Watts R G, Howard T H
Department of Pediatrics, University of Alabama at Birmingham 35233.
Cell Motil Cytoskeleton. 1994;28(2):155-64. doi: 10.1002/cm.970280207.
F-actin is a major component of the neutrophil (PMN) cytoskeleton. In basal PMNs, F-actin exists in two structurally and functionally distinct pools: Triton insoluble F-actin (TIF)--cold insensitive, not depolymerizable by dilution, and distributed in pseudopods and submembranous locations; and Triton soluble F-actin (TSF)--unstable in cold, diffusely distributed, and gelsolin enriched. The element(s) conferring these unique properties to the Triton insoluble F-actin pool are unknown, but logically include distinct actin regulatory proteins. To study the morphologic and functional determinants of the Triton insoluble F-actin pool, the distribution and quantity of three candidate regulatory proteins, alpha-actinin, tropomyosin (TM), and actin binding protein (ABP-280), were compared in F-actin (Triton insoluble and Triton soluble) and G-actin pools isolated from basal and chemotactic factor activated human PMNs in suspension, using immunoblots and ionic extraction. F-actin content was measured by NBDphallacidin binding and gel scans. The results show that: (1) alpha-actinin, actin binding protein 280, and tropomyosin are localized to TIF and excluded from TSF; (2) TM, alpha-actinin, and ABP 280 are required to stabilize fractions of Triton insoluble F-actin in PMNs; and (3) chemotactic factor activation results in release of a fraction of TM from the Triton insoluble F-actin pool in temporal association with F-actin polymerization in the Triton insoluble F-actin pool. Shifts in ABP 280 or alpha-actinin do not occur. The results suggest that TM, alpha-actinin, and ABP 280 provide structure to TIF and that TM release from TIF is involved in chemotactic factor induced actin polymerization in PMNs.
F-肌动蛋白是中性粒细胞（PMN）细胞骨架的主要成分。在基础PMN中，F-肌动蛋白存在于两个结构和功能不同的池中：Triton不溶性F-肌动蛋白（TIF）——对冷不敏感，不能通过稀释解聚，分布在伪足和膜下位置；以及Triton可溶性F-肌动蛋白（TSF）——在冷时不稳定，分布弥散，富含凝溶胶蛋白。赋予Triton不溶性F-肌动蛋白池这些独特性质的因素尚不清楚，但从逻辑上讲包括不同的肌动蛋白调节蛋白。为了研究Triton不溶性F-肌动蛋白池的形态和功能决定因素，使用免疫印迹和离子提取法，比较了悬浮状态下基础和趋化因子激活的人PMN中F-肌动蛋白（Triton不溶性和Triton可溶性）和G-肌动蛋白池中三种候选调节蛋白α-辅肌动蛋白、原肌球蛋白（TM）和肌动蛋白结合蛋白（ABP-280）的分布和数量。通过NBD鬼笔环肽结合和凝胶扫描测量F-肌动蛋白含量。结果表明：（1）α-辅肌动蛋白、肌动蛋白结合蛋白280和原肌球蛋白定位于TIF，而不存在于TSF中；（2）TM、α-辅肌动蛋白和ABP 280是稳定PMN中Triton不溶性F-肌动蛋白部分所必需的；（3）趋化因子激活导致一部分TM从Triton不溶性F-肌动蛋白池中释放，与Triton不溶性F-肌动蛋白池中的F-肌动蛋白聚合在时间上相关。ABP 280或α-辅肌动蛋白没有发生变化。结果表明，TM、α-辅肌动蛋白和ABP 280为TIF提供结构，并且TM从TIF的释放参与趋化因子诱导的PMN中的肌动蛋白聚合。
