microtubules

microtubule assembly

• assemble from microtubule organising centres (MTOCs) - spontaneous nucleation of microtubules energetically unfavourable

• MTOC in animal cells - centrosome:

centrosome consists of a pair of orthogonally arranged cylindrical centrioles surrounded by pericentriolar material - (-) end of microtubules remain anchored in centrosome

• MTOC in cilia/flagella - basal body

• Microtubules in neuron:

dendrites have mixed polarity

axon have non-continuous microtubules

tubulin

• monomers of microtubules - consist of 2 subunits

• 2 subunits: alpha-tubulin, beta-tubulin

• GTP binding site on alpha-tubulin trapped at interface between the alpha and beta subunits - not accessible, once GTP has bound it cannot be removed so alpha-tubulin always bound to GTP

• GTP binding site on beta-tubulin on surface of dimer - GTP can be hydrolysed to GDP so beta-tubulin can exist in GDP/GTP form, GDP can be exchanged for GTP

organisation of tubulin subunits in a microtubule

• tubulin dimer - heterodimer of alpha and beta tubulin

• microtubule consists of protofilaments (13 longitudinal repeating tubulin units) joined together

• protofilaments have polarity - all alpha/beta-tubulin dimers are oriented in the same direction so one end has the alpha-subunit whilst the other has the beta-subunit

• seam - where alpha-subunit in one filament meets beta-subunit in adjacent protofilament

steady state

• at steady state, tubulin dimers are preferentially added to (+) end and lost from the (-) end

• (-) ends anchored to MTOC - dynamic occur mostly at (+) end

• (-) end has higher Cc - needs higher concentration of free tubulin to grow, vice versa for (+) end

• (+) end not capped so individual dimers can be added and removed

centrosomes

• 9 triplet microtubules assembled on “cartwheel” structure

• surrounded by pericentriolar material

• gamma-tubulin ring - nucleates microtubules, has helical template to bind alpha,beta-tubulin dimers, (-) end of protofilaments associated with gamma-TuRC while (+) end free for further assembly

dynamic instability of microtubules

• dynamic instability - alternation between growing and shrinking phases

• catastrophe - transition to shrinking phase, microtubule depolymerizes

• rescue - depolymerizing microtubule begins growing again

• dynamic life of microtubule end determined by:

1. rate of growth

2. frequency of catastrophe

3. rate of depolymerisation

4. frequency of rescues

dynamic instability - why

• GTP-beta-tubulin slightly curved

• GDP-beta-tubulin curved

• GTP-beta-tubulin binds more stably and caps growing end

• GDP-beta-tubulin binds less stably - disassembly

• if GTP-beta-tubulin added faster than it hydrolyses GTP, tubule grows

• if addition is slower than hydrolysis, the cap is lost and tubules shrinks rapidly - catastrophe

• protofilament gets curved when beta-tubulin converts GTP to GDP - changes conformation which introduces tension

GTP cap

• protects growing (+) end

• GTP-beta-tubulin containing protofilaments zip up by lateral interactions to form straight protofilaments - creates cap

• lateral protofilament-protofilament interactions strong enough to prevent microtubule unpeeling at (+) end