Department of Cell Pathology, Faculty of Biotechnology, University of Wrocław, Poland.
Life Sci. 2012 Jun 6;90(21-22):851-61. doi: 10.1016/j.lfs.2012.03.039. Epub 2012 Apr 19.
Formation of different protrusive structures by migrating cells is driven by actin polymerization at the plasma membrane region. Gelsolin is an actin binding protein controlling the length of actin filaments by its severing and capping activity. The main goal of this study was to determine the effect of gelsolin expression on the migration of human colon adenocarcinoma LS180 and melanoma A375 cells.
Colon adenocarcinoma cell line LS180 was stably transfected with plasmid containing human cytoplasmic gelsolin cDNA tagged to enhanced green fluorescence protein (EGFP). Melanoma A375 cells were transfected with siRNAs directed against gelsolin. Real-time PCR and Western blotting were used to determine the level of gelsolin. The ability of actin to inhibit DNase I activity was used to quantify monomeric and total actin level and calculate the state of actin polymerization. Fluorescence confocal microscopy was applied to observe gelsolin and vinculin distribution along with actin cytoskeleton organization.
Increased level of gelsolin expression leads to its accumulation at the submembranous region of the cell accompanied by distinct changes in the state of actin polymerization and an increase in the migration of LS180 cells. In addition, LS180 cells overexpressing gelsolin form podosome-like structures as indicated by vinculin redistribution and its colocalization with gelsolin and actin. Downregulation of gelsolin expression in melanoma A375 cells significantly reduces their migratory potential.
Our experimental data indicate that alterations in the expression level of gelsolin and its subcellular distribution may be directly responsible for determining migration capacity of human cancer cells.
迁移细胞形成不同的突起结构是由质膜区域的肌动蛋白聚合驱动的。凝溶胶蛋白是一种肌动蛋白结合蛋白，通过其切割和盖帽活性控制肌动蛋白丝的长度。本研究的主要目的是确定凝溶胶蛋白表达对人结肠腺癌 LS180 和黑色素瘤 A375 细胞迁移的影响。
用含有与人细胞质凝溶胶蛋白 cDNA 标记增强型绿色荧光蛋白（EGFP）的质粒稳定转染结肠腺癌细胞系 LS180。用针对凝溶胶蛋白的 siRNA 转染黑色素瘤 A375 细胞。实时 PCR 和 Western blot 用于测定凝溶胶蛋白的水平。用肌动蛋白抑制 DNA 酶 I 活性的能力来定量单体和总肌动蛋白水平，并计算肌动蛋白聚合状态。荧光共聚焦显微镜用于观察凝溶胶蛋白和 vinculin 与肌动蛋白细胞骨架组织的分布。
凝溶胶蛋白表达水平的增加导致其在细胞亚膜区域的积累，伴随着肌动蛋白聚合状态的明显变化和 LS180 细胞迁移能力的增加。此外，LS180 细胞过表达凝溶胶蛋白形成类似足突的结构，如 vinculin 重新分布及其与凝溶胶蛋白和肌动蛋白的共定位所示。黑色素瘤 A375 细胞中凝溶胶蛋白表达的下调显著降低了其迁移能力。
我们的实验数据表明，凝溶胶蛋白表达水平的改变及其亚细胞分布的改变可能直接决定人类癌细胞的迁移能力。
