Polymerization kinetics of ADP- and ADP-P _i -actin determined by fluorescence microscopy

Abstract

We used fluorescence microscopy to determine how polymerization of Mg-ADP-actin depends on the concentration of phosphate. From the dependence of the elongation rate on the actin concentration and direct observations of depolymerizing filaments, we measured the polymerization rate constants of ADP-actin and ADP-Pi-actin. Saturating phosphate reduces the critical concentration for polymerization of Mg-ADP-actin from 1.8 to 0.06 M almost entirely by reducing the dissociation rate constants at both ends. Saturating phosphate increases the barbed end association rate constant of Mg-ADP-actin 15%, but this value is still threefold less than that of ATP-actin. Thus, ATP hydrolysis without phosphate dissociation must change the conformation of polymerized actin. Analysis of depolymerization experiments in the presence of phosphate suggests that phosphate dissociation near the terminal subunits is much faster than in the interior. Remarkably, 10 times more phosphate is required to slow the depolymerization of the pointed end than the barbed end, suggesting a weak affinity of phosphate near the pointed end. Our observations of single actin filaments provide clues about the origins of the difference in the critical concentration at the two ends of actin filaments in the presence of ATP.filaments ͉ rate constant ͉ treadmilling A full understanding of actin dynamics in cells will require a complete set of kinetic and equilibrium constants for the polymerization and depolymerization reactions for the three different nucleotide states of actin monomers and polymers, which can bind ATP, ADP-P i (ADP with inorganic phosphate [P i ] bound noncovalently in the ␥-phosphate position), or ADP. Each of these species can bind and dissociate at both the fast growing barbed end and the slow growing pointed end of a filament. Monomeric actin hydrolyzes ATP very slowly (1), but polymerization changes the conformation of the subunits so that they hydrolyze ATP irreversibly (2) at 0.3 s Ϫ1 (3), assuming hydrolysis is random. The ␥-phosphate dissociates slowly from the ADP-P i intermediate with a half time of Ϸ350 s (4). The reaction is reversible, but the affinity of polymerized ADP-actin for inorganic phosphate is low, with a K d in the millimolar range (5). Electron microscopy (6, 7) and limited proteolysis (8) indicate that P i dissociation is coupled to a conformation change in the actin filament.As a result of ATP hydrolysis, single actin filaments at steady state add fresh ATP-subunits at the barbed end balanced by dissociation of ADP-actin at the pointed end in a process known as treadmilling (9-12). Treadmilling in a medium containing ATP depends on hydrolysis of ATP bound to polymerized subunits and P i dissociation coupled to differences in the rate constants for subunit association and dissociation at the two ends (9, 13). The critical concentrations of ATP-actin and ADP-P i -actin are similar (14, 15), but some of the kinetic parameters required to model the steady state behavior of actin (16) were unknown, such as the associati...

Actin dynamics and the elasticity of cytoskeletal networks

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Vascular disease-causing mutation R258C in ACTA2 disrupts actin dynamics and interaction with myosin

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Identification of cation-binding sites on actin that drive polymerization and modulate bending stiffness

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