Watts R G
Department of Pediatrics, University of Alabama at Birmingham 35233, USA.
Blood. 1995 Apr 15;85(8):2212-21.
Structurally and functionally distinct F-actin pools coexist with globular (G)-actin in a variety of eukaryotic cells, including polymorphonuclear leukocytes (PMNs). In PMNs, a Triton-soluble F-actin pool (TSF) exists as short cytoplasmic filaments capped with gelsolin, while Triton-insoluble F-actin (TIF) is a three-dimensional meshwork of F-actin associated with actin-binding protein 280 (ABP-280), alpha-actinin, and tropomyosin. The unique association of gelsolin with the TSF suggests a role for gelsolin in creation or regulation of TSF. To evaluate gelsolin's role in TSF formation, the quantities of actin and gelsolin were determined by quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblots in uninduced HL-60 cells (U-HL-60) and in HL-60 cells induced to myeloid differentiation with 1.25% dimethyl sulfoxide for 4 to 5 days (I-HL-60). U-HL-60 cells contain 17.76 +/- 6.01 pmol actin per 10(6) cells (TIF, 5.3 +/- 1.5; TSF, 2.17 +/- 0.37; G, 10.3 +/- 5.7; n = 5) and 0.073 pmol gelsolin per 10(6) cells (TIF, 0; TSF, 0.002 +/- 0.005; G, 0.07 +/- 0.01; n = 3), representing molar actin to gelsolin (A:G) ratios of 1,085:1 for TSF and 147:1 for G. After myeloid differentiation, the actin content increases 1.80-fold (31.94 +/- 6.14 pmol/10(6) cells) equally in each actin pool (TIF, 9.36 +/- 2.35; TSF, 3.29 +/- 0.62; G, 19.29 +/- 4.83). Gelsolin increases 2.4-fold overall (0.178 +/- 0.02 pmol/10(6) cells) but 19-fold in TSF (0.038 +/- 0.009) and only 1.9-fold in G pool (0.139 +/- 0.006), resulting in A:G ratios of 87:1 in TSF and 139:1 in G. The findings of an increase in TSF gelsolin with decreased A:G ratios (1,085:1 v 87:1) with myeloid differentiation suggest shortening of TSF filaments, while the A:G ratios of unbound gelsolin are unchanged (147:1 v 139:1). Measurement of EGTA-resistant gelsolin/actin complexes in HL-60 cells shows that 95% to 100% of complexes exist in the TSF-actin pool only. These findings are consistent with a role for gelsolin in formation and organization of Triton-soluble F-actin. Furthermore, the apparent shortening of TSF-actin filaments with myeloid cellular differentiation and maturation may represent one mechanism of conversion of the nonmotile myeloblast to the motile PMN.
在包括多形核白细胞（PMN）在内的多种真核细胞中，结构和功能上不同的丝状肌动蛋白（F-肌动蛋白）池与球状肌动蛋白（G-肌动蛋白）共存。在PMN中，一种Triton可溶的F-肌动蛋白池（TSF）以短的细胞质细丝形式存在，细丝末端结合有凝溶胶蛋白，而Triton不溶的F-肌动蛋白（TIF）是与肌动蛋白结合蛋白280（ABP-280）、α-辅肌动蛋白和原肌球蛋白相关的F-肌动蛋白三维网络。凝溶胶蛋白与TSF的独特关联表明凝溶胶蛋白在TSF的产生或调节中起作用。为了评估凝溶胶蛋白在TSF形成中的作用，通过定量十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和免疫印迹法测定了未诱导的HL-60细胞（U-HL-60）以及用1.25%二甲基亚砜诱导髓系分化4至5天的HL-60细胞（I-HL-60）中肌动蛋白和凝溶胶蛋白的量。U-HL-60细胞每10⁶个细胞含有17.76±6.01 pmol肌动蛋白（TIF，5.3±1.5；TSF，2.17±0.37；G，10.3±5.7；n = 5），每10⁶个细胞含有0.073 pmol凝溶胶蛋白（TIF，0；TSF，0.002±0.005；G，0.07±0.01；n = 3），TSF中肌动蛋白与凝溶胶蛋白（A:G）的摩尔比为1085:1，G中为14:1。髓系分化后，肌动蛋白含量增加1.80倍（31.94±6.14 pmol/10⁶个细胞），每个肌动蛋白池增加量相同（TIF，9.36±2.35；TSF，3.29±0.62；G，19.29±4.83）。凝溶胶蛋白总体增加2.4倍（0.178±0.02 pmol/10⁶个细胞），但TSF中增加19倍（0.038±0.009），G池中仅增加1.9倍（0.139±0.006），导致TSF中A:G比为87:1，G中为139:1。髓系分化时TSF凝溶胶蛋白增加且A:G比降低（1085:1对87:1）的结果表明TSF细丝缩短，而未结合凝溶胶蛋白的A:G比不变（147:1对139:1）。对HL-60细胞中乙二醇双四乙酸（EGTA）抗性凝溶胶蛋白/肌动蛋白复合物的测量表明，95%至100%的复合物仅存在于TSF-肌动蛋白池中。这些发现与凝溶胶蛋白在Triton可溶F-肌动蛋白的形成和组织中的作用一致。此外，随着髓系细胞分化和成熟，TSF-肌动蛋白细丝明显缩短可能代表了非运动性原粒细胞向运动性PMN转化的一种机制。
