BIPHASIC FILAMENTOUS-ACTIN POLYMERIZATION IN ACTIVATED NEUTROPHILS

活化中性粒细胞中的双相丝状肌动蛋白聚合

• 批准号: 3804870
• 负责人: L HARVATH
• 金额: --
• 依托单位: CENTER BIOLOGICS EVALUATION RESEARCH HEMATOLOGY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3804870
• 关键词: SDS polyacrylamide gel electrophoresis; actins; cell motility; chemical kinetics; chemoattractants; flow cytometry; human tissue; leukocyte activation /transformation; neutrophil; polymerization; receptor binding; western blottings

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).

肌动蛋白是一种主要的细胞骨架蛋白，它以单体形式存在于细胞中。 肌动蛋白）形式以及聚合物或丝状形式（F-肌动蛋白）， 对细胞活化和运动至关重要。 肌动蛋白聚合 化学引诱物刺激的人中性粒细胞（PMN）进行了评价， 两种不同的技术。 一种技术使用荧光染料共轭 phallacidin特异性染色F-肌动蛋白。 激活的PMN 固定，透化，并用荧光鬼笔菌素染色， 流式细胞术分析F-肌动蛋白含量的变化。 在 第二种技术，用沉淀法沉淀细胞骨架相关肌动蛋白， 用Triton X-100洗涤剂萃取程序。 所提取的 聚丙烯酰胺凝胶电泳分析细胞骨架蛋白 蛋白质印迹法鉴定肌动蛋白。 PMN是 暴露于趋化性N-甲酰基肽和氧化的N-甲酰基 一种肽衍生物，与膜受体结合，但不能引起 趋化反应 肌动蛋白聚合的动力学是 使用这两种技术进行评估。 肌动蛋白的快速的初始阶段 在暴露于两种形式后15秒内发生聚合 N-甲酰基肽 N-甲酰肽的趋化形式 刺激双相反应，其中第二聚合相 发生在刺激后约2分钟，而 非趋化形式的肽不能引起双相反应。 这些结果表明，趋化因子刺激的PMN具有周期性的 肌动蛋白对化学引诱物刺激的反应。 的初始阶段 F-肌动蛋白反应与受体-配体结合有关，而 第二个F-肌动蛋白相似乎与伴随PMN的事件有关 能动性 一些结果发表在今年的论文（癌症投资。8 (6)：651 - 654，1990）。