Page 2: Learning Objectives

• Characterize actin filament structure and composition.

• Formation and dynamics of thin filaments: Understanding how they are formed in cells and the roles of associated motor proteins.

• Actin-associated proteins: List and distinguish their functions.

• Role of thin filaments: Distribution and diverse functions in cellular contexts.

• Muscle tissue structure and function: Analyzing actin's contribution to muscle activity.

• Cellular movement mechanisms: Outlining how actin facilitates cellular motility.

Actin Filaments

• Major Protein Component: Actin is integral to all cells, located at cell junctions and throughout cellular structures.

Cytoskeletal Filaments

• Types of cytoskeletal filaments:

  • Actin: Also known as the basic unit of thin filaments/microfilaments, vital for a multitude of functions. (side roads)

  • Intermediate filaments: Provide tensile strength against stress.

  • Microtubules (thick filaments): Serve various functions, acting as pathways for intracellular transport. (major highways)

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Actin Dynamics

• Dynamic structure: Actin constitutes 10% of muscle cell protein and 1-5% in non-muscle cells.

• Filament formation and disassembly: Actin filaments are continuously formed and disassembled, tailored to cellular needs and environmental stimuli.

• Actin genes: Humans have six actin genes that contribute to the diversity of actin functions.

Functions of Actin

• Diverse roles: Actin is involved in cellular structure, movement, phagocytosis (“eating”), and cell division.

• Three types of actins:

  • α-actin: Found in muscle fibers.

  • β-actin: Present in the leading edge of cells.

  • γ-actin: Associated with stress fibers.

Actin-Binding Proteins

• Table of Proteins: Overview of actin-binding proteins and their functions, e.g., Profilin (nucleotide exchange), Cofilin (disassembly), and various capping and cross-linking proteins.

• Functionality: Proteins that can promote actin polymerization, stabilize filaments, or sever actin structures. Proteins associated with actin can modify it by nucleating, stabilizing, serving, capping, etc.

Actin Monomers

• Actin exists as G-actin: Globular actin binds ATP and Mg2+.

• Polymerization: Forms filamentous actin (F-actin) with 2 helical structures wrapped around one another. .

Polarization of Actin Filaments

• Polar characteristics: Myosin S1 regions coat F-actin, creating a polarized structure with distinct ends for filament dynamics.

  • form arrow like structures:

    • (-) pointed end: end has cleft exposed to the solution

    • (+) barbed end: has the cleft contacting other subunits

Filament Dynamics

• End addition preference: G-actin adds more readily to the (+) end of the filament compared to the (-) end, influencing overall growth.

  • to form filaments G-actin-ATP units are added to both ends.

  • (+0) end addition is almost 10x greater than the (-) end

Actin Filaments need a Critical Concentration

• Requirement for filament growth: Actin filaments need a critical concentration of G-actin-ATP. Each different Cc which much lower [Cc] required in the (+) end.

• ATP is hydrolyzed quickly when actin monomers are added creating 3 distinct regions.

• Treadmilling is a dynamic equilibrium process in filament assembly and disassembly. Seen at steady state the (-) end loses monomers while new ones are added to the (+) end

Steps of Actin Filament Formation

• 1. Nucleation

  2. Elongation

  3. Steady State

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Actin Recycling

• Profilin's role: Enhances actin recycling and promotes the binding of ATP-actin at the (+) end, contributing to dynamic filament reassembly.

• Profilin binds G-actin lost from filaments and opens the cleft enhancing loss of ADP

  • Profilin-ATP-actin complex binds the (+) end

• Cofilin binds 2 ADP-actin towards the (-) end, causes a twist and a break

  • Generates more (-) ends for disassembly

• Thysomin - B4 binds free ATP-actin to keep it all from assembling needless into filaments

  • Forms dynamic equilibrium with the free subunits

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Capping Proteins

• To regulate the formation and length of filaments/dynamics

• CapZ binds the (+) end

  • Formed from two related proteins

• Cells regulate CapZ ability to stop growth

  • Phosphatidylinositol 4,5- bisphosphate (PIP2)

  • Regulator proteins

• Tropomodulin binds the (-) end and can module filament assembly and disassembly

  • Mostly found where actin needs to be stabilized

Actin-Nucleating Proteins

• 2 major classes of actin-nucleating proteins help in the assembly of F-actin cells.

  • Formin proteins: Facilitate long filament assembly.

  • Arp2/3 complex: Promotes branched filament networks.

Formins Homology 1, 2 Domains in Formins

• FH1 domains bind profilin to increase ATP-actin

• FH2 domains form a ring that rocks back and forth promoting addition of ATP actin to the (+) end

  

Regulatory Signaling

• Signal pathways: Actin nucleation and filament formation can be controlled through receptor-mediated signaling pathways, leading to the activation of formin proteins.

