Department of Quantitative Cell Biology, Institute of Molecular Cell Biology, University of Münster, Schlossplatz 5, Münster, 48149, Germany.
Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
Chembiochem. 2021 Oct 1;22(19):2872-2879. doi: 10.1002/cbic.202100301. Epub 2021 Jul 30.
Talin is a cell adhesion molecule that is indispensable for the development and function of multicellular organisms. Despite its central role for many cell biological processes, suitable methods to investigate the nanoscale organization of talin in its native environment are missing. Here, we overcome this limitation by combining single-molecule resolved PAINT (points accumulation in nanoscale topography) imaging with the IRIS (image reconstruction by integrating exchangeable single-molecule localization) approach, enabling the quantitative analysis of genetically unmodified talin molecules in cells. We demonstrate that a previously reported peptide can be utilized to specifically label the two major talin isoforms expressed in mammalian tissues with a localization precision of <10 nm. Our experiments show that the methodology performs equally well as state-of-the-art single-molecule localization techniques, and the first applications reveal a thus far undescribed cell adhesion structure in differentiating stem cells. Furthermore, we demonstrate the applicability of this peptide-PAINT technique to mouse tissues paving the way to single-protein imaging of endogenous talin proteins under physiologically relevant conditions.
塔林是一种细胞黏附分子，对于多细胞生物的发育和功能不可或缺。尽管塔林在许多细胞生物学过程中起着核心作用，但仍缺乏合适的方法来研究其天然环境中的塔林纳米级组织。在这里，我们通过结合单分子分辨 PAINT（点在纳米级形貌中的积累）成像和 IRIS（通过交换单分子定位进行图像重建）方法克服了这一限制，从而能够对细胞中未经基因修饰的塔林分子进行定量分析。我们证明，以前报道的一种肽可以特异性标记哺乳动物组织中表达的两种主要塔林同工型，定位精度<10nm。我们的实验表明，该方法与最先进的单分子定位技术同样有效，首次应用揭示了分化干细胞中一种迄今未被描述的细胞黏附结构。此外，我们证明了该肽-PAINT 技术在小鼠组织中的适用性，为在生理相关条件下对内源性塔林蛋白进行单蛋白成像铺平了道路。
