Real-time measurements of actin filament polymerization by total internal reflection fluorescence microscopy
Real-time measurements of actin filament polymerization by total internal reflection fluorescence microscopy
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DOI：
10.1529/biophysj.104.047399
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发表时间：
2005-02-01
影响因子：
3.4
通讯作者：
Pollard, TD
中科院分区：
文献类型：
--
作者：
Kuhn, JR;Pollard, TD
了解肌动蛋白聚合的机制及其相关蛋白的调节需要一种检测方法来监测聚合动力学。拓扑结构同时满足这些标准的唯一测定法是全内反射荧光显微术（Amann和Pollard，2001; Fujiwara等人，2002年）。的.用俄勒冈州绿色而不是罗丹明标记Cys-374上的肌动蛋白，荧光信号强四倍。为了区分倒刺和尖端的生长，我们使用图像漂移校正和最大强度投影来揭示单个N-乙基马来酰亚胺失活肌球蛋白附着的点。对着玻璃盖片哀叹。我们估计协会在高肌动蛋白浓度和解离率附近和低于临界肌动蛋白浓度。在倒刺末端，Mg-ATP肌动蛋白的结合速率常数为7.4 μ M（-1）s（-1），解离速率常数为0.89 s（-1）。在尖端，缔合和解离速率常数分别为0.56 μ M（-1）s（-1）和0.19 s（-1）。当维生素D结合蛋白螯合所有游离单体时，ADP-肌动蛋白在1.4 s（-1）和0.16 s（-1）时从倒刺末端解离，与缓冲液核苷酸无关。
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.