• Exam%3%will%be%held%Friday,%Nov%13th%in%the%Tes:ng%Center%Arjona%110%• You%must%sign%up%for%an%available%Ex am%Slot%on%HuskyCT%Exam%Registra:on%Link!%!• Exam%slots:%12:00-1:30,%1:30-3:00,%3:00-4:30,%4:30-6:00;%there%is%no%regular%class%on%exam%day.%• If%you%have%another%class%conflict%w ith%the%available%slots%you%must%provide%a%scr eenshot%of%your%UConn%schedule,%and%contact%me%ASAP!%• HuskyCT%Materials%will%be%unavailable%online%on%Friday.%Be%sure%to%download%any%materials%you%want%if%you%plan%on%last%minute%studying!%%• There%will%be%2%Review%sessions%before%the%Exam.!• 1.!Wednesday,!11/11,!6-8pm!TLS!154!• 2.!Thursday,!11/12,!6-8pm!TLS!154!• Bring!your!Problem!Sets!!• The%early%exam%for%people%with%legi:mate%reasons%that%have%been%cleared%with%me%will%be%on%Thursday,%11/12,%:me/place%TBA.%• If!you!need!to!take!the!early!exam!for!a!legiGmate!reason!and!have!NOT!contacted!me!yet,[email protected]!• Students%who%have%CSD%Exam%:me/place%accommoda:ons%will%be%taking%the%exam%at%the%CSD%office.!Please!get!any!Exam!AdministraGon!Forms!to!me!ASAP.!Cytoskeletal%Func:ons%During%Interphase%%%%Remember%the%direc:ons%that%MT%motors%move!Ques:ons%to%ponder%Where!are!the!microtubule!(+)!ends!and!(-)!ends!?!Are!Kinesins!or!Dyneins!important!for!melanosome!localizaGon!in!high!cAMP!?!!Are!Kinesins!or!Dyneins!important!for!melanosome!localizaGon!in!low!cAMP!?!!What!will!melanosome!organizaGon!look!like!if!you!treat!cells!with!taxol!?!What!will!melanosome!organizaGon!look!like!if!you!treat!cells!with!nocodazole!?!What!happens!when!you!wash!away!the!nozodazole!or!taxol!?!Today:%%Ac:n,%Microtubule,%and%Motor%Func:ons!Microtubules%in%a%radial%array%(Cytokinesis)%Ac:n%1. Membrane!Protrusion!(Lamellipodia!&!Filopodia)!2. Cell!Adhesion!!!!!!!!!!!!!!!!!!!!!!!!!(Stress!Fibers!&!Focal!Adhesions)!3. Endocytosis!and!Trafficking!!!!!(Clathrin!Pits!&!Endosomes)!4. Cytokinesis!à!Later!!Microtubules%1. Organelle!PosiGoning! !!!!!!!!!!!(ER!&!Golgi)!2. Anterograde!Transport!!!!!!!!!!!!!!(ER-Golgi-Plasma!Membrane)!3. Retrograde!Transport!!!!!!!(Endosome-Golgi)!4. Mitosis!à!Later!Lamellipodia%and%Filopodia%are%membrane%protrusions!Dynamic!cell!surface!extensions!containing!acGn!are!a!common!feature!of!animal!cells.!Many!cells!in!vertebrate!Gssues!are!capable!of!migraGon.!One!model!cell!type!used!in!many!studies!of!locomoGon!is!the!fibroblast,!a!cell!of!the!connecGve!Gssue.!The!leading!edge!of!a!fibroblast!regularly!extends!thin!sheet-like!structures!called!lamellipodia!and!thin!needle-like!projecGons!or!spikes!called!filopodia.!Aber!protruding!forward,!lamellipodia!and!filopodia!adhere!to!the!substrate!or!sweep!over!the!top!of!a!cell!as!a!ruffle.!As!we!will!see,!fibroblasts!also!contain!long!cables!of!acGn!called!stress%fibers%that!funcGon!in!cell!adhesion!and!contracGon.!D.Drubin!Figure 16-47 Molecular Biology of the Cell lamellipodium%The!behavior!of!acGn!filaments!in!lamellipodia!and!filopodia!can!be!traced!by!microinjecGng!the!cells!with!fluorescently!labeled!acGn.!Immediately!aber!microinjecGon,!fluorescent!acGn!is!incorporated!into!filaments!at!the!extreme!leading!edge!