Kiss Alexa, Gong Xiaowei, Kowalewski Jacob M, Shafqat-Abbasi Hamdah, Strömblad Staffan, Lock John G
Center for Innovative Medicine, Department of Biosciences and Nutrition, Karolinska Institutet, Novum, Hälsov. 7-9, G-building floor 6, S-141 83 Huddinge, Sweden.
Integr Biol (Camb). 2015 Oct;7(10):1171-85. doi: 10.1039/c4ib00291a. Epub 2015 May 22.
Talin is a key cell-matrix adhesion component with a central role in regulating adhesion complex maturation, and thereby various cellular properties including adhesion and migration. However, knockdown studies have produced inconsistent findings regarding the functional influence of talin in these processes. Such discrepancies may reflect non-monotonic responses to talin expression-level variation that are not detectable via canonical "binary" comparisons of aggregated control versus knockdown cell populations. Here, we deployed an "analogue" approach to map talin influence across a continuous expression-level spectrum, which we extended with sub-maximal RNAi-mediated talin depletion. Applying correlative imaging to link live cell and fixed immunofluorescence data on a single cell basis, we related per cell talin levels to per cell measures quantitatively defining an array of cellular properties. This revealed both linear and non-linear correspondences between talin expression and cellular properties, including non-monotonic influences over cell shape, adhesion complex-F-actin association and adhesion localization. Furthermore, we demonstrate talin level-dependent changes in networks of correlations among adhesion/migration properties, particularly in relation to cell migration speed. Importantly, these correlation networks were strongly affected by talin expression heterogeneity within the natural range, implying that this endogenous variation has a broad, quantitatively detectable influence. Overall, we present an accessible analogue method that reveals complex dependencies on talin expression-level, thereby establishing a framework for considering non-linear and non-monotonic effects of protein expression-level heterogeneity in cellular systems.
踝蛋白是一种关键的细胞与基质粘附成分，在调节粘附复合物成熟过程中起核心作用，进而影响包括粘附和迁移在内的各种细胞特性。然而，关于踝蛋白在这些过程中的功能影响，敲低实验得出了不一致的结果。这种差异可能反映了对踝蛋白表达水平变化的非单调反应，而这种反应通过对聚集的对照细胞群体与敲低细胞群体进行经典的“二元”比较是检测不到的。在这里，我们采用了一种“类似物”方法来绘制踝蛋白在连续表达水平范围内的影响，我们通过亚最大RNA干扰介导的踝蛋白耗竭来扩展这一范围。通过相关成像将活细胞和固定免疫荧光数据在单个细胞基础上联系起来，我们将每个细胞的踝蛋白水平与定量定义一系列细胞特性的每个细胞测量值相关联。这揭示了踝蛋白表达与细胞特性之间的线性和非线性对应关系，包括对细胞形状、粘附复合物与F-肌动蛋白的关联以及粘附定位的非单调影响。此外，我们证明了踝蛋白水平依赖性对粘附/迁移特性之间相关性网络的变化，特别是与细胞迁移速度有关。重要的是，这些相关网络受到自然范围内踝蛋白表达异质性的强烈影响，这意味着这种内源性变化具有广泛的、可定量检测的影响。总体而言，我们提出了一种易于使用的类似物方法，揭示了对踝蛋白表达水平的复杂依赖性，从而建立了一个框架，用于考虑细胞系统中蛋白质表达水平异质性的非线性和非单调效应。
