Ac#n:&Structure&and&Bioche m istry&&Ac#n:&Structure&and&Bioche m istry&&Self&Study:&An&overview&of&the&major&cytoskeletal&systems!D.Drubin!Today:&&How&are&ac#n&filaments&assembled&?!Ac#n!is!one!of!the!most!abundant!proteins!in!eukaryo5c!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!ac5n!has!been!highly!conserved!through!evolu5on.&Ac5ns!contain!~375!amino!acids!and!have!a!molecular!weight!of!~42!kDa.!Ac5ns!from!amoeba!and!humans!are!>80%!iden5cal!to!each!other!in!sequence.!Simple!eukaryotes!like!yeast!have!one!ac5n!gene.!Mammals!have!several!genes!that!produce!mul5ple!types!of!ac5n:!α-ac5n!in!muscle,!β-!and!γ-ac5n!in!non-muscle!cells.!!(Do#bacteria#have#a#cytoskeleton?)#Figure 16-12 Molecular Biology of the Cell (© Garland Science 2008) Ac#n&filaments&are&helical&polymers&composed&of&globular&ac#n&monomers&that&bind&ATP!• Ac5n!monomer!molecule!is!separated!into!two!lobes!by!a!deep!cleU!that!binds!ATP!or!ADP!!complexed!with!Mg+2.!!Ac5n!exists!in!two!forms!in!the!cell:!!as!a!monomer!called!G-ac#n&!(globular-ac5n)!and!as!a!filamentous!polymer!called!F-ac#n&(filamentous-ac5n).!!• F-ac5n!is!a!helical!polymer!of!G-ac5n!subunits!held!together!by!non-covalent!(hydrophobic!!and!ionic)!interac5ons.!!F-ac5n!is!a!polar!polymer.!All!of!the!subunits!within!an!ac5n!!filament!have!the!same!polarity:!they!are!all!oriented!in!the!same!direc5on.!Thus!the!!two!ends!of!a!filament!are!different.!At!one!end!(-)!the!ATP!cleU!is!exposed,!and!at!the!!other!end!(+)!it!contacts!a!neighboring!subunit.!D.Drubin!Structural&features&of&ac#n!!• The&polarity&of&ac#n&filaments&can&be&determined&by&decora#ng&the&filaments&with&the&&ac#n-binding&“head”&domain&of&the&myosin&II&molecule&(calle d&subf ragment-1&or&S1).&!When!S1!is!mixed!with!ac5n!filaments,!it!a_aches!to!the!sides!at!an!angle,!crea5ng!an!!arrowhead-like!pa_ern.!One!end!of!the!filament!is!called!the!pointed!end!(minus!(–)!end)!!and!the!other!is!called!the!barbed!end!(plus!(+)!end).!!• One!important!consequence!of!filament!polarity!is!that!the!two!ends!of!the!filament!have!!different!dynamic!proper5es.!D.Drubin!Structural&features&of&ac#n!• In#vitro,!ac5n!polymeriza5on!(assembly)!can!be!ini5ated!by!adding!salts!(Mg+2,!K+)!to!a!solu5on!of!pure!G-ac5n.!!The!process!is!reversible!–!if!salt!is!removed,!ac5n!will!depolymerize.!(However,!because!the!ionic!composi5on!of!the!cytoplasm!is!constant,!ac5n!polymeriza5on!cannot!be!regulated!by!this!mechanism!in!cells.!The!assembly!and!disassembly!proper5es!of!pure!ac5n!are!different!from!those!for!ac5n!in!the!cell,!as!we!will!see!later.)!!• Ac5n!polymeriza5on!can!be!monitored!by:!!!• (1)!measuring!the!sca_ering!of!light!(polymer!sca_ers!more!light!than!monomer).!!• (2)!a_aching!a!fluorescent!tag!(pyrene)!to!ac5n!which!fluoresces!more!brightly!when!it!is!incorporated!into!F-ac5n!than!when!it!is!!present!as!G-ac5n;!pyrene-ac5n!assembly!can!be!measured!using!a!spectrofluorometer.!! !!• (3)!visualizing!filaments!by!fluorescence!or!electron!microscopy.!!