MCB 2210 3 25 2015 o Actin nucleating proteins nucleators accelerate the initial kinetics polymerization to trigger actin polymerization Human cells express different actin nucleators that function during distinct processes in different regions of the cell Formins generate long unbranched filaments straight o Dimerize 2 formins come together o Facilitate barbed end growth which remaining attached to the end o 15 kinds Tandem actin monomer binding proteins nucleate unbranched filaments o May cooperate with formins during filament assembly Arp2 3 complex the only actin nucleator know to generate branched filaments o Creates branches at about 70 degrees relative to an already existing actin filament o 7 tightly associated subunits 2 actin related proteins Arp2 and Arp3 together with 5 other proteins ARPC1 5 o Works in conjunction with proteins from the WASP family complex cannot work on there own require these WASP proteins Actin Arp2 3 or actin or actin WASP would fluoresce slightly Actin Arp2 3 WASP fluoresces much brighter WASP proteins have an activation signal Humans express 8 WASP like proteins The binding of WASP proteins to the Arp2 3 complex activated its nucleation activity Alone neither the WASP nor the Arp2 3 complex possess strong nucleation activity Causes the formation of a new actin filament that s capped at its pointed end by the Arp2 3 complex but is free to elongate at its barbed end o Nucleation is accompanied by Arp2 3 mediated filament branching with the Arp2 3 complex at he branch point Actin nucleators control cell shape movement and division during health and disease They are essential for embryonic development defense against infection wound healing and metastasis of cancer cells Required for morphological changes like contraction of muscle cells elongation of nerve axons formation of surface protrusions and cytokinesis Genetic mutations can cause disease Many of the functions of the actin cytoskeleton have been determined by examining the effects of drugs that affect assembly and disassembly o Phalloidin is isolated from Amanita phalloides the death cap mushroom It binds at the interface between subunits in the filament and prevents filament depolymerization Even if phalloidin stabilized actin filaments are diluted below the critical concentration they will not disassemble Fluorescent phalloidin used to visualize cellular actin filaments o Cytochalasin is a fungal alkyloid that binds to the end of actin filaments it caps them and prevents elongation The critical concentration then shifts to that of the end and filaments eventually depolymerize Cytochalasin is membrane permeable and blocks cell locomotion and cytokinesis The effect of cytochalasin is reversible if removed from the cells by dilution the cytoskeleton will reassemble o Latrunculin is isolated from the sea sponge Latruncula magnifica It binds to actin monomers and prevents them from polymerizing into filaments it sequesters them Latrunculin is membrane permeable and addition of latrunculin to cells causes rapid disassembly of actin filaments Like cytochalasin the effects of latrunculin are reversible Differentiating zebrafish muscle cells show actin enrichment at attachments to smite boundaries Microtubules tubular polymers composed of tubulin heterodimers that bind GTP GDP 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 The tubulin heterodimer consists of the two closely related proteins tubulin and tubulin Tubulin sequences are highly conserved in eukaryotes and each one is 55kDa dimers do not come apart under physiological conditions o Mammals express multiple and tubulin genes Appear to be hollow structures with outer ridges available to bind other proteins Structure o Both tubulin and tubulin bind GTP tubulin binds irreversibly to GTP and cannot hydrolyze it In contrast tubulin hydrolyzes GTP to GDP and can exchange GTP for GDP o Microtubules are composed of 13 linear protofilaments varies from 1115 with 13 being the most prevalent Each of which is composed of alternating and 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 orientation the MT itself is polar o One end of the MT the minus end slow growing is ringed with tubulin The other the plus end fast growing is ringed with tubulin o 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 polymerization at the plus end How do we study microtubule dynamics o In vitro polymerization of pure tubulin can be ini3ated by raising its temperature to 37 C and depolymerization can be induced by cooling to 4 C o If the concentration of tubulin is above the critical concentration Cc it will assemble whereas if it is below the Cc it will not o Tubulin polymerization can be monitored by 1 measuring the scattering of light again polymer scatters more light than monomer dimer 2 attaching a fluorescent tag rhodamine or fluorescein to tubulin which is easier to visualize when it is incorporated into microtubules o During a time course examining bulk MT assembly there is a lag phase during which nucleation takes place Once nuclei are formed elongation occurs rapidly Microtubule polymerization in vitro proceeds in 3 steps o The first step is nucleation the formation of a stable seed nucleus of oligomers which can elongate to form a microtubule This lag phase is slow o After nucleation subunits add onto the nuclei leading to filament elongation or growth This elongation phase is fast o At longer time points the free tubulin concentra3on reaches the Cc and the reaction reaches steady state In this phase there is no net increase or decrease in the amount of polymerized microtubules elongation by subunit addition is balanced by shrinkage due to subunit loss Critical concentration Cc the concentration of tubulin in equilibrium with microtubules o At tubulin concentrations below the Cc polymerization doesn t take place o When polymerization is induced at tubulin concentrations above Cc microtubules assemble until a steady state is reached and the free tubulin concentration falls to the Cc o The polymers will continue to be made as long as the concentration is above the Cc The