Real-time measurements of actin filament polymerization by total internal reflection fluorescence microscopy.
Understanding the mechanism of actin polymerization and its regulation by associated proteins requires an assay to monitor polymerization dynamics and filament topology simultaneously. The only assay meeting these criteria is total internal reflection fluorescence microscopy (Amann and Pollard, 2001; Fujiwara et al., 2002). The fluorescence signal is fourfold stronger with actin labeled on Cys-374 with Oregon green rather than rhodamine. To distinguish growth at barbed and pointed ends we used image drift correction and maximum intensity projections to reveal points where single N-ethylmaleimide inactivated myosins attach filaments to the glass coverslip. We estimated association rates at high actin concentrations and dissociation rates near and below the critical actin concentration. At the barbed end, the association rate constant for Mg-ATP-actin is 7.4 microM(-1) s(-1) and the dissociation rate constant is 0.89 s(-1). At the pointed end the association and dissociation rate constants are 0.56 microM(-1) s(-1) and 0.19 s(-1). When vitamin D binding protein sequesters all free monomers, ADP-actin dissociates from barbed ends at 1.4 s(-1) and from pointed ends at 0.16 s(-1) regardless of buffer nucleotide.
Kuhn
Jeffrey R
JR
Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA.
Pollard
Thomas D
TD
D008841:Actin Cytoskeleton / Q000737:chemistry* / Q000648:ultrastructure*; D001665:Binding Sites; D003199:Computer Systems; D003460:Crystallization / Q000379:methods; D003461:Crystallography / Q000295:instrumentation / Q000379:methods; D019281:Dimerization; D004867:Equipment Design; D019544:Equipment Failure Analysis; D007089:Image Enhancement / Q000379:methods*; D007090:Image Interpretation, Computer-Assisted / Q000379:methods*; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000295:instrumentation* / Q000379:methods*; D046912:Multiprotein Complexes / Q000032:analysis / Q000737:chemistry / Q000648:ultrastructure; D011485:Protein Binding; D011487:Protein Conformation
D046912:Multiprotein Complexes