Mechanical forces in nanoscale biology: From hemostasis to single-molecule centrifugation

摘要 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。 决定细胞命运和指导组织形成的反应。这一机械生物学领域提供了至关重要的 对出血性疾病、癌症和传染病等情况的洞察，在这些情况下，情况变得越来越清楚 传统的生化和基因组特征不足以理解这种丰富的行为 关于生命系统或它们是如何失效的。相反，我们必须发现力是如何改变人的结构和功能的 分子，触发机械转导通路来改变细胞反应。技术发展 能够精确操纵单个分子和细胞的技术一直是发展的动力 但由于获得此类技术的机会有限，以及它们的限制，增长一直受到阻碍 能力，这限制了可以解决的科学问题的类型。 为了克服这些挑战，我们将开发机械生物学的方法，这些方法将(I)开辟新的领域 通过引入新的能力进行研究，以及(2)使单分子和纳米级民主化 方法，以便所有生物医学研究人员都可以使用这些强大的工具进行发现。我们将继续 为了开发离心力显微镜等仪器，离心力显微镜是一种微型显微镜，可以安装在 台式离心机，即使是非专业人员也能执行高通量单分子力 测量，以及可编程DNA纳米开关等纳米级设备。我们将开发DNA 纳米开关卡尺，一种能够以埃级精度测量单分子距离的工具 以实现单分子蛋白质鉴定和形状确定。我们还将开发功能 基于相互作用的纳米开关发现(FIND)，一种不基于传统方法的高通量筛选方法 机器人，但在分子设备上，将分子成分聚集在一起进行分析和筛选 感兴趣的互动。Find将能够筛选复杂的作用模式，以找到激活的化合物 特定的下游途径或变构稳定特定的蛋白质构象。 我们将应用我们的纳米级方法来回答机械生物学中的关键开放问题。例如，要 揭示止血的机械调节，我们将用单分子方法研究止血的力- 调节的von Willebrand因子的酶裂解及其流动诱导的延长和激活 粘合功能。我们还将利用单细胞培养技术研究听力和耳聋的分子基础。 分子力谱来探测毛细胞尖端链接的性质，并将这种方法与 单通道电导测量，可同时测量打开所需的力 机械转导通道和通道门控下的分子运动。总的来说，这些 应坚定地将力量确立为理解生命基本过程的关键参数，以及 为理解和治疗疾病提供了一个新的句柄。

Abstract Mechanical forces play key roles throughout biology, from governing the adhesion of leukocytes in the immune response, to determining cell fate and directing tissue formation. This field of mechanobiology is providing vital insights into conditions 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 changes the structure and function of molecules, triggering mechanotransduction pathways to modify cell responses. Technological developments that enable precise manipulation of single molecules and cells have been a driving force in the development of the field, but growth has been impeded by both limited access to such technologies and by constraints in their capabilities, which has restricted the types of scientific questions that can be addressed. To overcome these challenges, we will develop approaches in mechanobiology that will (i) open up new areas of study through the introduction of new capabilities, and (ii) democratize single-molecule and nanoscale methods so that all biomedical researchers can make discoveries using these powerful tools. We will continue to develop instruments such as the Centrifuge Force Microscope, a miniature microscope that fits into a benchtop centrifuge to enable even non-specialists to perform high-throughput single-molecule force measurements, and nanoscale devices such as programmable DNA nanoswitches. We will develop DNA nanoswitch calipers, a tool capable of measuring distances on single-molecules with angstrom-level precision to enable single-molecule protein identification and shape determination. We will also develop Functional Interaction-based Nanoswitch Discovery (FIND), a high-throughput screening assay based not on traditional robotics, but on molecular devices that bring together molecular components to analyze and screen for interactions of interest. FIND will enable screening of complex modes of action to find compounds that activate a specific downstream pathway or allosterically stabilize a particular protein conformation. We will apply our nanoscale approaches to answer key open questions in mechanobiology. For example, to uncover the mechanical regulation of hemostasis we will use single-molecule methods to study the force- regulated enzymatic cleavage of von Willebrand factor, and the flow-induced elongation and activation of its adhesive function. We will also investigate the molecular basis of hearing and deafness by using single- molecule force spectroscopy to probe the properties of the hair cell tip link, and combine this approach with single-channel conductance measurements to simultaneously measure the force required to open mechanotransduction channels and the molecular movements that underlie channel gating. 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.