Mechancial forces in nanoscale biology: from hemostasis to single-molecule centrifugation

机械力在整个生物学中发挥着关键作用，从控制免疫中白细胞的黏附。 对决定细胞命运和组织发育的反应。这个新兴的“机械生物学”领域是 提供对出血性疾病、癌症和传染病等疾病的重要见解 越来越清楚的是，传统的生化和基因组特征不足以理解 生命系统的丰富行为或它们如何失败。相反，我们必须发现武力是如何改变 分子的结构和功能，并触发机械转导通路来改变细胞的反应。 能够精确操纵单个分子和细胞的技术发展(例如，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，油田的增长是 受制于获取此类技术的机会有限，因为这些技术可能昂贵、具有技术挑战性，而且 吞吐量。这些挑战也限制了可以解决的科学问题的类型。 为了克服这些挑战，我们将开发高吞吐量和可访问的新方法 将(I)通过引入新的能力开辟新的研究领域，以及(Ii) 使单分子作用力测量大众化，以便所有生物医学研究人员都能发现 使用这些强大的工具。例如，我们将通过以下方式加速单分子测量 几乎所有生物医学研究人员都已经拥有的一种仪器：台式离心机。通过开发一种 可以放入标准离心机桶中的微型显微镜，我们将创造一种方便、廉价的 台式仪器将为非专业人员带来高通量的单分子操作，提供 与许多其他方法相比，效率提高1000倍，成本降低10-100倍。我们还将发展 自组装DNA纳米设备，促进种群异质性的单分子研究，以及 使无仪器作用力光谱学成为可能。值得注意的是，这些项目将开辟机械领域-- 生物学和单分子操纵给新的研究人员和系统，加快了发现的步伐。 此外，我们将应用我们的单分子方法来回答机械生物学中的关键开放问题。 关于(I)止血的机械调节，(Ii)免疫反应中的黏附分子，以及(Iii) 机械转导和听力和耳聋的分子基础。例如，我们将表演 用单分子离心法进行大规模平行力测量研究力调节酶 切割von Willebrand因子，并调查与von Willebrand病相关的突变，最 常见的遗传性出血性疾病。我们还将研究白细胞的细胞黏附，并调查 听力和耳聋的分子基础。总体而言，这些努力应牢固地将部队确立为关键 参数，为理解生命的基本过程提供了一个新的句柄- 以及治疗--疾病。

Mechanical forces play key roles throughout biology, from governing the adhesion of leukocytes in the immune response, to determining cell fate and tissue development. This emergent field of "mechanobiology" is providing vital insights into diseases such as bleeding disorders, cancer, and infectious diseases, where it is becoming clear that conventional biochemical and genomic characterizations are not sufficient to understand the rich behavior of living systems or how they fail. Rather, we must uncover how force can change the structure and function of molecules, and trigger mechanotransduction pathways to modify cell responses. 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 technologies as they can be expensive, technically challenging, and low- throughput. These challenges have also limited the types of scientific questions that can be addressed. To overcome these challenges, we will develop high-throughput and accessible new approaches in mechanobiology that will (i) open up new areas of study through the introduction of new capabilities, and (ii) democratize single-molecule force measurements so that all biomedical researchers can make discoveries using these powerful tools. For example, we will accelerate single-molecule measurements by building upon an instrument that almost all biomedical researchers already have: the benchtop centrifuge. By developing a miniature microscope that fits into a standard centrifuge bucket, we will create an accessible and inexpensive benchtop instrument that will bring high-throughput single-molecule manipulation to non-specialists, offering a 1000 fold efficiency boost and 10-100 fold cost improvement over many other methods. We will also develop self-assembled DNA nanoscale devices that facilitate single-molecule studies of population heterogeneity, and that enable instrument-free force spectroscopy. Significantly, these projects will open the fields of mechano- biology and single-molecule manipulation to new researchers and systems, accelerating the pace of discovery. Additionally, we will apply our single-molecule approaches to answer key open questions in mechanobiology regarding (i) the mechanical regulation of hemostasis, (ii) adhesion molecules in the immune response, and (iii) mechanotransduction and the molecular basis for hearing and deafness. For example, we will perform massively-parallel force measurements using single-molecule centrifugation to study force-regulated enzymatic cleavage of von Willebrand factor, and investigate mutations related to von Willebrand Disease, the most common inheritable bleeding disorder. We will also study cellular adhesion of leukocytes, and investigate the molecular basis of hearing and deafness. Overall, these efforts should firmly establish force as a key parameter for understanding the basic processes of life, and provide a new handle for both understanding— and treating—disease.