Introduction to MCB 252 Topic 17 Properties and Dynamics of ActinProf David Rivier MCB 252 Spring 2015 Topic 2 Properties and Dynamics of Actin Reading Lodish 17 0 17 2 Outline Actin Cytoskeleton A Properties of Actin B Role of Actin in Cell Movement C Regulation of Actin Assembly in Cells D Stable Actin Structures E Muscle Cells and Myosin Motors The cytoskeleton early studies Electron microscopy defined 3 types of filaments in cells 1950s 60s The subunits comprising these filaments were purified biochemically late 60s early 70s Antibodies against the components were used in fluorescence microscopy to examine filament structure in the whole cell Cytoskeletal Polymers microfilaments intermediate filaments microtubules Pollard and Earnshaw 35 2 Image Cora Ann Schonenberger Actin Cytoskeleton Properties of Actin Actin assembly Role of ATP Treadmilling Role of actin in cell movement Actin is required for movement Actin assembly can drive movement Regulation of actin assembly in cells Proteins involved in actin assembly disassembly Turning the protein involved in assembly and disassembly ON and OFF Building stable actin structures Actin Stable Structures and Dynamic Structures Actin Subunit of microfilaments One of the most abundant intracellular proteins in eukaryotes 10 of muscle protein Highly conserved throughout evolution 90 identity between widely divergent species e g flies and humans Different isoforms have different functions humans have six different actin genes alphas muscle beta leading edge of moving cells gamma stress fibers Actin continued 42 kD globular protein ATP binding protein Monomer G actin Spontaneous polymerization to filament F actin in presence of Mg2 K or Na Polymerization is reversible Reversible assembly is critical for many cell movements ATP Structure of G actin Actin Filament Structure By EM looks like twisted beads on a string 7 9 nm diameter Subunits arranged as a tightly wound helix short pitch every subunit yellow green orange blue pattern 2 long pitch helicesyellow orange and blue green Filaments have polarity what does this mean 72 nm Pollard and Earnshaw 4 5 Actin Filament Polarity Experimentally demonstrated by electron microscopy decoration experiments 1 Mix myosin head domain S1 globular head binds actin more later with actin filaments 2 Allow to bind process for EM Revealed different morphologies at each end one barbed end one pointed end Image courtesy Dr John Heuser Wash U Med School Actin Filament Polarity Experiment add myosin decorated filaments as seeds to nucleate Gactin polymerization Conclusion actin can grow at both ends it grows faster at the plus end it grows slower at the minus end pointed end seed barbed end See also Lodish Fig 17 9 Pollard and Earnshaw 36 9 Now let s look at assembly starting with G actin monomers without any F actin present at the beginning How to study the KINETICS of assembly How to study the rate of Actin polymerization ATP G actin can assemble into filaments in the presence of Mg but not in the absence of Mg Have a relatively high amount of ATP G actin in your test tube Add Mg to trigger F actin assembly and measure the rate of assembly by microscopy or change in fluorescence for instance G actin F actin G actin Net F actin Assembly F actin 1 No Net F actin Assembly 2 At Equilibrium 3 Ends still exchanging monomers 4 Still some free G actin Dynamics of Actin Assembly Critical Concentration 100 actin subunits in filaments time after initiating polymerization Cc concentration monomers at steady state Critical Concentration F actin Assembly polymerization occurs when G actin Cc F actin Disassembly depolymerization occurs when G actin Cc Cc At Time 0 at Cc Cc Cc Cc Cc At Time 0 At Equilibrium at Cc Cc Cc Cc Critical Concentration F actin Assembly polymerization occurs when G actin Cc F actin Disassembly depolymerization occurs when G actin Cc Critical Concentration The rate of association may be fast at one end and slow at the other Critical Concentration Above Cc both ends grow G actin associates Below Cc both ends shrink G actin dissociates At Cc the rates of association and dissociation balance For a HOMOGENEOUS polymer The equilibrium value K on rate off rate K same at both ends but Plus end fast on rate and fast off rate Minus end slow on rate and slow off rate For a HOMOGENEOUS polymer These two polymers products are chemically identical Addition to plus or minus end is the same overall reaction There the energy released is identical in both cases Therefore the equilibrium value is the same for both Recall that Actin is an ATPase What is the role of ATP hydrolysis What are the possible roles of ATP hydrolysis Is ATP hydrolysis required for actin filament assembly What experiments would let you address this question Is ATP hydrolysis required for actin filament assembly What experiments would let you address this question 1 Use ADP G actin in polymerization reaction Filaments assemble 2 Use non hydrolyzable ATP analogues Filaments assemble If ATP hydrolysis is not required for F actin assembly what is the role of ATP hydrolysis Actin is NOT a homogeneous polymer It s ends are different Plus end has ATP actin Minus end has ADP actin Therefore the critical concentrations for the two ends are different ATP hydrolysis results in different types of ends and therefore different Cc for each end The ends of actin polymers are chemically different They therefore have different critical concentrations The ends are different because Rate of ATP hydrolysis rate of assembly at the minus end Rate of ATP hydrolysis rate of assembly at the plus end The and ends of actin have different Ccs rate of polymerization Plus end B C 0 A D E Actin concentration Minus end Consequence of different Ccs rate of polymerization Plus end Minus end 0 Actin concentration So at concentrations between Cc and Cc actin filaments can treadmill Treadmilling occurs when the concentration of G actin is between the Cc of the plus end and the Cc of the minus end Treadmilling Treadmilling is a consequence of equilibria If ATP hydrolysis is not required for F actin assembly what is the role of ATP hydrolysis To change the critical concentration value for the plus end vs the minus end The ATP form adds onto the ends After addition ATP is hydrolyzed to ADP which stays bound to the actin subunits within the polymer The ADP forms dissociate from the polymer more readily than the ATP form The ATP form associates more readily with the plus end and the