Department of Chemistry, Emory University, Atlanta, Georgia 30322, United States.
Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia 30322, United States.
J Am Chem Soc. 2024 Aug 21;146(33):23034-23043. doi: 10.1021/jacs.4c03629. Epub 2024 Aug 12.
Cells apply forces to extracellular matrix (ECM) ligands through transmembrane integrin receptors: an interaction which is intimately involved in cell motility, wound healing, cancer invasion and metastasis. These small (piconewton) integrin-ECM forces have been studied by molecular tension fluorescence microscopy (MTFM), which utilizes a force-induced conformational change of a probe to detect mechanical events. MTFM has revealed the force magnitude for integrin receptors in a variety of cell models including primary cells. However, force dynamics and specifically the force loading rate (LR) have important implications in receptor signaling and adhesion formation and remain poorly characterized. Here, we develop an LR probe composed of an engineered DNA structure that undergoes two mechanical transitions at distinct force thresholds: a low force threshold at 4.7 pN (hairpin unfolding) and a high force threshold at 47 pN (duplex shearing). These transitions yield distinct fluorescence signatures observed through single-molecule fluorescence microscopy in live cells. Automated analysis of tens of thousands of events from eight cells showed that the bond lifetime of integrins that engage their ligands and transmit a force >4.7 pN decays exponentially with a τ of 45.6 s. A subset of these events mature in magnitude to >47 pN with a median loading rate of 1.1 pN s and primarily localize at the periphery of the cell-substrate junction. The LR probe design is modular and can be adapted to measure force ramp rates for a broad range of mechanoreceptors and cell models, thus aiding in the study of molecular mechanotransduction in living systems.
细胞通过跨膜整合素受体向细胞外基质（ECM）配体施加力：这种相互作用与细胞迁移、伤口愈合、癌症侵袭和转移密切相关。这些小（皮牛顿）的整合素-ECM 力已通过分子张力荧光显微镜（MTFM）进行研究，该显微镜利用探针的力诱导构象变化来检测机械事件。MTFM 已在多种细胞模型（包括原代细胞）中揭示了整合素受体的力大小。然而，力动力学，特别是力加载率（LR），在受体信号转导和粘附形成中具有重要意义，但仍未得到充分描述。在这里，我们开发了一种 LR 探针，由一种经过工程设计的 DNA 结构组成，该结构在两个不同的力阈值下经历两次机械转变：低力阈值为 4.7 pN（发夹展开）和高力阈值为 47 pN（双链剪切）。这些转变在活细胞中的单分子荧光显微镜下产生了不同的荧光特征。对 8 个细胞中的数万次事件进行的自动分析表明，与配体结合并传递力>4.7 pN 的整合素的键寿命呈指数衰减，τ 为 45.6 s。这些事件中有一部分在大小上成熟到>47 pN，平均加载率为 1.1 pN s，主要定位于细胞-基质连接的外围。LR 探针设计具有模块化，可以适应测量广泛的机械感受器和细胞模型的力上升率，从而有助于研究活系统中的分子机械转导。
