Arsenovic Paul T, Bathula Kranthidhar, Conway Daniel E
Department of Biomedical Engineering, Virginia Commonwealth University;
Department of Biomedical Engineering, Virginia Commonwealth University.
J Vis Exp. 2017 Apr 11(122):54902. doi: 10.3791/54902.
The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. Nesprin-2G is a key component of the LINC complex that connects the actin cytoskeleton to membrane proteins (SUN domain proteins) in the perinuclear space. These membrane proteins connect to lamins inside the nucleus. Recently, a Förster Resonance Energy Transfer (FRET)-force probe was cloned into mini-Nesprin-2G (Nesprin-TS (tension sensor)) and used to measure tension across Nesprin-2G in live NIH3T3 fibroblasts. This paper describes the process of using Nesprin-TS to measure LINC complex forces in NIH3T3 fibroblasts. To extract FRET information from Nesprin-TS, an outline of how to spectrally unmix raw spectral images into acceptor and donor fluorescent channels is also presented. Using open-source software (ImageJ), images are pre-processed and transformed into ratiometric images. Finally, FRET data of Nesprin-TS is presented, along with strategies for how to compare data across different experimental groups.
LINC复合物被认为是介导机械力从细胞骨架传递到细胞核的关键结构。Nesprin-2G是LINC复合物的关键组成部分，它将肌动蛋白细胞骨架与核周空间中的膜蛋白（SUN结构域蛋白）相连。这些膜蛋白与细胞核内的核纤层蛋白相连。最近，一种荧光共振能量转移（FRET）力探针被克隆到微型Nesprin-2G（Nesprin-TS（张力传感器））中，并用于测量活的NIH3T3成纤维细胞中Nesprin-2G上的张力。本文描述了使用Nesprin-TS测量NIH3T3成纤维细胞中LINC复合物力的过程。为了从Nesprin-TS中提取FRET信息，还介绍了如何将原始光谱图像光谱解混为受体和供体荧光通道的概述。使用开源软件（ImageJ）对图像进行预处理并转换为比率图像。最后，给出了Nesprin-TS的FRET数据，以及如何比较不同实验组数据的策略。
