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

机械力在整个生物学中起着关键作用，从控制免疫系统中白细胞的粘附， 反应，决定细胞命运和组织发育。这个新兴的“机械生物学”领域是 为出血性疾病、癌症和传染病等疾病提供重要见解， 越来越清楚的是，传统的生物化学和基因组表征不足以理解 生命系统的丰富行为或它们如何失败。相反，我们必须揭示力是如何改变 分子的结构和功能，并触发机械转导途径以改变细胞反应。 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AFM）已经成为该领域发展的驱动力。然而，该领域的增长是 由于获得这些技术的机会有限，这些技术可能昂贵，技术上具有挑战性， 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，我们将开发高通量和可访问的新方法， 机械生物学将（i）通过引入新的能力开辟新的研究领域，以及（ii） 使单分子力测量民主化，以便所有生物医学研究人员都能做出发现 使用这些强大的工具。例如，我们将加速单分子测量， 一种几乎所有生物医学研究人员都已经拥有的仪器：台式离心机。通过开发一个 微型显微镜，适合到一个标准的离心机桶，我们将创造一个方便和廉价的 台式仪器，将带来高通量单分子操作的非专业人士，提供了一个 1000倍的效率提升和10-100倍的成本改善超过许多其他方法。我们还将开发 自组装DNA纳米级装置，促进群体异质性的单分子研究，以及 这使得无需仪器的力谱法成为可能。重要的是，这些项目将打开机械领域， 生物学和单分子操纵的新的研究人员和系统，加快发现的步伐。 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）免疫应答中的粘附分子，和（iii） 机械传导和听力和耳聋的分子基础。例如，我们将执行 利用单分子离心的平行力测量来研究力调节的酶 切割血管性血友病因子，并研究与血管性血友病相关的突变， 常见的遗传性出血性疾病我们还将研究白细胞的细胞粘附， 听力和耳聋的分子基础。总的来说，这些努力应牢固地确立武力是一个关键， 理解生命的基本过程的参数，并提供了一个新的处理，既理解- 和治疗疾病。

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.