CAREER: High-throughput multi-axial tension-inducing DNA origami device for investigating mechanosensitive signaling pathways and protein structures under defined tension

我们体内微小的机械力调节着机械敏感蛋白的功能，而机械敏感蛋白随后又控制着细胞的行为和反应。通过研究整合素（一种微小的蛋白质，作为细胞锚点和来自环境的机械信号的传感器），可以发现控制细胞如何将它们感受到的机械力转化为有关其行为的决定的新规则。这种见解将促进对生命系统的理解，并在理解癌症等疾病方面具有巨大的潜力，在这些疾病中，细胞的粘性和机械特性起着关键作用。除了科学价值之外，这项研究的影响将超越实验室。将设计一个创新的，动手的课程，使本科生沉浸在生物物理研究中，培养下一波科学家。此外，艺术家将帮助创作以科学为灵感的漫画，使复杂的科学概念对所有年龄段的儿童都容易理解和兴奋。该项目将开发一种新的方法来理解整合素蛋白的机械转导途径和结构生物学，整合素蛋白是细胞粘附和信号传导的关键因素。该项目的核心是环形折纸的多轴熵弹簧镊子（MAESTRO），这是一种低成本、高通量的技术，可以从多个方向对单个整合素施加受控的机械张力。MAESTRO利用单链DNA的熵弹性来产生机械张力，DNA折纸的精细位置控制来控制应用张力的方向，以及设备小型化来降低成本和提高吞吐量。为了验证MAESTRO的性能，将通过评估机械张力下Holliday结的动力学来对张力诱导工具进行基准测试。测量结果将与发表的光镊实验数据和由玻尔兹曼分布得出的理论预测进行比较。先进的单分子方法，包括共聚焦单分子荧光共振能量转移（smFRET）和全内反射显微镜，将被用来解剖整合素在响应这些多向机械刺激时的复杂信号。最后，冷冻电子显微镜将用于可视化张力诱导的整合素状态变化，为其信号作用提供结构见解。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Minuscule mechanical forces within us regulate the functions of mechanosensitive proteins, which subsequently control how cells act and react. By studying integrins, the tiny proteins that serve as cellular anchors and sensors for the mechanical cues from their environments, new rules governing how cells translate mechanical forces they feel into decisions regarding their behavior can be uncovered. This insight will advance the understanding of living systems and has enormous potential in understanding diseases like cancer, where the stickiness and mechanical properties of cells play key roles. Beyond its scientific merit, the research's impact will stretch beyond the laboratory. An innovative, hands-on course that immerses undergraduate students in Biological Physics research, fostering the next wave of scientists, will be designed. Additionally, artists will help create science-inspired cartoons, making complex scientific concepts accessible and thrilling for children of all ages.This project will develop a novel approach to understanding the mechanotransduction pathways and structural biology of integrin proteins, a critical factor for cell adhesion and signaling. At the heart of this project is the Multi-Axial Entropic Spring Tweezers along Ring-shaped Origami (MAESTRO), a low-cost, high-throughput technology for applying controlled mechanical tensions to individual integrins from multiple directions. MAESTRO leverages the entropic elasticity of single-stranded DNA to generate mechanical tension, the exquisite positional control of DNA origami to control the directions of applied tensions, and device miniaturization to reduce cost and increase throughput. To validate the performance of MAESTRO, the tension-inducing tool will be benchmarked by assessing the kinetics of Holliday junctions under mechanical tension. Measurements will be compared with published data from optical tweezer experiments and theoretical predictions derived from the Boltzmann distribution. Advanced single-molecule methods, including confocal single-molecule Fluorescence Resonance Energy Transfer (smFRET) and total internal reflection microscopy, will be used to dissect the intricate signaling of integrins in response to these multi-directional mechanical stimuli. Finally, cryo-electron microscopy will be used to visualize the tension-induced state changes of integrins, offering structural insights into their signaling roles.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.