The Position of the GFP Tag on Actin Affects the Filament Formation in Mammalian Cells
The Position of the GFP Tag on Actin Affects the Filament Formation in Mammalian Cells
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DOI：
10.1247/csf.17016
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发表时间：
2017-01-01
影响因子：
1.5
通讯作者：
Uyeda, Taro Q. P.
中科院分区：
文献类型：
--
作者：
Nagasaki, Akira;Kijima, Saku T.;Uyeda, Taro Q. P.
肌动蛋白是微丝的主要组成部分，参与真核生物的多种细胞功能。在过去的二十年里，肌动蛋白与荧光蛋白融合被用作检测活体真核细胞中肌动蛋白细胞骨架的组织和动力学的探针。一般认为，荧光蛋白融合蛋白的表达不会干扰内源性肌动蛋白在细胞内的分布，融合蛋白的分布反映了内源性肌动蛋白的分布。然而，我们注意到egfp - β -肌动蛋白在一些哺乳动物细胞系中引起了微丝的过度形成。为了研究EGFP标签在肌动蛋白上的位置是否影响细丝的形成，我们构建了一个含有β -actin-EGFP基因的表达载体。与egfp - β -肌动蛋白相比，表达β -肌动蛋白- egfp的细胞在细胞质中显示出高背景的肌动蛋白丝。此外，洗涤剂不溶性实验显示，大部分来自表达egfp - β -肌动蛋白的细胞的洗涤剂不溶性细胞骨架在颗粒中被回收。此外，我们发现egfp - β -actin的表达影响NBT-L2b细胞的迁移和U2OS细胞的机械刚度。这些结果表明EGFP融合到肌动蛋白的n端容易形成过多的肌动蛋白丝。此外，与肌动蛋白- egfp相比，EGFP-actin对细胞形态和生理表型都有影响。
Actin, a major component of microfilaments, is involved in various eukaryotic cellular functions. Over the past two decades, actin fused with fluorescent protein has been used as a probe to detect the organization and dynamics of the actin cytoskeleton in living eukaryotic cells. It is generally assumed that the expression of fusion protein of fluorescent protein does not disturb the distribution of endogenous actin throughout the cell, and that the distribution of the fusion protein reflects that of endogenous actin. However, we noticed that EGFP-beta-actin caused the excessive formation of microfilaments in several mammalian cell lines. To investigate whether the position of the EGFP tag on actin affected the formation of filaments, we constructed an expression vector harboring a beta-actin-EGFP gene. In contrast to EGFP-beta-actin, cells expressing beta-actin-EGFP showed actin filaments in a high background from the monomer actin in cytosol. Additionally, the detergent insoluble assay revealed that the majority of the detergent-insoluble cytoskeleton from cells expressing EGFP-beta-actin was recovered in the pellet. Furthermore, we found that the expression of EGFP-beta-actin affects the migration of NBT-L2b cells and the mechanical stiffness of U2OS cells. These results indicate that EGFP fused to the N-terminus of actin tend to form excessive actin filaments. In addition, EGFP-actin affects both the cellular morphological and physiological phenotypes as compared to actin-EGFP.