Real-time measurements of actin filament polymerization by total internal reflection fluorescence microscopy

被引:315
作者
Kuhn, JR [1 ]
Pollard, TD [1 ]
机构
[1] Yale Univ, Dept Mol Cellular & Dev Biol, New Haven, CT 06520 USA
关键词
D O I
10.1529/biophysj.104.047399
中图分类号
Q6 [生物物理学];
学科分类号
071011 ;
摘要
Understanding the mechanism of actin polymerization and its regulation by associated proteins requires an assay to monitor polymerization dynamics and. lament topology simultaneously. The only assay meeting these criteria is total internal reflection fluorescence microscopy (Amann and Pollard, 2001; Fujiwara et al., 2002). The. uorescence 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. laments 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-ATPactin is 7.4 muM(-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 muM(-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.
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收藏
页码:1387 / 1402
页数:16
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