unit 2

• Microtubules are straight, hollow cylinders of varied length that consist of usually 13 longitudinal arrays of polymers called protofilaments
• the basic subunit of a protofilament is heterodimer of tubulin, one alpha-tubulin and one beta-tubulin
• alpha and beta-tubulin bind noncovalently to form an alpha-beta-heterodimer, which does not normally dissociate
• cytoplasmic microtubules pervade the cytosol and are responsible for a variety of functions:

  • maintaining axons
  • formation of mitotic and meiotic spindles
  • maintaining or altering cell shape
  • placement and movement of vesicles
• singlet: one hollow tube with 13 protofilaments
• axonemal microtubules include the organized and stable microtubules found in structures such as cilia, flagella, and basal bodies to which cilia and flagella attach
• the axoneme, the central shaft of a cilium or flagellum, is a highly ordered bundle of MTs
• doublets: found in cilia and flagella
• triplets: found in basal bodies
• doublets and triplets contain one 13 protofilament tubule (the A tubule) and one or two additional incomplete rings (B and C tubules) of 10 or 11 protofilaments. they are bound to each other and will never separate
• in vitro microtuble assembly (alpha-tubulin, beta-tubulin, GTP)

  1. a-B tubulin come together and form heterodimers
  2. heterodimers form oligomers (short protofilaments)
  3. MT assembly - adding heterodimers
  4. MT growth -> can occur in this experiment at both ends
• Kinetics of MT assembly

  1. lag phase: slow assembly of dimers -> oligomers -> protofilaments
  2. elongation phase: established MT structures elongate quickly
  3. plateau phase: free tubulin concentration falls and MTs reach structural equilibrium

  
• critical concentration: tubulin heterodimer concentration at which assembly and disassembly is balanced. [heterodimer] at which growth stops

  • higher at the (-) than the (+) end
• if the concentration of tubulin monomers is above the critical concentration of the plus end but below the critical concentration of the minus end, what will happen to in vitro microtubule assembly?

  microtubule length will remain generally constant, but cellular position will shift towards the plus end
• addition of tubulin dimers occurs more quickly at the plus ends of microtubules
• the two ends of a MT differ chemically, and one can grow or shrink much faster than the other.

  • this can be visualized by mixing basal bodies (structures found at the base of cilia) within tubulin heterodimers
  • the rapidly growing MT end is the plus end, and the other is the minus end
• MT originate from a microtubule-organizing center (MTOC)
• many cells have an MTOC called a centrosome near the nucleus
• in animal cells, the centrosome is associated with two centrioles surrounded by pericentriolar material
• centriole walls are formed by nine triplets of microtubules
• the two centrioles of a centrioles of a centrosome are oriented at right angles to each other
• Centrioles are involved in basal body formation for cilia and flagella
• cells without centrioles have poorly organized mitotic spindles
• in order to form a basal body, a centrosome breaks up
• centrosomes have large ring-shape protein complexes in them; these contain gamma-tubulin
• gamma-tubulin is only found in centrosomes
• • gamma-tubulin ring complexes (γ-TuRCs) nucleate the assembly of new MTs away from the centrosomes
• the loss of γ-TuRCs prevents a cell from nucleating MTs
• drugs effecting MT function are called antimitotic drugs because they interfere with spindle assembly and disassembly and thus inhibit cell division
• Negative regulator of MT assembly

  • stop MT growth
  • colchicine and nocodazole: both bind B-tubulin and inhibit MT assembly
  • mitotic spindle cannot assemble
• positive regulators of MT assembly and stability

  • block MT disassembly
  • taxol: binds tightly to MTs and stabilizes them
  • used as an anti-cancer drug
• Microtubule stability is tightly regulated in cells by a variety of microtubule binding proteins

  • cells regulate MTs with great precision
  • some MT-bind proteins use ATP to bind vesicle or organelle transport or to generate sliding forces between MTs
  • others regulate MT structures and stability
  • stabilize MTs (MAPs and +TIP proteins)
  • destabilize MTs (catastrophins)
• microtubule-associated proteins (MAPs): bind at regular intervals along a microtubule wall, allowing for interaction with other cellular structures and filaments

  • a MAP called Tau causes MTs to form tight bundles in axons
  • MAP2 promotes the formation of looser bundles in dendrites
• • MAPs such as Tau and MAP2 have two regions
  • one region bind to the MT wall, and another part of the protein extends at right angles to the MT to allow for interaction with other proteins
  • the length of the extended "arm" controls the spacing of MTs in the bundle
• +TIP proteins

  • MTs can be stabilized by proteins that capture and protect the growing plus ends
  • these are +TIP proteins ( +-end tubulin interacting proteins)
  • proteins such as EB1 (end-binding protein 1) decrease the likelihood that MTs will undergo catastrophic subunit loss
• some proteins promote the depolarization of MTs

  • catastrophins act at the end of MTs and promote the spelling of subunits from the ends
  • mitotic centromere-associated kinesin (MCAK) regulates the shortening of the mitotic spindle during telophase
• dynamic assembly and disassembly of a cytoplasmic microtubule in a cell occurs primarily at its plus end, because its minus end is usually anchored to a microtubule organizing center
• microfilaments are the smallest of the cytoskeletal filaments

  • they are best known for their role in muscle contractions
  • they are involved in cell migration, amoeboid movement, and cytoplasmic streaming
  • cell motility
  • movement associated with the plasma membrane (changes in cellular shape)
  • microfilament driven
  • made of a single protein (actin)
• Actin is the protein building block of microfilaments

  • actin is a very abundant protein in all eukaryotic cells
  • once synthesized, it folds into a globular-shaped molecule that can bind ATP or ADP (G-actin; globular actin)
  • G-actin molecules polymerize to form microfilaments, F-actin
• cytosolic MTs are responsible for a variety of cellular functions

  • in animal cells crosslinked bundles of cytosolic MT are required to maintain axons
  • in plant cells, cytosolic MTs govern the orientation of cellulose microfibrils deposited during the growth of cell walls