• 

ARP2/3 and NPFs

• Complex function: The ARP2/3 complex promotes nucleation and creation of branched F-actin structures, activated by nucleation-promoting factors. This binds to existing F-actin and promotes branch formation.

WASp Activation

• WASp's (Wiskott-Aldrich syndrome) role: Serves as a nucleation-promoting factor by activating signaling cascades that stimulate Arp2/3 near cellular membranes.

Listeria Monocytogenes

• Overview of Gram-positive bacteria: Causes listeriosis with significant health risks, particularly for pregnant women and immunocompromised individuals.

• ActA protein mimcs an NPF and activated and recruits Arp2/3. Actin polymerization propels the bacteria through the cell.

Actin Role in Membrane Dynamics

• Membrane mechanisms: Actin polymerization generates force and movement and aids in endocytosis, with contributions from signaling factors that assist in membrane dynamics.

• Bursts of Arp 2/3 activation recruits other factors that can help power this energetically unfavorable membrane dynamic

• 

Phagocytosis

• Immune function: Describes how actin aids immune cells in engulfing pathogens through receptor-mediated signaling and network formation.

  • Opsonized pathogen are immobilized by the cell and receptor mediate signaling activates local Arp2/3 activation and network formation. creates a contractile network that leads to isolation of the pathogen and subsequent destruction.

Cross-Linking Proteins

• Structural diversity: Actin cross-linking proteins mold filaments into varied structures, essential for functions such as sound perception in stereocilia.

Erythrocyte Structure

• Stress handling: Actin networks are crucial for erythrocyte integrity, maintaining its shape and structure under stress. Actin membrane is anchored by actin associated proteins such as a/B spectrin. Actin gives cell shape and structure.

Actin Filaments and Muscle

• Myosin II was the first discovered motor protein, integral in muscle cell function by converting chemical energy to mechanical work. All family motor proteins can bind to actin.

• Immobilized ATPase containing mysoin S1 subunits will move actin filaments under experimental conditions. 40 myosin genes in the human genome.

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• Myosin Classes

  • Myosin I – only one with asingle head domain and it is associated with the membrane

  • Myosin II – mostly in muscle (thick fibers) but also non-muscle in less organized fashion, used for contraction

  • Myosin V – motor protein to hall things towards the membrane

  • Myosin VI – only member that moves towards the negative end, moving things away from the membrane

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Page 29: Myosin-Actin Interaction

• Power stroke dynamics: Myosin-ADP binds to F-actin. ATP binds releasing myosin from F-actin Hydrolysis cocks the head domain. Head domain binds actin. Release of Pi moves it back into its original position. In muscle 10% of the myosin is contacting f-actin when activated.

• Myosin Steps

  • Step size of myosin head is relative to the length of the neck domain. (longer legs = bigger step),

    • Myosin II moves about 8nm

    • Myosin V takes longer 72 nm steps, hauling cargo 36nm ata. step

• Muscle Fiber Composition

  • muscles —> muscle fibers (multinucleated muscle cells) —> myofibril structures composed of sacromere

  • Z disk structures flank: Myosin II thick filaments alternating with F-actin thin filaments attached to the Z disk

  • Thick filaments are bipolar with heads out toward Z disks. F-actin is attached to Z-disk. During contraction the mysoin II moves towards the (+) shortening the distance between the (-) end of F-actin.

    • Controlled by ATP availability and Ca2+ influx

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Accessory Proteins in Muscle

• Structural support: CapZ, Tropomodulin, Titin, and Nebulin play crucial roles in maintaining muscle structure and function.

  • CapZ helps bind F-actin (+) and Tropomodulin (-)

  • a giant elastic titin protein binds Z disk and extends toward the M band on either side (holds the thick filaments in place)

  • Giant nebulin protein wraps around the F-actin

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Cytokinesis

• Role of actin: Highlighting the involvement of actin-myosin in cytokinesis, forming contractile rings that assist in cell division.

• Sarcoplasmic reticulum (SR) holds a high store of Ca2+ for skeletal muscle regulation

• Neural impulse travels down the transverse tubules and opens voltage- gated Ca2+ channels in the SR

• The release of Ca2+ allows muscle contraction by binding Troponin

  • Troponin shifts Tropomyosin

• Actin-Mysoin II is involved in Cytokinesis. A contractile ring forms after chromosomal partition and eventually pinches off.

Smooth Muscle Contraction

• Regulatory mechanisms: Smooth muscle contractions are controlled by signaling pathways rather than calcium levels alone.

  • Myosin is inactive during relaxation

• Phosphorylation activates it forming filaments that can induce contraction in smooth muscle.

  • Dephosphorylation relaxes it again.