Mechanical detection of interactions between proteins related to intermediate filament and transcriptional regulation in living cells
Mechanical detection of interactions between proteins related to intermediate filament and transcriptional regulation in living cells
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机械检测活细胞中与中间丝相关的蛋白质和转录调控之间的相互作用
DOI：
10.1016/j.bios.2022.114603
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发表时间：
2022
影响因子：
12.6
通讯作者：
Nakamura Chikashi
中科院分区：
文献类型：
--
作者：
Yamagishi Ayana;Mizusawa Mei;Uchida Koki;Iijima Masumi;Kuroda Shun’ichi;Fukazawa Kyoko;Ishihara Kazuhiko;Nakamura Chikashi
中间丝（IF）结合各种蛋白质并调节细胞质中的细胞功能。最近，IFs被发现通过作为转录相关蛋白的捕获支架并阻止其易位到细胞核中来调节基因表达。为了揭示IFs调控的转录调控机制，需要一种分析IFs与转录相关蛋白相互作用的方法。虽然有许多方法可以观察活细胞中的相互作用，但在未修饰和天然状态下测量活细胞中的蛋白质-蛋白质相互作用仍然具有挑战性。在这项研究中，我们利用了一种纳米针，它可以通过插入细胞进入细胞溶质。识别转录相关蛋白的抗体的修饰允许针检测在针从细胞缩回期间解除抗体和与IF相互作用的靶蛋白之间的相互作用所需的机械力。我们专注于IF波形蛋白，上皮间质转化的标志物，机械检测波形蛋白丝捕获的转录相关蛋白。选择转录相关因子Prohibitin 2（PHB 2）作为波形蛋白结合蛋白的候选蛋白。我们使用原子力显微镜和抗PHB 2抗体修饰的纳米针在表达波形蛋白的小鼠乳腺癌和波形蛋白敲除（VKO）细胞中进行PHB 2的机械检测。在波形蛋白表达细胞中检测到比VKO细胞中显著更大的解结合力。结果表明，该方法是有用的，在细胞内的机械检测的IF结合蛋白。
Intermediate filaments (IF) bind to various proteins and regulate cell function in the cytoplasm. Recently, IFs were found to regulate gene expression by acting as capture scaffolds for transcription-related proteins and preventing their translocation into the nucleus. To reveal such transcriptional regulatory mechanisms controlled by IFs, a method to analyze the interaction between IFs and transcription-related proteins is necessary. Although there are many methods to observe interactions in living cells, it is still challenging to measure protein-protein interactions in living cells in their unmodified and native state. In this study, we utilized a nanoneedle that can access the cytosol by insertion into the cell. Modification of antibody recognizing transcription-related proteins allows the needle to detect mechanical force required to unbind the interaction between antibody and target proteins interacting with IFs during retraction of the needle from the cell. We focused on IF vimentin, a marker of epithelial-mesenchymal transition, to mechanically detect transcription-related proteins trapped by vimentin filaments. Prohibitin 2 (PHB2), a transcription-related factor, was selected as the candidate vimentin-binding protein. We conducted mechanical detection of PHB2 using atomic force microscopy and anti-PHB2 antibody-modified nanoneedles in vimentin-expressing mouse breast cancer and vimentin-knockout (VKO) cells. Significantly larger unbinding forces were detected in the vimentin-expressing cells than in the VKO cells. The results demonstrate that this method is useful for in-cell mechanical detection of IF-binding proteins.