Norgauer J, Just I, Aktories K, Sklar L A
Department of Immunology, Scripps Clinic and Research Foundation, La Jolla, California 92037.
J Cell Biol. 1989 Sep;109(3):1133-40. doi: 10.1083/jcb.109.3.1133.
Stimulation of human neutrophils with the chemotactic N-formyl peptide causes production of oxygen radicals and conversion of monomeric actin (G-actin) to polymeric actin (F-actin). The effects of the binary botulinum C2 toxin on the amount of F-actin and on neutrophil cell responses were studied. Two different methods for analyzing the actin response were used in formyl peptide-stimulated cells: staining of F-actin with rhodamine-phalloidin and a transient right angle light scatter. Preincubation of neutrophils with 400 ng/ml component I and 1,600 ng/ml component II of botulinum C2 toxin for 30 min almost completely inhibited the formyl peptide-stimulated polymerization of G-actin and at the same time decreased the amount of F-actin in unstimulated neutrophils by an average of approximately 30%. Botulinum C2 toxin preincubation for 60 min destroyed approximately 75% of the F-actin in unstimulated neutrophils. Right angle light scatter analysis showed that control neutrophils exhibited the transient response characteristic of actin polymerization; however, after botulinum C2 toxin treatment, degranulation was detected. Single components of the binary botulinum C2 toxin were without effect on the actin polymerization response. Fluorescence flow cytometry and fluorospectrometric binding studies showed little alteration in N-formyl peptide binding or dissociation dynamics in the toxin-treated cells. However, endocytosis of the fluorescent N-formyl peptide ligand-receptor complex was slower but still possible in degranulating neutrophils treated with botulinum C2 toxin for 60 min. The half-time of endocytosis, estimated from initial rates, was 4 and 8 min in control and botulinum C2 toxin-treated neutrophils, respectively.
用趋化性N-甲酰肽刺激人中性粒细胞会导致氧自由基的产生以及单体肌动蛋白（G-肌动蛋白）向多聚肌动蛋白（F-肌动蛋白）的转化。研究了二元肉毒杆菌C2毒素对F-肌动蛋白量和中性粒细胞反应的影响。在甲酰肽刺激的细胞中使用了两种不同的方法来分析肌动蛋白反应：用罗丹明-鬼笔环肽对F-肌动蛋白进行染色以及瞬态直角光散射。用400 ng/ml肉毒杆菌C2毒素的组分I和1600 ng/ml组分II对中性粒细胞进行预孵育30分钟，几乎完全抑制了甲酰肽刺激的G-肌动蛋白聚合，同时未刺激的中性粒细胞中的F-肌动蛋白量平均减少了约30%。肉毒杆菌C2毒素预孵育60分钟可破坏未刺激的中性粒细胞中约75%的F-肌动蛋白。直角光散射分析表明，对照中性粒细胞表现出肌动蛋白聚合的瞬态反应特征；然而，肉毒杆菌C2毒素处理后，检测到了脱颗粒现象。二元肉毒杆菌C2毒素的单个组分对肌动蛋白聚合反应没有影响。荧光流式细胞术和荧光光谱结合研究表明，毒素处理的细胞中N-甲酰肽的结合或解离动力学几乎没有改变。然而，在用肉毒杆菌C2毒素处理60分钟的脱颗粒中性粒细胞中，荧光N-甲酰肽配体-受体复合物的内吞作用较慢，但仍然可能。根据初始速率估计，对照和肉毒杆菌C2毒素处理的中性粒细胞中内吞作用的半衰期分别为4分钟和8分钟。
