Lecture 15

• Actin

  Globular protein (G-actin) that polymerizes into filamentous actin (F-actin), forming a helical structure with a distinct polarity
• 1. Actin Dynamics

  • ATP-bound G-actin adds preferentially to the plus end
  • ADP-actin disassembles from the minus end
  • Results in treadmilling, where subunits are continuously added at one end and lost at the other
• Mechanism of Actin Filament Assembly and Disassembly

  1. Nucleation

  2. Elongation and Treadmilling

  3. Regulatory Proteins
• Nucleation

  Initial actin assembly is nucleated by proteins like the Arp2/3 complex and formins, which provide a template for adding actin monomers
• Elongation and Treadmilling

  Once nucleated, actin filaments elongate primarily at the plus end, with the energy from ATP hydrolysis driving the dynamic instability of filament growth and shrinkage
• Regulatory Proteins

  Proteins such as profilin promote the addition of actin monomers by facilitating ADP-ATP exchange on G-actin, whereas thymosin-β4 sequesters actin monomers, inhibiting their polymerization
• Phalloidin


  Binds and stabilizes F-actin, commonly used in conjunction with fluorescent labeling to visualize actin filaments in cells
• Cytochalasin D and Latrunculin A

  Bind to actin monomers and inhibit their polymerization, effectively blocking filament formation
• Cofilin and Aip1

  Act cooperatively to disassemble actin filaments, with cofilin severing filaments into fragments and Aip1 further breaking these into monomers
• Myosin II

  Involved in muscle contraction, myosin II forms bipolar thick filaments that interact with actin filaments to generate contractile force through a power stroke mechanism facilitated by ATP hydrolysis
• Myosin V

  Known for its role in organelle transport, myosin V walks along actin filaments, carrying cargo such as vesicles to specific locations within the cell, critical for processes like endocytosis and exocytosis
• Skeletal Muscle Mechanics

  Myosin II motors in muscle cells pull on actin filaments to shorten sarcomeres, the basic unit of muscle contraction. This interaction is regulated by the cyclic binding and hydrolysis of ATP, coupled with conformational changes in the myosin head
• Cell Motility

  • Actin polymerization and myosin motor activity are key drivers of cell movement, enabling cells to extend and retract protrusions like lamellipodia and filopodia during migration
• Cell Division

  • Actin-myosin interactions are crucial during cytokinesis, where they help form the contractile ring that divides the mother cell into two daughter cells
• Actin Binding Proteins

  • Monomer binding proteins
  • Filament severing proteins
  • Capping proteins
  • Nucleators
  • Bundling proteins
  • Side binders
  • Molecular motors
• Profilin


  Binds to actin monomers and promotes the exchange of ADP for ATP, facilitating actin assembly at the fast-growing (+) end of filaments
• Thymosin β4


  Sequesters actin monomers, preventing their polymerization and thereby regulating the pool of actin available for filament formation
• Cofilin


  Binds along actin filaments and induces breaks, increasing the turnover of actin filaments and contributing to their dynamic restructuring
• CapZ


  Caps the barbed (+) end of actin filaments, stabilizing them by preventing the addition or loss of actin monomers
• Tropomodulin


  Caps the pointed (-) end, regulating the length and disassembly of actin filaments
• Formin


  Promotes the nucleation and elongation of unbranched actin filaments. It is activated by Rho GTPases and plays a critical role in the formation of long straight actin filaments
• Arp2/Arp3 Complex


  Induces the formation of branched actin networks, essential for cellular processes like cell motility and the maintenance of cell cortex structure
• Fimbrin and α-Actinin


  Organize filaments into tight or loose bundles, respectively. Fimbrin tightly packs filaments, excluding myosin II and facilitating the formation of rigid structures like microvilli. α-Actinin forms looser bundles that allow access for myosin II, crucial in contractile structures such as stress fibers and the contractile ring during cytokinesis
• Tropomyosin


  Stabilizes actin filaments by binding along their sides, influencing the interaction of actin with other proteins including myosins
• Myosins

  • Myosin I
  • Myosin II
  • Myosin V
• Myosin II


  Forms dimers with coiled-coil tails, creating a structure conducive to filament formation. The heads of these dimers are active sites for ATP hydrolysis, driving the motor function
• Muscle Architecture

  Myosin II molecules are central to the formation of sarcomeres—the functional units of muscle fibers—where they interact with actin filaments to facilitate contraction
• Contraction Mechanics

  In muscle cells, myosin II heads bind to actin filaments and pull them closer together, shortening the sarcomere and thus contracting the muscle. This process is regulated by the troponin-tropomyosin complex, which responds to calcium ion levels to expose or hide the actin binding sites on myosin
• Power Stroke Mechanism

  Binding of ATP to the myosin head initiates a conformational change that allows the myosin to bind to an actin filament. Upon ATP hydrolysis and subsequent release of ADP and phosphate, the myosin head pivots, pulling the actin filament along—this is the power stroke
• Resetting the Myosin Head

  Binding of a new ATP molecule releases myosin from actin, allowing the head to reset to its original position and ready it for another cycle of movement
• Cellular Functions of Myosins

  • Maintaining cell integrity
  • Segregating chromosomes during cell division
  • Facilitating intracellular transport
• Fluorescent Actin Filament Movement Assays

  • Observing fluorescently labeled actin filaments as they move over myosin-coated surfaces, providing insights into the mechanics of myosin action
• Role in Organelle Organization

  • Myosins organize organelles within the cell by transporting them along the cytoskeleton
• Contribution to Body Mechanics

  • Beyond the cellular level, myosins play essential roles in processes like blood circulation, digestion, and overall body movement
• Myosin Motor Functions in Cells and the Body

  • Organizing organelles within cells
  • Segregating chromosomes during cell division
  • Transporting proteins and RNA
  • Facilitating cell motility and chemotaxis
  • Maintaining structural integrity of cells
• Non-muscle Myosin II

  Important for forming contractile bundles of actin filaments which are essential in various cellular activities like cytokinesis and maintaining cell tension
• Myosin V


  Known for its role in transporting vesicles along actin filaments towards their plus ends, crucial for processes such as exocytosis
• Step Size and Duty Ratio

  Myosin movement along actin filaments is characterized by its step size and the percentage of time a myosin head remains bound to actin (duty ratio). These factors are critical for determining the effectiveness and type of motion myosin can perform
• Myosin II

  Typically takes smaller steps (5-10 nm) and has a low duty ratio, meaning it spends less time attached to actin, which is suitable for its role in muscle contraction where many myosin heads work collectively
• Myosin V

  Described as a processive motor that takes larger, successive 36 nm steps with a high duty ratio, ensuring that at least one of its heads is always in contact with the actin filament, ideal for transporting cargo over long distances within cells