microtubules

• microtubules are rigid polymers of alpha and beta tubulin that provide structural support and tracks for movement if organelles and proteins
• microtubules are composed of polymers of alpha and beta tubulin heterodimers
• microtubules are 25nm in diameter and can be as long as 20 um in vivo
• microtubules have polarity with growing plus end and a slower growing minus end

  • plus end is peripheral and the minus end is interior (usually anchored to microtubules organizing center (MTOC))
• microtubules organizing centers (MTOC) are centrosomes/centrioles (cytoplasm) or basal bodies (axonemes in cilia)
• motor proteins move along microtubules (dynein and kinesin)
• every single microtubule starts its life in the centrosome and moves out adding tubulin dimers into the cell
• dynein and kinesin are the inly 2 motor proteins that can bind to microtubules (one goes one direction, the other goes in the other)
• alpha and beta tubulin each bind GTP or GDP
• dimers are very stable and rarely dissociate
• GTP on alpha tubulin is not exchanged readily (usually stays GTP)
• GTP on Beta tubulin is exchanged more quickly
• when microtubule is formed, beta tubulin GTP is hydrolyzed
• assembly of microtubules is much faster with GTP tubulin than GDP tubulin
• association and dissociation occurs only at ends of microtubules
• alpha GTP is buried in dimer - not readily active

  beta GTP is exposed - actively hydrolyzed
• microtubules are cylinders composed of longitudinally arranged protofilaments composed of tubulin dimers
• most common microtubules have 13 protofilaments but some have more (15, 16) or less (11)
• all microtubules have beta tubulin at plus end and alpha tubulin at minus end
• if enlarged million fold, microtubules would have diameter of 25nm (same mechanical properties of steel pipe)
• catastrophe: dimers start peeling off of microtubules

  • losing instead of gaining
  • can be stopped before it hits zero as long as dimer is bonded to BDP
  • can start binding out again
• dynamic instability:

  • axoneme microtubules (in cilia) are stable for days/weeks
  • spindle microtubules are turned over in minutes/seconds
  • undergo rapid depolymerization and then regrowth in minutes (leads to movement of chromosomes in mitosis)
  • growing and shrinking microtubules exist in steady state
  • microtubules resist compression, thus provide cytoskeletal support
  • accessory proteins can bind tubulin dimers, stabilize microtubules, associate with microtubule ends or server microtubules (microtubule associated proteins (MAPs))
• dynamic instability:

  • at steady state, individual microtubules grow slowly until they undergo rapid shortening (catastrophe)
  • microtubule polymer loses tubulin at rate of 1000 dimers/sec
  • protofilaments peel away from microtubule
  • rapid shortening is terminated by random event (rescue) where GTP tubulin caps the plus end which is followed by further growth
• • hydrolysis of beta tubulin GTP to GDP drives dynamic instability
  • dimeric tubulin hydrolyses beta tubulin GTP slowly, but it increases when incorporated into microtubule (250X)
  • alpha tubulin GTP does not hydrolyze rapidly, therefore microtubules usually have GDP bound to beta tubulin and GTP bound to alpha tubulin
  • GTP tubulin at the microtubule ends stabilizes the filament (GTP caps) through direct exchange of GTP onto beta tubulin at plus end
  • loss of GTP cap is thought to cause a catastrophe
  • frequency of catastrophe is inversely proportional to concentration of GTP tubulin dimers
• • microtubules are not formed from spontaneous nucleation from tubulin dimers (very slow)
  • cellular microtubules originate from microtubule organizing centers (MTOCs) (e.g., centrioles and centrosomes)
  • gamma tubulin ring complex (gamma TuRC) in centrioles nucleates microtubule and caps their minus end (exists separate from centrioles)
• gamma is the first tubulin put in place to initiate the growth of the microtubule
• microtubule associated proteins (MAPs):

  • proteins regulate microtubule stability, assembly and structure
  • presence of MAPs is determined by cell type and shape
  • microtubules composed of 80% tubulin and 20% MAPs
  • many MAPs are neuronal (vertebrates)
  • play critical role in regulation
  • cell type specific
• MAPs - microtubule stabilizing:

  • many MAPs bind along length of the microtubule
  • some rod shaped MAPs (MAP1A) radiate from microtubule and some lie parallel to the protofilament (e.g. tektins- found in sperm and cilia)
• Tau family of MAPs:

  • includes MAP2 and MAP4 and Tau (all rod shaped)
  • contain several microtubule binding domains (18aa) separated by flexible linkers (13aa long)
  • MAP2 keeps microtubules apart from each other
• Tau in disease:

  • in presence of tau, microtubules grow 3X faster adn have only 2% catastrophe rates
  • tau microtubule binding sites can be phosphorylated (which inhibits microtubule binding and destabilizes microtubules)
  • tau is major MAP in axons in brain
  • one tau gene gives 6 isoforms (alternate splicing)
  • tau gene has 16 exons: exons 2, 3 and 10 are differentially spliced: (2-3-10-; 2+3-10-; 2+3+10-; 2-3-10+; 2+3-10+; 2+3+10+) ( + = in final mRNA)
  • including partial phosphorylation, up to 30 tau species can be present in brain from one gene
  • mice with tau knock out are fine
• Alzheimer's disease and tau:

  • patients have 'neurofibrillary tangles' that are detected by light microscopy - more = worse diagnosis
  • tau forms paired helical filaments that aggregate in tangles
  • 'tangles' contain highly phosphorylated tau that is proteolytically truncated and cross linked via disulfide bonds
  • resistant to proteolysis and insoluble
  • may cause dementia or may be by product of disease
  • some mutations in tau are genetically inherited as dementias
• CTE (chronic traumatic encephalopathy):

  • 202 deceased athletes who had played American football from a brain donation program, CTE was neuro pathologically diagnosed in 177 players across all levels of play (87%), including 110 of 111 former National Football League players (99%)
• microtubule destabilizing MAPs:

  1. promote catastrophe: stathmin
  2. remove tubulin subunits: MCAK (kinesin 13 family)
  3. actively severs microtubules: katanin
• promote catastrophe: stathmin

  • sequesters (binds) tubulin dimers and blocks polymerization
  • phosphorylated in G2/M (inactive as enters mitosis) and dephosphorylated in G1
  • some cancer cells have high levels of mutated stathmin
• remove tubulin subunit: MCAK (kinesin 13 family)

  • kinesin like (motor protein) that bind microtubules and ATP hydrolysis of motor domain stresses microtubule at plus enf and leads to disassembly (catastrophe)
  • loss of MCAK leads to block in anaphase (mitotic centromere associated kinesin)
  • acts to control the rates of catastrophe
  • if lost, cells can't get through mitosis
• actively severs microtubules: katanin (sword in japenese)

  • katain is heterodimer severs microtubules into short fragments (active in mitosis to sever non spindle microtubules)
  • short fragments depolymerize into tubulin dimers
  • severing requires ATP hydrolysis by katanin
• growing microtubule can be with other proteins, can either lead to catastrophe or be bound to and stabilize some of the MAPs and Taus
• • several anti cancer drugs target cytoskeletal elements
  • taxol (paclitaxel) inhibits microtubule disassembly (isolated from bark of yew tree)
  • blocks cancer cells in mitosis