Microtubules:-Structure-and-Bioche m istry-----Ac7n!is!one!of!the!most!abundant!proteins!in!eukaryo3c!cells.!!It!comprises!from!1-20%!of!the!total!protein!in!cells.!Cytoskeletal!subunit!proteins!span!the!cell!and!are!generally!abundant.-The!sequence!of!ac3n!has!been!highly!conserved!through!evolu3on.-Ac3ns!contain!~375!amino!acids!and!have!a!molecular!weight!of!~42!kDa.!Ac3ns!from!amoeba!and!humans!are!>80%!iden3cal!to!each!other!in!sequence.!Simple!eukaryotes!like!yeast!have!one!ac3n!gene.!Mammals!have!several!genes!that!produce!mul3ple!types!of!ac3n:!α-ac3n!in!muscle,!β-!and!γ-ac3n!in!nonmuscle!cells.!!(Do#bacteria#have#a#cytoskeleton?)#Last-Class:--How-are-ac7n-filaments-assem bled-?!Today:--How-are-microtubules-assembled-?!A!microtubule-(MT)!is!a!polymer!of!tubulin!subunits!that!are!arranged!in!a!cylindrical!filament!~24nm!in!diameter.!The!subunits!found!within!microtubules!are!tubulin!heterodimers.!!(How!does!this!compare!to!ac3n?)!The!tubulin!heterodimer!consists!of!the!two!closely!related!proteins!α-tubulin!and!β-tubulin.!Tubulin!sequences!are!highly!conserved!in!eukaryotes!and!each!one!is!~55kDa.!αβ-dimers!do!not!come!apart!under!physiological!condi3ons.!Mammals!express!mul3ple!α-!and!β-tubulin!genes.!Figure 16-11 Molecular Biology of the Cell (© Garland Science 2008) Microtubules-are-tubular-polymers-composed-of-αβ-tubulin-heterodimers!Microtubules-appear-to-be-hollow-structures-with-outer-ridges-available-to-bind-other-proteins-Q.Shen!&!H.Wang!MT!binding!protein!Microtubules-are-tubular-polymers-composed-of-αβ-tubulin-heterodimers-that-bind-GT P/GDP!• Both!α-tubulin!and!β-tubulin!bind!GTP.!!α-tubulin!binds!irreversibly!to!GTP!and!cannot!!hydrolyze!it.!!In!contrast!β-tubulin!hydrolyzes!GTP!to!GDP!and!can!exchange!GTP!for!GDP.!!(How!do!these!proper3es!compare!to!those!of!ac3n?)!!• Microtubules!are!composed!of!13!linear!protofilaments!(varies!from!11-15,!with!13!being!!the!most!prevalent),!each!of!which!is!composed!of!alterna3ng!α!and!β!subunits.!The!!protofilaments!interact!laterally!to!form!a!cylinder.!!• Each!protofilament!has!an!intrinsic!polarity,!and!since!all!of!the!protofilaments!are!aligned!in!parallel!with!the!same!orienta3on,!the!MT!itself!is!polar.!• One!end!of!the!MT,!the!minus!end!(slow!growing),!is!ringed!with!α-tubulin. !The!oth er,!the!!plus!end!(fast!growing),!is!ringed!with!β-tubulin.!Inside!the!cell,!the!minus!ends!of!MTs!!are!usually!capped!and!embedded!in!the!MT-organizing-center-(MTOC-or-centrosome),!!so!in!discussing!MT!assembly!we!will!concentrate!on!the!dynamics!of!polymeriza3on!at!!the!plus!end.!D.Drubin!Structural-features-of-microtubules!Purifica7on-of-tubulin-from-pig-brains!hcp://www.youtube.com/watch?v=77D_X0wAZo8!• In#vitro,!polymeriza3on!of!pure!tubulin!can!be!ini3ated!by!raising!its!temperature!to!37°C,!!and!depolymeriza3on!can!be!induced!by!cooling!to!4°C.!(How!did!we!polymerize!ac3n?)!• If!the!concentra3on!of!tubulin!is!above!the!cri3cal!concentra3on!(Cc)!it!will!assemble,!!whereas!if!it!is!below!the!Cc!it!will!not.!(Does!this!sound!familiar?)!• Tubulin!polymeriza3on!can!be!monitored!by!!(1)!measuring!the!scacering!of!light!(again,!!polymer!scacers!more!light!than!monomer/dimer).!!