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机械敏感蛋白的功能，随后控制细胞的行为和反应。通过研究整合素，这种微小的蛋白质充当细胞锚和来自环境的机械线索的传感器，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。这一见解将推进对生命系统的理解，并在理解癌症等疾病方面具有巨大潜力，其中细胞的粘性和机械特性发挥着关键作用。除了其科学价值，这项研究的影响将超越实验室。一个创新的，动手课程，沉浸在生物物理研究本科生，培养下一波科学家，将被设计。此外，艺术家们还将帮助创作具有科学灵感的卡通，使所有年龄段的儿童都能理解复杂的科学概念，并感到兴奋。该项目将开发一种新的方法来理解机械转导途径和整合素蛋白的结构生物学，整合素蛋白是细胞粘附和信号传导的关键因素。该项目的核心是多轴熵弹簧镊子沿着环形折纸（MAESTRO），这是一种低成本，高通量的技术，用于从多个方向向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，利用DNA折纸的精密位置控制来控制施加张力的方向，并利用设备小型化来降低成本并提高产量。为了验证MAESTRO的性能，将通过评估机械张力下Holliday连接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数据从光镊实验和理论预测来自玻尔兹曼分布。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，将被用来解剖这些多方向的机械刺激响应的整合素的复杂信号。最后，低温电子显微镜将被用于可视化的张力诱导的状态变化的整合素，提供结构的见解，他们的信号作用。这个奖项反映了NSF的法定使命，并已被认为是值得的支持，通过评估使用基金会的智力价值和更广泛的影响审查标准。

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.