Watts R G, Howard T H
Department of Pediatrics, University of Alabama, Birmingham 35233.
Cell Motil Cytoskeleton. 1992;21(1):25-37. doi: 10.1002/cm.970210104.
Filamentous (F) actin is a major cytoskeletal element in polymorphonuclear leukocytes (PMNs) and other non-muscle cells. Exposure of PMNs to agonists causes polymerization of monomeric (G) actin to F-actin and activates motile responses. In vitro, all purified F-actin is identical. However, in vivo, the presence of multiple, diverse actin regulatory and binding proteins suggests that all F-actin within cells may not be identical. Typically, F-actin in cells is measured by either NBDphallacidin binding or as cytoskeletal associated actin in Triton-extracted cells. To determine whether the two measures of F-actin in PMNs, NBDphallacidin binding and cytoskeletal associated actin, are equivalent, a qualitative and quantitative comparison of the F-actin in basal, non-adherent endotoxin-free PMNs measured by both techniques was performed. F-actin as NBDphallacidin binding and cytoskeletal associated actin was measured in cells fixed with formaldehyde prior to cell lysis and fluorescent staining (PreFix), or in cells lysed with Triton prior to fixation (PostFix). By both techniques, F-actin in PreFix cells is higher than in PostFix cells (54.25 +/- 3.77 vs. 23.5 +/- 3.7 measured as mean fluorescent channel by NBDphallacidin binding and 70.3 +/- 3.5% vs. 47.2 +/- 3.6% of total cellular actin measured as cytoskeletal associated actin). These results show that in PMNs, Triton exposure releases a labile F-actin pool from basal cells while a stable F-actin pool is resistant to Triton exposure. Further characterizations of the distinct labile and stable F-actin pools utilizing NBDphallacidin binding, ultracentrifugation, and electron microscopy demonstrate the actin released with the labile pool is lost as filament. The subcellular localization of F-actin in the two pools is documented by fluorescent microscopy, while the distribution of the actin regulatory protein gelsolin is characterized by immunoblots with anti-gelsolin. Our studies show that at least two distinct F-actin pools coexist in endotoxin-free, basal PMNs in suspension: 1) a stable F-actin pool which is a minority of total cellular F-actin, Triton insoluble, resistant to depolymerization at 4 degrees C, gelsolin-poor, and localized to submembranous areas of the cell; and 2) a labile F-actin pool which is the majority of total cellular F-actin, Triton soluble, depolymerizes at 4 degrees C, is gelsolin-rich, and distributed diffusely throughout the cell. The results suggest that the two pools may subserve unique cytoskeletal functions within PMNs, and should be carefully considered in efforts to elucidate the mechanisms which regulate actin polymerization and depolymerization in non-muscle cells.
丝状（F）肌动蛋白是多形核白细胞（PMN）和其他非肌肉细胞中的主要细胞骨架成分。PMN暴露于激动剂会导致单体（G）肌动蛋白聚合成F-肌动蛋白，并激活运动反应。在体外，所有纯化的F-肌动蛋白都是相同的。然而，在体内，多种不同的肌动蛋白调节和结合蛋白的存在表明细胞内所有的F-肌动蛋白可能并不相同。通常，细胞中的F-肌动蛋白通过NBD鬼笔环肽结合或作为Triton提取细胞中的细胞骨架相关肌动蛋白来测量。为了确定PMN中F-肌动蛋白的两种测量方法，即NBD鬼笔环肽结合和细胞骨架相关肌动蛋白是否等效，对通过这两种技术测量的基础、非黏附、无内毒素PMN中的F-肌动蛋白进行了定性和定量比较。在用甲醛固定细胞后进行细胞裂解和荧光染色（预固定），或在用Triton裂解细胞后进行固定（后固定）的情况下，测量了作为NBD鬼笔环肽结合的F-肌动蛋白和细胞骨架相关肌动蛋白。通过这两种技术，预固定细胞中的F-肌动蛋白均高于后固定细胞（通过NBD鬼笔环肽结合测量的平均荧光通道为54.25±3.77对23.5±3.7，作为细胞骨架相关肌动蛋白测量的总细胞肌动蛋白的百分比为70.3±3.5%对47.2±3.6%）。这些结果表明，在PMN中，Triton暴露会从基础细胞中释放出一个不稳定的F-肌动蛋白池，而一个稳定的F-肌动蛋白池对Triton暴露具有抗性。利用NBD鬼笔环肽结合、超速离心和电子显微镜对不同的不稳定和稳定F-肌动蛋白池进行的进一步表征表明，与不稳定池一起释放的肌动蛋白会以细丝形式丢失。通过荧光显微镜记录了两个池中F-肌动蛋白的亚细胞定位，而肌动蛋白调节蛋白凝溶胶蛋白的分布则通过抗凝溶胶蛋白免疫印迹进行表征。我们的研究表明，至少两个不同的F-肌动蛋白池共存于悬浮的无内毒素基础PMN中：1）一个稳定的F-肌动蛋白池，它是总细胞F-肌动蛋白的少数部分，Triton不溶性，在4℃下抗解聚，凝溶胶蛋白含量低，定位于细胞的亚膜区域；2）一个不稳定的F-肌动蛋白池，它是总细胞F-肌动蛋白的多数部分，Triton可溶性，在4℃下解聚，凝溶胶蛋白含量高，在细胞内广泛分布。结果表明，这两个池可能在PMN中发挥独特的细胞骨架功能，并且在阐明调节非肌肉细胞中肌动蛋白聚合和解聚机制的努力中应仔细考虑。
