Howard T H, Oresajo C O
Cell Motil. 1985;5(6):545-57. doi: 10.1002/cm.970050609.
The studies presented here characterize a simple, quantitative NBDphallacidin extraction assay for determining the F-actin content of fMLP-activated neutrophils. The NBDphallacidin extraction assay is based upon the specificity of NBDphallacidin binding to F-actin and the solubility of NBDphallacidin in methanol. Cells are fixed, permeabilized, and stained with NBDphallacidin; the cells are then pelleted, the bound NBDphallacidin is extracted into methanol, and the RFI (excite 465; emit 535) of the solution is determined. Binding of NBDphallacidin to neutrophils is saturable and 90% of bound NBDphallacidin is displaced by nonfluorescent phalloidin. The extraction of bound NBDphallacidin into methanol is complete and the excitation/emission characteristics of NBDphallacidin are not altered by extraction. The assay is relatively inexpensive, applicable to the study of cells in suspension or on substratum, allows kinetic studies with 5-10s time resolution, and is not affected by the shape of the cell or the distribution of the probe. We used the NBDphallacidin extraction assay to study the kinetics of fMLP-induced change in the F-actin content of neutrophils and the effect of tBOC peptide, an inhibitor of fMLP binding, on these changes. The extraction assay reveals a rapid, sequential fMLP-induced increase followed by a decrease in F-actin content. The tBOC peptide inhibits fMLP-induced actin polymerization. Addition of tBOC during fMLP-induced polymerization or at times when F-actin content is maximal enhances F-actin depolymerization. The rate of F-actin depolymerization is greater than or equal to fourfold faster in the presence than in the absence of tBOC. The results show that The NBDphallacidin extraction assay is useful for studying the kinetics of change in F-actin content of nonmuscle cells; fMLP receptor occupancy is required for fMLP-dependent polymerization but not depolymerization; and both the actin polymerizing and depolymerizing processes are active in the cell within 5 s after fMLP stimulation. Implications of these observations for understanding the observed increase and, then, decrease in F-actin content of fMLP-activated cells are discussed.
本文介绍的研究描述了一种简单的定量NBD鬼笔环肽提取测定法，用于测定fMLP激活的中性粒细胞的F-肌动蛋白含量。NBD鬼笔环肽提取测定法基于NBD鬼笔环肽与F-肌动蛋白结合的特异性以及NBD鬼笔环肽在甲醇中的溶解性。细胞经固定、通透处理后用NBD鬼笔环肽染色；然后将细胞离心成沉淀，将结合的NBD鬼笔环肽提取到甲醇中，并测定溶液的相对荧光强度（激发波长465；发射波长535）。NBD鬼笔环肽与中性粒细胞的结合是可饱和的，90%结合的NBD鬼笔环肽可被非荧光鬼笔环肽取代。结合的NBD鬼笔环肽向甲醇中的提取是完全的，且提取不会改变NBD鬼笔环肽的激发/发射特性。该测定法相对便宜，适用于悬浮细胞或贴壁细胞的研究，允许进行时间分辨率为5 - 10秒的动力学研究不，且不受细胞形状或探针分布的影响。我们使用NBD鬼笔环肽提取测定法研究fMLP诱导的中性粒细胞F-肌动蛋白含量变化的动力学以及fMLP结合抑制剂tBOC肽对这些变化的影响。提取测定法揭示了fMLP诱导的F-肌动蛋白含量先快速、相继增加然后减少。tBOC肽抑制fMLP诱导的肌动蛋白聚合。在fMLP诱导的聚合过程中或在F-肌动蛋白含量最大时添加tBOC会增强F-肌动蛋白解聚。在有tBOC存在时，F-肌动蛋白解聚速率比不存在时快四倍或更高。结果表明，NBD鬼笔环肽提取测定法可用于研究非肌肉细胞中F-肌动蛋白含量变化的动力学；fMLP依赖性聚合需要fMLP受体占据，但解聚不需要；并且在fMLP刺激后5秒内，肌动蛋白的聚合和解聚过程在细胞内均有活性。讨论了这些观察结果对于理解fMLP激活细胞中观察到的F-肌动蛋白含量先增加然后减少的意义。