(mostly!in!the!region!highlighted!in!yellow),!indicaGng!that!acGn!polymerizaGon!is!taking!place!at!this!locaGon.!!Ac:n%ac:vely%polymerizes%near%the%leading%edge%Ac:n%assembly%drives%membrane%protrusion!D.Drubin!T.Pollard!Because!acGn!polymerizaGon!at!the!leading!edge!is!coupled!to!membrane!protrusion,!it!has!been!hypothesized!that!the!assembly!of!acGn!filaments!in!the!network!abueng!the!plasma!membrane!generates!the!force!that!pushes!the!membrane!forward.!The!energy!that!drives!this!process!comes!from!the!hydrolysis!of!ATP!by!acGn.!!Aber!acGn!filaments!are!nucleated!at!the!leading!edge!of!a!fibroblast,!the!enGre!network!of!filaments!is!thought!to!flux!backwards!towards!the!interior!of!the!cell!in!a!process!called!retrograde!flow.!During!this!process,!depolymerizaGon!of!filaments!begins.!Retrograde!flow!of!filaments!is!thus!coupled!to!depolymerizaGon.!Cofilin!is!thought!to!be!the!major!acGn!depolymerizing!acGvity!behind!the!leading!edge.!!Ac:n%assembly%drives%membrane%protrusion!hMp://www.youtube.com/watch?v=8a0SOM9EFJI!membrane%RFP-ac:n%Fluorescence%speckle%microscopy%highlights%the%retrograde%flow%phenomenon%!journal.pone.0003234.s003.mov!phase%fluorescence%F-ac:n%organiza:on%in%protrusive%structures%AcGn!filaments!are!oriented!with!their!barbed!(+)!ends!facing!the!membrane!in!the!direcGon!of!protrusion.!In!lamellipodia,!filaments!are!organized!as!a!branched!network.!In!filopodia,%they!are!organized!into!parallel!bundles.!filopodia%lamellipodium%T.Svitkina!Ac:n%also%assembles%into%stress%fibers!Stress%fibers%are!long!bundles!of!acGn!filaments!that!lie!along!the!lower!surface!of!the!cell.!Their!ends!terminate!at!integrin-containing!focal%adhesions,!special!structures!that!aMach!the!cell!to!the!underlying!substrates.!We!will!learn!more!about!this!when!discussing!the!extracellular!matrix!and!cell!moGlity.!Stress%fibers%contrac t%in%r esponse%to%phosphoryla:on%of%non-muscle%myosin%II%light%chain%by%MLCK!A%refresher%on%Rho-family%GTPase%signaling!Rho-family%GTPases%(such%as%Rho,%Rac,%&%Cdc42)%are!members!of!a!large!family!of!GTP-binding!and!hydrolyzing!proteins!called!the!Ras!superfamily.!These!proteins!are!small!(about!20kDa)!molecular!switches!that!cycle!between!an!acGve!form!that!is!bound!to!GTP!and!an!inacGve!form!bound!to!GDP.!The!acGve!GTP-bound!form!can!interact!with!effector!molecules!that!carry!out!downstream!cellular!funcGons.!Guanine%nucleo:de%exchange%factors%(GEFs)!mediate!the!conversion!of!the!inacGve!GDP-bound!form!to!the!acGve!GTP-bound!form!by!catalyzing!the!exchange!of!bound!GDP!for!free!GTP.!Conversely,!GTPase%ac:va:ng%proteins%(GAPs)%catalyze!the%conversion!of!the!acGve!GTP-bound!form!to!the!inacGve!GDP-bound!form.!Guanine%nucleo:de%dissocia:on%inhibitors%(GDIs)!stabilize!the!inacGve!GDP-bound