温度跃迁显微镜和 Hsp70 热休克蛋白与体内客户蛋白的相互作用。

IF 4.2 3区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Methods Pub Date : 2024-10-01 DOI:10.1016/j.ymeth.2024.09.019
Aniket Ravan , Samuel Procopio , Yann R. Chemla , Martin Gruebele
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引用次数: 0

摘要

蛋白质-蛋白质相互作用等生物分子过程在很大程度上取决于细胞类型,甚至在单一细胞类型内也会发生变化。在这里,我们开发了一种带有珀尔帖(Peltier)控温平台、激光温度跃迁以诱导热应力、自动聚焦功能以减轻实验过程中的温度漂移的显微镜,用于研究活生物体(斑马鱼幼体)内所选细胞类型中的蛋白质-蛋白质相互作用。作为该仪器的一项应用,我们发现热休克蛋白 Hsp70 与其客户之一磷酸甘油酸激酶的结合在活体细胞间存在相当大的差异。我们调整了先前折叠研究的一个关键特征,即幼虫体内细胞的罕见转化,这样就可以对单个细胞进行成像和分化,以了解细胞间的反应。我们的方法可扩展到其他生物体和细胞类型,而非本研究中展示的生物体和细胞类型。
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Temperature-jump microscopy and interaction of Hsp70 heat shock protein with a client protein in vivo
Biomolecular processes such as protein–protein interactions can depend strongly on cell type and even vary within a single cell type. Here we develop a microscope with a Peltier-controlled temperature stage, a laser temperature jump to induce heat stress, and an autofocusing feature to mitigate temperature drift during experiments, to study a protein–protein interaction in a selected cell type within a live organism, the zebrafish larva. As an application of the instrument, we show that there is considerable cell-to-cell variation of the heat shock protein Hsp70 binding to one of its clients, phosphoglycerate kinase in vivo. We adapt a key feature from our previous folding study, rare transformation of cells within the larva, so that individual cells can be imaged and differentiated for cell-to-cell response. Our approach can be extended to other organisms and cell types than the ones demonstrated in this work.
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来源期刊
Methods
Methods 生物-生化研究方法
CiteScore
9.80
自引率
2.10%
发文量
222
审稿时长
11.3 weeks
期刊介绍: Methods focuses on rapidly developing techniques in the experimental biological and medical sciences. Each topical issue, organized by a guest editor who is an expert in the area covered, consists solely of invited quality articles by specialist authors, many of them reviews. Issues are devoted to specific technical approaches with emphasis on clear detailed descriptions of protocols that allow them to be reproduced easily. The background information provided enables researchers to understand the principles underlying the methods; other helpful sections include comparisons of alternative methods giving the advantages and disadvantages of particular methods, guidance on avoiding potential pitfalls, and suggestions for troubleshooting.
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