a Biomedical Engineering Department , University of California , Davis, Davis , CA , USA.
Cell Adh Migr. 2017 Sep 3;11(5-6):504-513. doi: 10.1080/19336918.2016.1276147. Epub 2017 Jan 27.
Physical force has emerged as a key regulator of tissue homeostasis, and plays an important role in embryogenesis, tissue regeneration, and disease progression. Currently, the details of protein interactions under elevated physical stress are largely missing, therefore, preventing the fundamental, molecular understanding of mechano-transduction. This is in part due to the difficulty isolating large quantities of cell lysates exposed to force-bearing conditions for biochemical analysis. We designed a simple, easy-to-fabricate, large-scale cell stretch device for the analysis of force-sensitive cell responses. Using proximal biotinylation (BioID) analysis or phospho-specific antibodies, we detected force-sensitive biochemical changes in cells exposed to prolonged cyclic substrate stretch. For example, using promiscuous biotin ligase BirA* tagged α-catenin, the biotinylation of myosin IIA increased with stretch, suggesting the close proximity of myosin IIA to α-catenin under a force bearing condition. Furthermore, using phospho-specific antibodies, Akt phosphorylation was reduced upon stretch while Src phosphorylation was unchanged. Interestingly, phosphorylation of GSK3β, a downstream effector of Akt pathway, was also reduced with stretch, while the phosphorylation of other Akt effectors was unchanged. These data suggest that the Akt-GSK3β pathway is force-sensitive. This simple cell stretch device enables biochemical analysis of force-sensitive responses and has potential to uncover molecules underlying mechano-transduction.
物理力已成为组织动态平衡的关键调节因子，在胚胎发生、组织再生和疾病进展中发挥着重要作用。目前，在高物理压力下蛋白质相互作用的细节在很大程度上仍然未知，因此阻碍了对机械转导的基本分子理解。部分原因是难以分离大量暴露于受力条件下用于生化分析的细胞裂解物。我们设计了一种简单、易于制造的大规模细胞拉伸装置，用于分析受力敏感的细胞反应。使用邻近生物素化（BioID）分析或磷酸特异性抗体，我们检测到暴露于长时间周期性基质拉伸下的细胞中受力敏感的生化变化。例如，使用广谱生物素连接酶 BirA*标记的α-连环蛋白，肌球蛋白 IIA 的生物素化随拉伸而增加，表明在受力条件下肌球蛋白 IIA 与α-连环蛋白的接近。此外，使用磷酸特异性抗体，拉伸时 Akt 磷酸化减少，而Src 磷酸化不变。有趣的是， Akt 途径下游效应物 GSK3β 的磷酸化也随拉伸而减少，而其他 Akt 效应物的磷酸化不变。这些数据表明 Akt-GSK3β 途径对力敏感。这种简单的细胞拉伸装置能够进行受力敏感反应的生化分析，并有潜力揭示机械转导的分子基础。