• (4)!conduc5ng!sedimenta5on!analysis;!because!F-ac5n!is!larger!than!G-ac5n,!F-ac5n!sediments!more!readily!by!centrifuga5on.!How&do&we&study&ac#n&assembly?!D.Drubin!Ac#n&assembly&kine#cs!Ac5n!polymeriza5on!proceeds!in!3!steps!in#vitro.&The!first!step!is!nuclea#on,!the!forma5on!of!a!stable!seed!(‘nucleus’)!consis5ng!of!three!ac5n!monomers,!which!can!elongate!to!form!a!filament.!This!‘lag’!phase!is!slow!because!ac5n!dimers!are!very!unstable.!The!lag!can!be!eliminated!by!adding!nuclea5ng!factors!(or!ac5n!filaments!themselves;!‘seeds’).!AUer!nuclea5on,!subunits!add!onto!the!nuclei,!leading!to!filament!elonga#on!or!growth.!This!elonga5on!phase!is!fast.!At!longer!5me!points,!the!reac5on!reaches!steady&state.!In!this!phase!there!is!no!net!increase!or!decrease!in!the!amount!of!polymerized!ac5n:!elonga5on!by!monomer!addi5on!is!balanced!by!shrinkage!due!to!subunit!loss.!D.Drubin!Figure 16-10 Molecular Biology of the Cell (© Garland Science 2008) The&concentra#on&of&G-ac#n&in&equilibrium&with&F-ac#n&is&the&Cri#cal&concentra#on&(Cc)!As!ac5n!filaments!elongate,!the!concentra5on!of!free!subunits!(G-ac5n)!decreases!un5l!the!system!reaches!steady!state.!The!concentra5on!of!free!ac5n!at!steady!state!is!called!the!cri#cal&concentra#on&(Cc).!At!this!concentra5on,!the!rate!of!subunit!addi5on!(kon[C])!equals!the!rate!of!subunit!loss!(koff),!ie.!kon[C]=!koff.!Thus!Cc!=!koff!/!kon.!The!Cc!for!ac5n!polymeriza5on!under!typical!condi5ons!in#vitro#is!about!0.2μM.!If!the!free!subunit!concentra5on!is!above!Cc,!subunits!will!add!onto!the!ends!of!filaments.!If!the!free!subunit!concentra5on!is!below!Cc,!subunits!will!be!lost!from!the!ends!of!filaments.!D.Drubin!The&concentra#on&of&G-ac#n&in&equilibrium&with&F-ac#n&is&the&Cri#cal&concentra#on&(Cc)!At!monomer!concentra5ons!below!the!Cc,!polymeriza5on!does!not!take!place.!!When!polymeriza5on!is!induced!at!monomer!concentra5ons!above!the!Cc,!filaments!assemble!un5l!steady!state!is!reached!and!the!monomer!concentra5on!falls!to!the!Cc.!Ini#al&concentra#on&of&Ac#n&Monomers&At!monomer!concentra5ons!below!the!Cc,!polymeriza5on!does!not!take!place.!!When!polymeriza5on!is!induced!at!monomer!concentra5ons!above!the!Cc,!filaments!assemble!un5l!the!monomer!concentra5on!falls!to!the!Cc!and!steady!state!is!reached.!Ini#al&concentra#on&of&Ac#n&Monomers&The&concentra#on&of&G-ac#n&in&equilibrium&with&F-ac#n&is&the&Cri#cal&concentra#on&(Cc)!The&+&and&–&ends&of&ac#n&filaments&are&physically&and&kine#cally&different!D.Drubin!This! can! be! demonstrated! experimentally! by!mixing!G-ac5n!with!F-ac5n!seeds!that!have!been!decorated! with! myosin! S1! fragment! to! mark! the!filament!polarity.!The!newly!assembled!filaments!are!much!longer!at!the!+!end.)!!• The!polymeriza5on!rate!is!different!at!the!two!ends!of!an!ac5n!filament.!Because!ac5n!is!!a!polar!polymer!the!two!ends!have!different!proper5es.!The!barbed!end!(plus!(+)!end)!!elongates!up!to!ten!5mes!faster!than!the!pointed!end!(minus!(–)!end).!!!A&Cri#cal&concept:&&Cri#cal&concentra#on!D.Drubin!!•