Actin filaments

• Helical polymers G-actin
• Flexible, linear bundles, 2D networks
• Concentrated at cortex; beneath cell membrane
• Strength and shape to plasma membrane
• Structures - microvilli, extremely stable
• Cell migration - lamellipodia and filopodia
• Motor proteins- myosin (will move on actin fibers and transport cargo. Allow for sliding of adjacent actin fibers and is important during muscle contraction)

Microtubules

• Long and hollow cylinder of tubulin
• Rigid, long, and straight
• Function - internal framework for organelles (tracks for vesicular trafficking), structural support and motion (flagella and cilia), mitotic spindle formation during cellular division
• Motor proteins- kinesin and dynein

Intermediate filaments

• Rope-like fibers instead of helical structure, stable
• Heterogenous family (tissue specific)- laminin, keratin
• Animal cell nucleus- nuclear lamina
• Flexible and strong
• Function - create nuclear lamina, mechanical strength for epithelial cells and layers
  • Hair and nails

Accessory proteins

• Dynamic framework
• Regulated by accessory proteins
  • Cell can regulate length, stability, number, and geometry
  • Can bind stabilize disrupt and create junctions of those networks
• Cell changes cytoskeleton organization in response to signals
• If migrating, will adjust inside of cytoskeleton that allows and facilitates movement of the cell

Actin microfilaments and actin binding proteins

• Microfilaments are assembled into diverse structures (by means of actin binding proteins), usually associated with the plasma membrane
• Cross-linked bundles and networks: microvilli, cell cortex, adherens belt, filopodia, lamellipodium/leading edge, stress fibers, phagocytosis, moving endocytotic vesicles, contractile ring

Actin structures

• Highly conserved proteins across eukarya
  • Signifies that it is extremely important
• Binds to ATP and ADP
  • ATP hydrolysis in F-form
    • Slow ATP hydrolysis
    • G-actin in f-actin able to hydrolyze ATP
• Reversible assembly into filaments- two helices of actin subunits oriented in the same direction
• Monomer - G-actin (globular)
• Polymer - F-actin (filamentous)
  • Plus (barbed) and minus (pointed) polarity
  • Nucleotide binding site on minus end, where the ATP/ADP will bind
• Able to self assemble to form a longer filament

Dynamics of actin filaments

• Microfilaments are dynamic structures
• Assembly and disassembly regulated process- actin binding proteins
  • Plus end - fast growing end (highly dynamic, when actin is bound to ATP it's likely to bind to the filamentous actin on the plus end. The ATP is hydrolyzed into ADP and eventually dissociates from actin filament)
  • Minus end - slow growing end
• ATP hydrolysis promotes F-actin depolymerization
• Actin bound to ADP less binding strength among monomers

Actin nucleation - rate limiting step structures

In vitro (test tube)

• Nucleation- initial aggregate that allows for elongation
• Polymerization- spontaneous (faster) in the presence of seeds (preformed filament)
• Critical concentration (Cc) - concentration of G-actin at steady state (rate of polymerization = rate dissociation)
  • In cells, concentration of free G-actin much higher than Cc
  • Allows for dramatic and fast changes in the actin cytoskeleton
  • Has proteins that sequester G actin, liberates them when the cell needs them, allows for reshaping of cytoskeleton

Actin polymerization

• Steady state - rate of polymerization = dissociation(depolymerization)
• F-actin
  • T form - ATP
  • D form - ADP
• G-actin critical concentration (Cc)
  • High in t form
  • Low in D form
  • Suggests the plus end is the growing end and the minus end is the slow growing end
• Intermediate G-actin concentration- growth in plus end and shrinkage in negative end

Actin treadmilling

• At steady state, rate of growth in plus end is equal to shrinkage in minus end (Net addition of actin subunits at the plus end while simultaneously losing subunits at minus end)
• Size of F-filament remains constant
• Allows filament to move

Inhibitors of actin

All are toxic to cells- suggesting dynamic equilibrium G-actin - F-actin are essential

Latrunculin

Depolymerizes, binds actin subunits

Cytochalasin B

Depolymerizes, caps filament plus ends

Phalloidin

Stabilizes, binds along filaments

Actin binding proteins

Important in the regulation of this dynamic equilibrium between G-actin and F-actin

Actin monomers, monomer-sequestering protein, bundling protein, myosin motor protein, side-binding protein (tropomyosin), capping (plus end blocking) protein, cross-linking protein, severing protein, nucleating protein

Actin treadmilling- regulation

Actin turnover and filament length regulated by actin-binding proteins

Monomer availability: Thymosin

Binds G-actin to provide reserve G-actin when it is needed

Sequesters free G-actin (releases it when the cell needs to reshape actin cytoskeleton)

Monomer availability: Profilin

Enhances the exchange of ADP for ATP on G-actin

Promotes polymerization and F-actin formation on plus end

Actin-nucleating proteins

Key proteins from which an actin filament can grow- straight of branched filaments

Formins family

• Straight filaments (promote polymerization of plus end and make it faster)
• Location- plus end of F-actin
• Long actin filaments (stress fivers, contractile ring)

Arp2/3 complex

• Branched filaments
• Location- minus end of F-actin
• Act most efficiently on existing filaments (act as nucleation factor, promote growth and association of new G-actin)
• Branched F-actin networks (ex: leading edge of motile cells)

Filament: Gelsolin

• Actin severing protein- fragments of F-actin
• Fate of fragments depends on other actin binding proteins

Filament: Cofilin

• Enhances the rate of loss of ADP-actin from the minus end
• Promotes depolymerization on the minus end

Filament: Capping proteins

• Stabilize F-actin (prevent polymerization and depolymerization)
• If attached to plus end, can't grow any longer

Higher structural levels- accessory proteins

• Higher structural levels of actin filaments can be achieved with accessory proteins
• Crosslink different actin filaments
• Attach to other proteins, associations with each other or other types of proteins

Myosin

• Motor proteins that move along actin filaments
• Crosslink and slide filaments relative to one another or transport cargo
• Powered to ATP hydrolysis -> conformational change
• Move towards plus end of actin

Myosin structure

• Two heavy chains + 4 light chains
• Head: motor region (actin binding and ATP hydrolysis)
• Neck: lever-arm/hinge region
• Tail: tail-tail interaction and cargo binding

Myosin class 2

Assembles into bipolar fragments- muscle contraction

Myosin class 1

Facilitates binding actin filaments to the membrane -> important during endocytosis

Myosin class 4

Transport vesicle on the F-actin

Myosin mechanical work

• Myosin converts ATP hydrolysis to mechanical work by amplifying a small conformational change in the head when bound to F-actin
• ATP hydrolysis- change in conformation- reduced affinity for actin
• Propels forward (power stroke)

Sarcomere

• Skeletal muscle: contractile myofibirils are composed of thousands of repeating units called sarcomeres
• Myosin thick filaments and actin thin filaments

Accessory actin binding proteins

Stabilize actin filaments

CapZ

plus end cap

Tropomodulin

minus end cap

Nebulin

Regulates length of thin filament, surrounds F-actin

Titin

Molecular spring

Enables during muscle relaxation back to achieve the normal size of the a-band

Skeletal muscle contraction

ATP-dependent sliding of myosin thick filaments along actin thin filaments to shorten the sarcomere and hence the myofibril