Powerpoint

• Nucleation and actin web formation by Arp complex


  Arp complex attaches to the side of another actin filament

  Forms a treelike actin filament web

  NRP activation can induce Arp dependent nucleation
• Capping proteins


  prevents assembly and disassembly at positive ends of actin filaments
• Tropomyosin


  stabilizes filaments and binds accessory proteins
• Gelsolin


  severs filaments and binds to positive ends of actin filaments
• Myosin: actin binding motor proteins

  • all actin dependent motor proteins belong to the myosin family
  • myosin binds to and hydrolyze ATP proving energy for movement along actin filaments
  • Myosin's move along actin filaments from the negative to positive end
• Myosin II


  2 globular heads bent backwards and a long tail

  Four heavy chains; two of each per myosin head

  Heavy chains form a coiled-coil

  Hydrophobic amino acids
• The myosin II bipolar thick filaments in muscle

  Clusters of myosin II molecules bind to each other through their coiled- coil tails, forming a bipolar myosin filament (like a double headed arrow)
• Structural changes enabling myosin II movement along actin filament

  1. Binding: Myosin II binds to actin filament
  2. Power Stroke: Myosin head pivots, pulling actin filament towards center of sarcomere
  3. Release: ATP binds to myosin, causing detachment from actin
  4. Reset: ATP hydrolysis triggers myosin head to return to original position
  5. Rearrangement: Myosin head rebinds to actin, ready for next power stroke
• Skeletal muscle: myofibrils

  Z disc: attachment site for the plus ends of actin filaments

  The actin and myosin filaments slide past one another without shortening during contraction

  Sarcomere: the contractile unit of muscle. Highly organized assemblies of actin filaments and myosin-II filaments
• T tubules and the sarcoplasmic reticulum surround the myofibrils

  • The Ca2+ channels in the sarcoplasmic reticulum are triggered open when the cell membrane is depolarization and Ca2+ is released into the cytosol
• Signaling during muscle contraction: Excitation-contraction coupling

  • Muscle contraction is triggered by a sudden rise of cytosolic [Ca2+]
  • Ca2+ interacts with accessory proteins that are closely associated with actin filaments
  • Tropomyosin and troponin are two such proteins, each functions as a molecular switch
  • Tropomyosin: binds to actin filaments (covers 7 actin monomers), preventing the myosin heads from associating with the actin filaments
  • Troponin: a protein complex containing a Ca2+-sensitive protein, and is associated with the end of a tropomyosin
• The control of skeletal muscle contraction by troponin:

  [Ca2+] increases -> Ca2+ binds to troponin -> tropomyosin movement

  -> Myosin-binding site exposed
• Muscle contraction by a sliding-filament mechanism:

  1. Myosin heads walking toward the (+) end of the adjacent actin filament(without shortening)
  2. The actin and myosin filaments slide past each other without shortening
  3. Simultaneous shortening of all the sarcomeres
  4. Muscle contraction
• Phalloidin


  Stabilizes filaments by binding [example: amanita muschroom]
• Microtubules: usually grow out of an organizing center (MTOC)

  • Play crucial organizing roles in all eukaryotic cells
  • Can also form permanent structures (e.g. eukaryotic cilia and flagella)
  • Form mitotic spindles during mitosis
  • Guiding intracellular transport (track system in a cell)
  • Anchoring organelles in place
  • Highly dynamic: disassemble and reassemble at different cell locations
• Microtubule structure and subunits

  • Most microtubules are ~25nm hollow tubes comprised of 13 protofilaments arranged side-by-side
  • The α and β subunits share a similar structure
  • Polarity of a protofilament: the minus-end starts with an α and the plus-end ends with a β-subunit (-)αβαβαβαβαβ(+)
  • The αβ-tubulin heterodimer forms as soon as the α and β subunits are synthesized, forming the fundamental subunit
  • N- terminal domain: GTP-binding site
  •  C- terminal domain: binding site for associated proteins
• Dynamic instability due to the structural differences between a growing and shrinking microtubule end

  • GTP hydrolysis control the growth of microtubules
  • Tubulin dimers carrying GTP bind more tightly to one another
  • Tubulin dimers carrying GDP bind less strongly to one another
  • Tublulin dimer region is less stable
  • GTP-containing subunits form GTP cap at microtubule end for rapid growth
  • If the cap is lost microtubules shrink [catastophe]
• Microtubule dynamics: How do microtubules grow?