(2)!acaching!a!fluorescent!tag!!(rhodamine!or!fluorescein)!to!tubulin!which!is!easier!to!visualize!when!it!is!incorporated!!into !microtubules.!!• During!a!3me!course!examining!bulk!MT!assembly,!there!is!a!lag!phase!during!which!!nuclea3on!takes!place.!Once!nuclei!are!formed,!elonga3on!occurs!rapidly.!!How-do-we-study-microtubule-dynamics-?!D.Drubin!MT-assembly-kine7cs-resemble-those-of-ac7n!Microtubule!polymeriza3on!in#vitro#proceeds!in!3!steps.!The!first!step!is!nuclea3on,!the!forma3on!of!a!stable!seed!(‘nucleus’)!of!oligomers,!which!can!elongate!to!form!a!microtubule.!This!‘lag’!phase!is!slow.!Amer!nuclea3on,!subunits!add!onto!the!nuclei,!leading!to!filament!elonga3on!or!growth.!This!elonga3on!phase!is!fast.!At!longer!3me!points,!the!free!tubulin!concentra3on!reaches!the!Cc!and!the!reac3on!reaches!steady!state.!In!this!phase!there!is!no!net!increase!or!decrease!in!the!amount!of!polymerized!microtubules!–!elonga3on!by!subunit!addi3on!is!balanced!by!shrinkage!due!to!subunit!loss.!!(How!could!you!es3mate!the!Cri3cal!concentra3on!from!this!graph?)!The-concentra7on-of-tubulin-in-equilibrium-with-MTs-is-the-Cri7cal-concentra7on-(Cc)!At!tubulin!concentra3ons!below!the!Cc,!polymeriza3on!does!not!take!place.!!When!polymeriza3on!is!induced!at!tubulin!concentra3ons!above!the!Cc,!microtubules!assemble!un3l!steady!state!is!reached!and!the!free!tubulin!concentra3on!falls!to!the!Cc.!Ini7al-concentra7on-of-Tubulin-dime r s-The-+-and-–-ends-of-microtubules-are-physically-and-kine7cally-different!!The!polymeriza3on!rate!is!different!at!the!two!ends!of!a!microtubule.-Because!microtubules!are!polar!polymers,!the!two!ends!have!different!proper3es.!The!plus!(+)!end!elongates!faster!than!the!minus!(–)!end.!!Which!box!points!to!a!microtubule!seed!?!!Which!box!points!to!newly!formed!microtubules?!!!Which!box!points!to!the!plus!end!of!the!seed?!Which!box!points!to!the!minus!end!of!the!seed?!!!Which!end!is!likely!to!have!the!lower!Cc?!Which!has!the!higher!Cc?!1!2!3!4!5!Microtubules-can-also-undergo-treadmilling!Figure 16-15 Molecular Biology of the CellThe-role-of-nucleo7des-and-MT-dynamics:!The-GTP-cap-stabilizes-the-plus-end-Figure 16-16c Molecular Biology of the CellLoss-of-the-GTP-cap-destabilizes-the-m icrotubule!Figure 16-16b Molecular Biology of the Cell (© Garland Science 2008)The-role-of-nucleo7des-in-MT-dynamics!• GTP!hydrolysis!accompanies!microtubule!assembly.!!During!elonga3on,!GTP-containing!!tubulin!subunits!add!onto!the!(+)!ends!of!MTs.!!GTP!is!hydrolyzed!to!GDP!amer!the!!tubulin!subunit!incorporates!into!the!MT.!!Thus,!a!growing!(+)!end!of!a!MT!will!consist!of!a!!cap!of!GTP!subunits!–!a!GTP-cap-–!while!the!bulk!of!the!MT!will!contain!GDP!subunits.!• At!concentra3ons!of!unpolymerized!tubulin!above!the!Cc!the!rate!of!subunit!addi3on!is!!faster!than!the!rate!of!GTP!hydrolysis!or!the!dissocia3on!of!GTP-tubulin!from!the!end.!!Thus!the!MT!will!con3nue!to!grow.!However,!at!concentra3ons!of!unpolymerized!!tubulin!near!the!Cc,!the!rate!of!addi3on!of!GTP-tubulin!to!the!end!slows.!In!this!case,!the!!GTP!cap!can!be!lost!by!dissocia3on!of!GTP!subunits!from!the!MT!end!or!by!GTP!!hydrolysis.!Loss!of!GTP-subunits!from!the!end!exposes!GDP-tubulin!at!MT!ends.!!•