BIPHASIC FILAMENTOUS-ACTIN POLYMERIZATION IN ACTIVATED NEUTROPHILS

肌动蛋白是一种主要的细胞骨架蛋白，以单体形式存在(G- 肌动蛋白)形式以及聚合物或丝状形式(F-肌动蛋白)， 对细胞的激活和运动是必不可少的。肌动蛋白聚合 用化学诱导剂刺激的人中性粒细胞(PMN) 两种不同的技术。一种使用氟铬偶联的技术 Pallacidin可以对F-肌动蛋白进行特殊染色。被激活的PMN是 固定，渗透，荧光孔雀素和染色 流式细胞术分析F-肌动蛋白含量的变化。在 第二种技术，细胞骨架相关肌动蛋白的沉淀 使用Triton X-100洗涤剂的提取过程。提取的 用聚丙烯酰胺凝胶分析细胞骨架蛋白 用免疫印迹法鉴定肌动蛋白的表达。PMN是 接触趋化性N-甲酰肽和氧化N-甲酰基 与膜受体结合但不能诱导的多肽衍生物 趋化反应。肌动蛋白聚合的动力学为 使用这两种技术进行评估。肌动蛋白的一个快速的初始阶段 在接触这两种形式后的15秒内发生聚合 N-甲酰基多肽。N-甲酰基肽的趋化作用 激发了两相反应，其中第二聚合阶段 发生在刺激后大约2分钟，而 非趋化形式的多肽未能引起双相反应。 这些结果表明，趋化因子刺激的PMN具有周期性。 F-肌动蛋白对趋化物质刺激的反应。的初始阶段 F-肌动蛋白的反应与受体-配体结合有关，而 第二个F-肌动蛋白阶段似乎与伴随PMN的事件有关 能动性。 其中一些结果发表在今年的论文(癌症投资。8 (6)：651-654,1990年)。

Actin is a major cytoskeletal protein which exists in a monomeric (G- actin) form as well as a polymer or filamentous form (F-actin) and is essential for cell activation and motility. Actin polymerization in chemoattractant-stimulated human neutrophils (PMN) was evaluated with two different techniques. One technique used fluorochrome-conjugated phallacidin to specifically stain F-actin. The activated PMN were fixed, permeabilized, and stained with the fluorescent phallacidin and analyzed flow cytometrically for changes in F-actin content. In the second technique, cytoskeletal associated actin was precipitated using an extraction procedure with triton X-100 detergent. The extracted cytoskeletal proteins were analyzed by polyacrylamide gel electrophoresis and actin was identified by Western blotting. PMN were exposed to chemotactic N-formyl peptide and to an oxidized N-formyl peptide derivative that binds to membrane receptors but fails to elicit chemotactic responsiveness. The kinetics of actin polymerization were evaluated using both techniques. A rapid, initial phase of actin polymerization occurred within 15 seconds after exposure to both forms of the N-formyl peptide. The chemotactic form the N-formyl peptide stimulated a biphasic response in which a second polymerization phase occurred approximately 2 minutes after stimulation, whereas the nonchemotactic form of the peptide failed to elicit a biphasic response. These results indicate that chemoattractant-stimulated PMN have cyclic F-actin responses to chemoattractant stimulation. The initial phase of the F-actin response is related to receptor-ligand binding, whereas the second F-actin phase appears to be related to events accompanying PMN motility. Some of the results were published in paper this year (Cancer Invest. 8 (6): 651-654, 1990).