trap): Measures step size, processivity, and force generation.
  • Experiment: Myosin is bound to an immobilized bead; an actin filament held by lasers is lowered towards the bead; ATP stimulates movement.

Myosin II vs. Myosin V

  • Myosin II:
    • Step size: 5-10 nm.
    • Duty ratio: 10%.
    • Multiple heads in muscle ensure continuous binding.
  • Myosin V:
    • Step size: 36 nm.
    • Duty ratio: 70% (processive).

Myosin V Mechanism

  • Hand-over-Hand: Step size of 72 nm for a single motor head.
  • Inchworm: Step size of 36 nm for a single motor head.
  • Findings: Myosin V moves by hand-over-hand mechanism, taking 72 nm steps (distance between helical turns on actin).

Vesicle Transport

  • Vesicles can bind both Kinesin/Dynein (microtubule transport) and Myosin V (actin filament transport).
  • Mechanism: Vesicles move from microtubules to actin filaments near the cell cortex using both Kinesin/Dynein and Myosin V.