Bringing mechanobiology to the benchtop with single-molecule centrifugation

描述(申请人提供)：从控制免疫反应中白细胞的黏附和招募，到决定细胞命运和组织发育，机械力在整个生物学中发挥着关键的调节作用。随着我们认识到力学可以在分子、细胞和组织水平的生理反应中发挥重要作用，这一新兴领域正在引导人们对疾病的新理解，如出血性疾病、癌症、骨质疏松症和哮喘。能够精确操控单个分子和细胞的技术发展(如光学镊子和原子力显微镜)一直是该领域发展的推动力。然而，由于获得此类技术的机会有限，该领域的发展受到阻碍，因为这可能是昂贵的、具有技术挑战性的和低吞吐量的。为了克服这些挑战，我们将开发一种大胆的方法，以廉价、易于使用和高通量的方式，以单分子精度对微观样品施加受控力。通过将离心机和显微镜集成在一起，我们已经展示了一台离心力显微镜(CFM)的原型，可以并行执行数千个单分子力实验。我们建议通过开发一种强大的、多功能的、可供各种生物医学研究人员使用的机械生物学台式仪器，将单分子离心机的概念提升到一个新的水平。我们将通过以下方式实现这一目标：1)通过将高分辨率成像集成到CFM中，实现对结构转变(例如蛋白质展开)的大规模并行单分子测量；2)通过将高分辨率CFM微型化以直接安装到标准台式离心机中，提高单分子操作技术的可及性；3)通过在单分子和单细胞水平上测量分子内和分子间的键强度来展示这种CFM的多功能性。总而言之，该项目将产生一个可获得的、高通量和高分辨率的新平台，用于在纳米尺度上测量在机械力作用下的生物系统，具有广泛的应用，从测量单个分子的结构转变到测量单个细胞的亲和力，再到测量软样品的顺应性。该仪器利用最常见的实验室设备之一-台式离心机，将极大地降低成本并提高性能和安全性。这种方法将显著降低研究人员进行单分子操纵实验的门槛，因为它几乎不需要技术专业知识，而且与许多其他方法相比，效率提高了1000倍，成本提高了10-100倍。这个项目意义重大，因为它将为许多新的研究人员和系统打开机械生物学和单分子操纵领域，加快发现的步伐。

DESCRIPTION (provided by applicant): From governing the adhesion and recruitment of leukocytes in the immune response to determining cell fate and tissue development, mechanical forces play a key regulatory role throughout biology. This emergent field of "mechanobiology" is leading to new understandings of disease such as bleeding disorders, cancer, osteoporosis and asthma, as we recognize that mechanics can play a large role in physiological responses at the molecular, cellular and organismic levels. Technological developments that enable precise manipulation of single molecules and cells (e.g. optical tweezers and AFM) have been a driving force in the development of the field. However, growth of the field is impeded by limited access to such technology as it can be expensive, technically challenging, and low-throughput. To overcome these challenges, we will develop a bold approach for applying controlled forces to microscopic samples with single-molecule precision that is inexpensive, simple to use, and high-throughput. By integrating a centrifuge and a microscope, we have demonstrated a prototype Centrifuge Force Microscope (CFM) to perform thousands of single-molecule force experiments in parallel. We propose to take this concept of single-molecule centrifugation to the next level, by developing a powerful, multi- purpose, benchtop instrument for mechanobiology that can be used by a variety of biomedical researchers. We will accomplish this by 1) Enabling massively parallel single-molecule measurements of structural transitions such as protein unfolding by integrating high-resolution imaging into the CFM; 2) Increasing the accessibility of single-molecule manipulation techniques by miniaturizing the high-resolution CFM to be incorporated directly into a standard benchtop centrifuge; 3) Demonstrating the versatility of this CFM by measuring intra- and inter-molecular bond strengths at both the single-molecule and single-cell levels. In summary, this project will result in an accessible, high-throughput and high-resolution new platform for measuring biological systems under mechanical force at the nanoscale with a broad range of applications, ranging from measuring structural transitions within individual molecules, to measuring the affinity of single cells, to measuring the compliance of soft samples. This instrument will dramatically reduce cost and improve performance and safety by leveraging one of the most common pieces of laboratory equipment - the benchtop centrifuge. This approach will remarkably lower the barrier for researchers to do single- molecule manipulation experiments by requiring little technical expertise and by offering a 1000 fold efficiency boost and a 10-100 fold cost improvement from many other methods. This project is significant since it will open up the fields of mechanobiology and single-molecule manipulation to many new researchers and systems, accelerating the pace of discovery.