Exam 4

• Cytoskeleton


  • Cells need to be able to organise themselves in space (both interior and exterior) and mechanically interact with their environment and each other
  • Move and take things up
  • Support their membrane structures
  • Organize their internal compartments
  • Grow and divide properly
  • Adapt to change
• Cytoskeleton


  • Move towards nutrients, and away from toxins
• Cytoskeleton components


  • Actin filaments
  • Microtubules
  • Intermediate filaments
• Cytoskeleton


  Dynamic, components are constantly in flux, more like ant trails than highways
• Regulating dynamic behavior and assembly allows cells to build a wide range of structures from the same basic building blocks
• Cytoskeletal filaments assemble from smaller protein subunits
• Cytoskeletal filaments


  Held together by weak noncovalent interactions, can rapidly assemble and disassemble since they don't need to form or break covalent bonds
• Cytoskeletal subunits


  Asymmetrical and always bind head-to-tail, they all point in one direction
• Subunits can rapidly diffuse throughout the cytoplasm but the large filaments cannot
• Cells can rapidly disassemble filaments at one site and reassemble at a distant site
• Actin and tubulin subunits


  Catalyze ATP and GTP hydrolysis respectively, the energy from this allows these filaments to rapidly remodel
• Cytoskeletal filaments


  Regulated by accessory proteins, length, stability, geometry are all regulated
• Hundreds of accessory proteins bind the filaments or their subunits
• What accessory proteins do


  • Determine assembly sites
  • Change assembly kinetics/disassembly
  • Regulate partitioning
  • Harness energy to generate force
  • Link filaments to each other or to organelles/membranes
• Accessory proteins bring the cytoskeleton under the control of signals
• Motor proteins


  Accessory proteins that bind cytoskeletal filaments and use the energy derived from ATP/GTP hydrolysis to move along them
• Motor proteins


  Different motor proteins bind different types of filaments, move in different "directions," and can transport different cargoes
• Some motor proteins cause the filaments to exert tension, e.g. muscle contraction, cell division
• Actin filaments


  The actin cytoskeleton performs a wide range of functions in different cell types
• Types of actin


  • α: only in muscle cells
  • β and γ: found together in almost all non-muscle cells
• Actin subunits


  Assemble "head-to-tail" and form a tight, right-handed helix, filamentous or F-actin
• Actin filaments


  Polar, have a slow-growing minus-end and a fast-growing plus-end
• Actin filaments bundle together to form structures more rigid than an individual actin filament
• Nucleation


  The rate-limiting step in filament formation, small oligomers can spontaneously assemble but they are unstable and quickly disassemble
• Filament formation requires subunits to form an initial aggregate/nucleus stabilized by multiple subunit-subunit contacts (Nucleation)
• Nucleating factors


  Actin-related proteins (ARPs)
• Actin filament ends


  The plus-end polymerizes faster than the minus-end
• If the plus-end grows faster, it must also shrink faster (if the only consideration is spontaneous polymerization)
• Treadmilling


  Actin subunits can catalyze ATP hydrolysis, two forms of actin: D-form = ADP-bound, T-form = ATP-bound, subunits dissociate from D-form polymers faster than T-form, at certain free subunit concentrations D-form polymers shrink while T-form polymers grow
• Soluble ("free") subunits tend to be in the T-form, this ATP gets hydrolyzed when they are in a filament (gradual process: the longer they have been in the filament, the more likely they are to be in the D-form)
• Treadmilling: growth at plus-end exactly balances shrinkage at minus-end
• Actin polymerization


  Can generate force, above the critical concentration polymerization is energetically favorable, releases energy that cells can use to power other things, like mechanical work
• In vitro, actin polymerization is controlled simply by concentration (plus pH, salt, ATP), in cells accessory proteins tightly regulate its behavior
• Monomer availability


  In most non-muscle cells ~50% of actin is soluble and ~50% is in filaments, soluble monomer is well above the critical concentration but doesn't form filaments, actin-binding proteins make polymerization less favorable
• Actin-binding proteins


  • Thymosin (keeps monomers in a "locked" state)
  • Profilin (competes with thymosin and promotes association with the plus-end of a filament)
• Classes of actin-filament-binding proteins


  • "Side-binders" (e.g. tropomyosin)
  • "End-binders" (e.g. CapZ, tropomodulin)
• Tropomyosin


  Stabilizes and stiffens filament, prevents interactions with other filaments (important for control of muscle contraction)
• Capping proteins


  • CapZ: binds and stabilize the plus-ends, reducing both growth and de-polymerization rates
  • Tropomodulin: binds the minus-ends, of tropomyosin-coated filaments
• Severing proteins


  Break actin filaments into smaller pieces (generate new ends), can either accelerate assembly of new filament structures from these or speed up de-polymerization, change physical and mechanical properties of cytoplasm
• Cofilin


  (also called actin de-polymerization factor) binds along the filament and forces it to twist more tightly, this mechanical stress weakens the subunit-subunit contacts, preferentially binds ADP-containing filaments (tends to dismantle older filaments), can be blocked by tropomyosin-binding