Live Cell Protein Imaging of Tandem Complemented-GFP11-Tagged Coiled-Coil Domain-Containing Protein-124 Identifies this Factor in G3BP1-Induced Stress-Granules

IF 1.9 4区 生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY
Kübra Hacibeyoğlu, Merve Tuzlakoğlu Öztürk, Özge Arslan, Uygar Halis Tazebay
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引用次数: 0

Abstract

Coiled-coil domain-containing 124 protein is a multifunctional RNA-binding factor, and it was previously reported to interact with various biomolecular complexes localized at diverse subcellular locations, such as the ribosome, centrosome, midbody, and nucleoli. We aimed to better characterize the subcellular CCDC124 translocation by labelling this protein with a fluorescent tag, followed by laser scanning confocal microscopy methods. As traditional GFP-tagging of small proteins such as CCDC124 often faces limitations like potential structural perturbations of labeled proteins, and interference of the fluorescent-tag with their endogenous cellular functions, we aimed to label CCDC124 with the smallest possible split-GFP associated protein-tagging system (GFP11/GFP1-10) for better characterization of its subcellular localizations and its translocation dynamics. By recombinant DNA techniques we generated CCDC124-constructs labelled with either single of four tandem copies of GFP11 (GFP11 × 1::CCDC124, GFP11 × 4::CCDC124, or CCDC124::GFP11 × 4). We then cotransfected U2OS cells with these split-GFP constructs (GFP11 × 1(or X4)::CCDC124/GFP1-10) and analyzed subcellular localization of CCDC124 protein by laser scanning confocal microscopy. Tagging CCDC124 with four tandem copies of a 16-amino acid short GFP-derived peptide-tag (GFP11 × 4::CCDC124) allowed better characterization of the subcellular localization of CCDC124 protein in our model human bone osteosarcoma (U2OS) cells. Thus, by this novel methodology we successfully identified GFP11 × 4::CCDC124 molecules in G3BP1-overexpression induced stress-granules by live cell protein imaging for the first time. Our findings propose CCDC124 as a novel component of the stress granule which is a membraneless organelle involved in translational shut-down in response to cellular stress.

Abstract Image

Abstract Image

对串联补体-GFP11-标记的含卷曲盘旋结构域蛋白-124 的活细胞蛋白质成像发现了 G3BP1 诱导的应激颗粒中的这一因子。
含盘旋卷曲结构域的124蛋白是一种多功能RNA结合因子,以前曾报道过它与定位于不同亚细胞位置(如核糖体、中心体、中体和核仁)的各种生物分子复合物相互作用。我们的目的是通过用荧光标签标记该蛋白,然后用激光扫描共聚焦显微镜方法更好地描述 CCDC124 的亚细胞转位特征。由于对 CCDC124 这样的小蛋白进行传统的 GFP 标记往往会面临一些限制,如被标记蛋白的潜在结构扰动,以及荧光标记对其内源细胞功能的干扰,因此我们的目标是用尽可能小的分裂-GFP 相关蛋白标记系统(GFP11/GFP1-10)标记 CCDC124,以更好地表征其亚细胞定位及其转运动态。通过 DNA 重组技术,我们生成了标记有单个或四个串联拷贝 GFP11 的 CCDC124 结构体(GFP11 × 1::CCDC124、GFP11 × 4::CCDC124 或 CCDC124::GFP11×4)。然后,我们用这些分裂-GFP构建体(GFP11 × 1(或 X4)::CCDC124/GFP1-10)共转染 U2OS 细胞,并通过激光扫描共聚焦显微镜分析 CCDC124 蛋白的亚细胞定位。用四个串联拷贝的 16 氨基酸短 GFP 衍生肽标签(GFP11 × 4::CCDC124)标记 CCDC124,可以更好地表征 CCDC124 蛋白在我们的人骨骨肉瘤(U2OS)模型细胞中的亚细胞定位。因此,通过这种新颖的方法,我们首次利用活细胞蛋白成像技术成功鉴定了 G3BP1 表达诱导的应激颗粒中的 GFP11 × 4::CCDC124 分子。我们的研究结果表明,CCDC124 是应激颗粒的一种新成分,应激颗粒是一种无膜细胞器,在细胞应激反应中参与翻译关闭。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
The Protein Journal
The Protein Journal 生物-生化与分子生物学
CiteScore
5.20
自引率
0.00%
发文量
57
审稿时长
12 months
期刊介绍: The Protein Journal (formerly the Journal of Protein Chemistry) publishes original research work on all aspects of proteins and peptides. These include studies concerned with covalent or three-dimensional structure determination (X-ray, NMR, cryoEM, EPR/ESR, optical methods, etc.), computational aspects of protein structure and function, protein folding and misfolding, assembly, genetics, evolution, proteomics, molecular biology, protein engineering, protein nanotechnology, protein purification and analysis and peptide synthesis, as well as the elucidation and interpretation of the molecular bases of biological activities of proteins and peptides. We accept original research papers, reviews, mini-reviews, hypotheses, opinion papers, and letters to the editor.
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