Dependence of fibroblast migration on actin severing activity of gelsolin
Dependence of fibroblast migration on actin severing activity of gelsolin
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DOI：
10.1074/jbc.271.34.20516
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发表时间：
1996-08-23
影响因子：
4.8
通讯作者：
McCulloch, CAG
中科院分区：
文献类型：
--
作者：
Arora, PD;McCulloch, CAG
凝溶胶蛋白使肌动蛋白丝组装成核，阻断肌动蛋白丝的快速交换末端，并切断丝，这些过程可能在细胞移动性中发挥重要作用。我们研究了人牙龈成纤维细胞中细胞迁移、离子、凝溶胶蛋白含量和肌动蛋白切断活性之间的关系。这些细胞呈角蛋白阴性和结蛋白阴性，但表达波形蛋白和肌球蛋白 II。根据细胞响应趋化剂刺激的迁移能力来分离细胞。 mRNA 的 Northern 分析、[S-35]蛋氨酸掺入免疫沉淀凝溶胶蛋白、细胞裂解物的免疫印迹和定量共聚焦显微镜显示，与迁移细胞相比，非迁移细胞中的凝溶胶蛋白水平高 1.4-2 倍。由于细胞内凝溶胶蛋白的浓度似乎不是细胞迁移的中心决定因素，因此我们评估了其对运动性的要求，与用不相关抗体处理的对照相比，电注射凝溶胶蛋白抗体的细胞在体外抑制肌动蛋白被凝溶胶蛋白切断的细胞显示出迁移细胞数量减少了 72%。与电注射游离凝溶胶蛋白的细胞相比，电注射游离凝溶胶蛋白的细胞的迁移增加了 33%牛血清白蛋白。与非迁移细胞相比，迁移细胞含有丰富的游离凝溶胶蛋白，并表现出凝溶胶蛋白依赖性F-肌动蛋白切断活性，这需要Ca2+。细胞迁移的血清刺激需要增加 [Ca2+](i)，因为用 3 μM 1,2-双-(邻氨基苯氧基)-乙烷-N,N,N',N'-四乙酸四(乙酰氧基甲基)-酯孵育会阻碍钙流动和细胞迁移，血清还刺激凝溶胶蛋白募集到来自迁移细胞而非非迁移细胞的裂解物上清液中。在成纤维细胞中，仅凝溶胶蛋白浓度不会刺激细胞迁移。显然决定了迁移能力，相反，游离凝溶胶蛋白的 Ca2+ 依赖性肌动蛋白切断活性似乎是细胞迁移的主要决定因素。
Gelsolin nucleates actin filament assembly, blocks the fast-exchanging ends of actin filaments, and severs filaments, processes that may play an important role in cell mobility. We studied the relationship between cell migration, ion, gelsolin content, and actin severing activity in human gingival fibroblasts. These cells were keratin negative and desmin negative but expressed vimentin and myosin II. Cells were separated by their ability to migrate in response to a chemoattractant stimulus. Northern analysis of mRNA, [S-35]methionine incorporation into immunoprecipitated gelsolin, immunoblots of cell lysates, and quantitative confocal microscopy showed 1.4-2-fold higher levels of gelsolin in nonmigrant compared with migrant cells. Because the concentration of intracellular gelsolin did not appear to be a central determinant of cell migration, we assessed its requirement for motility, Cells that were electroinjected with a gelsolin antibody that inhibits actin severing by gelsolin in vitro showed a 72% reduction of the number of migrant cells compared with controls treated with an irrelevant antibody, Cells that were electroinjected with free gelsolin exhibited a 33% increase in migration compared with cells electroinjected with bovine serum albumin. Compared with nonmigrant cells, migrant cells contained abundant free gelsolin and exhibited gelsolin-dependent F-actin severing activity, which required Ca2+. Serum stimulation of cell migration required increases in [Ca2+](i) because incubation with 3 mu M 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)-ester blocked calcium flux and cell migration, Serum also stimulated the recruitment of gelsolin into the supernatants of lysates from migrant but not from nonmigrant cells, In fibroblasts, gelsolin concentration alone does not apparently determine migratory capacity, Instead, the Ca2+-dependent actin severing activity of free gelsolin appears to be a major determinant of cell migration.