  • Formation of an initial ring of 13 tubulin molecules
  • Tubulin dimers are then added to build up the “hollow tube”

  In vitro : Under high [tubulin], αβ dimers add to either end of a growing microtubule (add more rapidly to the plus end)

  
  

  In vivo : [tubulin] is too low for spontaneous polymerization

  A nucleation site is required to initiate the assembly

  This is provided by special organizing centers in vivo
• The centrosome: the primary microtubule-organizing center (MTOC) in animal cells

  • Centrosomes contain hundreds of γ-tubulin rings
  • Each γ-tubulin ring serves as the nucleation site for the growth of one microtubule
  • The orientation of adding αβ-tubulin dimers to γ-tubulin rings
• Plectin


  links to intermediate filaments
• Microtubules are maintained by balance of assembly and disassembly

  • Evidenced by certain drug effects on mitotic spindle
  • Colchicine binds to tubulin dimers, inhibits further polymerization once the colchicine-bound dimer is added to a microtubule
  • Taxol binds to microtubule and stabilizes them by inhibiting disassembly
  • Both drugs have the same overall effect on the cell: cause arrest of mitosis
• Colchine


  depolymerizes filaments by capping both filament ends
• Taxol (paclitaxel)


  stabilizes filaments by binding
• Chemical Inhibitors of Actin?

  1. taxol
  2. colchicine
• Microtubule associated motor proteins enable cargoes to move along microtubules

  Two families of motor proteins that move along cytoplasmic microtubules:

  ● Kinesin: Move toward the plus end

  ● Dynein: Move toward the minus end
• • Kinesin-1:
  • Motor Domain: N-terminus of heavy chain
  • Movement Direction: Towards microtubule plus end
  • Middle Domain: Long coiled-coil for dimerization
  • C-terminal Domain: Tail for cargo attachment
  • Kinesin-5:
  • Structure: Forms tetramers
  • Activity: Slides two microtubules past each other
  • Analogous Activity: Similar to bipolar thick filaments of myosin II
  • Kinesin-13:
  • Motor Domain: Located in middle of heavy chain
  • Activity: Binds to microtubule ends, promotes depolymerization
  • Kinesin-14:
  • Type: C-terminal kinesin
  • Movement Direction: Toward microtubule minus end
• cytoplasmic dynein moves towards the minus end of microtubules

  *AAA ATPase: ATPases Associated with Diverse Cellular Activity
• Motile cilia and flagella are built from microtubules and dyneins

  • The arrangement of microtubules within the flagellum or cilium, known as the "9 + 2" arrangement, which refers to nine outer doublet microtubules surrounding two central singlet microtubules.
  • the bending of axoneme in microtubules is caused by dynein and contributes to overall shape and structure
• Functions of Intermediate Filaments

  • Perform primarily a structural role: reinforce and protect cells and organize them into tissue (e.g. skin, hair, and fingernails)
  • Provide mechanical support for the plasma membrane, where it contacts other cells and the extracellular matrix
  • Cytoplasmic network: distributed throughout the cytoplasm (surrounding the nucleus and extending out to the cell periphery)
  • Nuclear lamina: a mesh of intermediate filaments underlies and strengthens the nuclear envelope in all eukaryotic cells
  • Plays NO role in cell motility or organelle transport
• Properties of Intermediate Filaments

  ● Size (Ф 8 - 12nm), so intermediate between microfilaments and microtubules

  ● Form braided rope-like fibers, not tubes (microtubules) or beaded-strings (microfilaments)

  ● Do NOT bind nucleotides● Highly stable – no dynamic instability model applies to

  the CYTOPLASMIC intermediate filaments

  ● Play no role in directed movements

  ● Diversity: intermediate filament proteins constitute a large & heterogeneous family
• Intermediate filaments strengthen cells against mechanical

  Cells that are subjected to mechanical stress have large # of intermediate filaments

  • nerve cells
  • epithelial cells
  • nuclear lamina (mesh like network)
• Intermediate filaments from the cytoplasmic --> Keratins!

  
• Keratin filaments in epithelial cells

  ● Kratins have the most diverse subunit family● Different epithelia have different sets of keratins

  ● Keratin filaments associate laterally with other cell components through their globular head & tail domains

  ● Mutations in the keratin genes cause human disease (epidermolysis bullosa simplex [EBS], in which skins become very fragile and blister easily)
• Intermediate filaments keep cells and their membranes from breaking in response to mechanical shear