Seetharaman Shailaja, Sala Stefano, Gardel Margaret L, Oakes Patrick W
Department of Physics, Institute for Biophysical Dynamics, and James Franck Institute, University of Chicago, Chicago, IL, USA.
Department of Cell & Molecular Physiology, Stritch School of Medicine, Loyola University Chicago, Maywood, IL, USA.
Methods Mol Biol. 2023;2600:169-182. doi: 10.1007/978-1-0716-2851-5_11.
A family of proteins have been identified that recognize damaged, strained actin filaments in stress fibers. These proteins are often referred to as strain- or force-sensing and trigger downstream signaling mechanisms that can facilitate repair at these strain sites. Here we describe a method using high-resolution microscopy to screen and quantify the mechanosensitive recruitment of proteins to these stress fiber strain sites. Strain sites are induced using spatially controlled illumination of UV light to locally damage actin stress fibers. Recruitment of potential strain-sensing proteins can then either be compared to (Blanchoin, Physiol Rev 94, 235-263, 2014) a known control (e.g., zyxin-GFP) or (Hoffman, Mol Biol Cell 23, 1846-1859, 2012) the pre-damaged stress fiber protein distribution. With this method, strain-sensing proteins and their dynamic association with stress fiber strain sites can be reproducibly measured and compared.
已鉴定出一类蛋白质，它们能识别应力纤维中受损、受拉的肌动蛋白丝。这些蛋白质通常被称为应变或力传感蛋白，并触发下游信号机制，从而促进这些应变位点的修复。在这里，我们描述了一种使用高分辨率显微镜来筛选和量化蛋白质向这些应力纤维应变位点的机械敏感募集的方法。通过对紫外光进行空间控制照射以局部损伤肌动蛋白应力纤维来诱导应变位点。然后，可以将潜在的应变传感蛋白的募集情况与（布兰乔因，《生理学评论》94卷，235 - 263页，2014年）已知对照（例如，zyxin - GFP）或（霍夫曼，《分子生物学细胞》23卷，1846 - 1859页，2012年）损伤前应力纤维蛋白分布进行比较。使用这种方法，可以可重复地测量和比较应变传感蛋白及其与应力纤维应变位点的动态关联。
