MCB 2210 001 3 27 2015 Mictotubules Continued The role of nucleotides in MT dynamics o GTP hydrolysis accompanies microtubule assembly o During elongation GTP containing tubulin subunits add onto the ends of MTs o GTP is hydrolyzed to GDP after the tubulin subunit incorporates into the MT o 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 o At concentrations of unpolymerized tubulin above the Cc the rate of subunit addition is faster than the rate of GTP hydrolysis or the dissociation of GTP tubulin from the end o Thus the MT will continue to grow However at concentrations of unpolymerized tubulin near the Cc the rate of addition of GTP tubulin to the end slows In this case the GTP cap can be lost by dissociation of GTP subunits from the MT end or by GTP hydrolysis o Loss of GTP subunits from the end exposes GDP tubulin at MT ends o MTs tend to elongate if they possess a GTP cap o But loss of the GTP cap destabilizes the polymer This is because GTP tubulin more readily makes lateral interactions with other protofilaments that maintain the cylindrical structure of the end If MTs possess a GDP cap these lateral interactions are weakened and the protofilaments tend to splay apart o Protofilament splaying destabilizes the MT and leads to depolymerization or shrinkage o Because the off rate of GDP tubulin is high disassembly of the MT can be rapid and can omen result in the complete disassembly of that MT For this reason it is called catastrophic disassembly or catastrophe o MTs can also regrow This is called rescue Individual microtubules have dynamic instability o Dynamic instability is a property of individual MTs in which they rapidly change length o At concentrations near the Cc some MTs in a population will grow and others will shrink o Although individual MTs can persist in the growing or shrinking state a MT can also change from growth to shrinkage catastrophe or from shrinkage to growth rescue This property of MTs is called dynamic instability o The alternation between growing and shrinking states occurs randomly and is due to conversions between a GTP and GDP cap at the plus end Thus at steady state the total amount of polymer remains constant but individual MTs can either elongate or shorten o Can be visualized in cells Proteins that interact with the ends of microtubules are able to influence microtubule stability and the organization of microtubule arrays involved in diverse functions like chromosome segregation and neuronal axon extension Cells have way to control microtubule dynamics o The total tubulin concentration in cells is 10 20 M a value much higher than the Cc of 0 03 M so polymerization is highly favored o But microtubules do not form randomly in cells in part because of the kinetic barrier to nucleation o Under physiological conditions microtubule assembly also involves microtubule associated proteins MAPs Cells regulate where and when microtubules polymerize o Special nucleation sites allow immediate polymerization of microtubules they eliminate the lag phase Microtubule Organizing Center MTOC nucleates and organizes cellular microtubules o In cells MTs radiate from a structure called the microtubule organizing center MTOC o In non mitotic interphase cells the MTOC is also called the centrosome o The minus end of the MT is embedded in the MTOC and the plus end faces out into the cytosol o If tubulin is depolymerized by cooling cells to 4 C or by adding a MT destabilizing drug and then allowed to re polymerize by warming or washing out the drug new MTs grow out from the centrosome and the normal microtubule distribute on returns Taxol is extracted from the bark of the yew tree o It binds to MTs and stabilizes the polymer preventing disassembly o Taxol blocks MT dependent processes such as mitotic spindle assembly and is a very effective chemotherapeutic agent in treating ovarian and breast cancers Colchicine is isolated from a flower o It binds to the tubulin dimer such that when the dimer polymerizes into a MT the colchicine prevents further polymerization onto the MT end o This promotes the formation of a GDP cap and thus MT destabilization o Its effects are reversible o Colchicine has been used to relieve pain from gout Nocodazole is a synthetic compound that binds to tubulin subunits and triggers MT depolymerization o Its effects are reversible o Nocodazole is also an anti neoplastic agent The centrosome consists of a pair of centrioles surrounded by pericentriolar material PCM o MT minus ends are embedded in the PCM and the plus ends face outward into the cytoplasm tubulin is a microtubule nucleator o Microtubules are nucleated at the centrosome by a third member of the tubulin superfamily tubulin gamma tubulin o tubulin is conserved in all eukaryotes and is functionally distinct from tubulin and tubulin o tubulin does not polymerize into MTs but instead exists in a protein complex called the tubulin ring complex TURC This complex consists of other proteins besides tubulin and forms a ring like structure that can be seen by electron microscopy to be embedded in the pericentriolar material of the centrosome o Purified TURC nucleates the assembly of pure tubulin although the mechanism of nuclea3on is not understood Microtubules function in cell polarity membrane trafficking and mitosis o They are crucial for organelle organization rapid vesicle transport and chromosome segregation o They have specialized functions in flagella and nerve axons o Drugs that affect microtubule assembly are used to treat several diseases including cancer Actin Binding Proteins and Microtubule Associated Proteins Actin Binding Proteins ABPs control the assembly disassembly organization and movement of actin filaments o Control of polymerization and depolymerization monomer sequestering proteins nucleating factors capping proteins stabilizing proteins d epolymerizing proteins severing proteins o Organization of filaments network forming crosslinking proteins bundle forming crosslinking proteins membrane linker proteins o Movement of filaments motor proteins ABPs cap stabilize sever and depolymerize filaments o Capping proteins stabilize filaments by binding to their ends Filaments are stabilized because the capping proteins prevent addition or loss of subunits from the filament ends Examples include CapZ which caps the end of filaments and tropomodulin which caps the ends of actin filaments o