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

纳米生物学中的机械力：从止血到单分子离心

• 批准号: 10413060
• 负责人: Wesley Philip Wong
• 金额: $ 48.68万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10413060
• 关键词: Address; Adhesions; Adhesives; Area; Behavior; Biochemical; Biological Assay; Biology; Blood Coagulation Disorders; Cells; Centrifugation; Communicable Diseases; Complex; DNA; Development; Devices; Disease; Genomics; Growth; Hair Cells; Hearing; Hemostatic function; Immune response; Immunology; Leukocytes; Life; Link; Malignant Neoplasms; Measurement; Measures; Mechanics; Methods; Microscope; Molecular; Movement; Organism; Pathway interactions; Play; Problem Solving; Process; Property; Protein Conformation; Proteins; Regulation; Research Personnel; Robotics; Role; Shapes; Spectrum Analysis; Structure; Technology; Tissues; base; centrifuge force microscope; deafness; driving force; high throughput screening; insight; instrument; instrumentation; interest; mechanical force; mechanotransduction; nanoscale; nanoswitch; programs; response; screening; single molecule; tool; von Willebrand Factor

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.

抽象的 机械力在整个生物学中发挥着关键作用，从控制免疫系统中白细胞的粘附到 反应，决定细胞命运并指导组织形成。机械生物学这一领域提供了至关重要的 对出血性疾病、癌症和传染病等疾病的深入了解，这些情况正在变得越来越清楚 传统的生化和基因组特征不足以理解丰富的行为 生命系统或它们如何失败。相反，我们必须揭示力如何改变物体的结构和功能 分子，触发机械转导途径来改变细胞反应。技术发展 能够精确操纵单分子和细胞一直是发展的驱动力 该领域的发展受到阻碍，因为获得此类技术的机会有限，而且这些技术的限制 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方法，这些方法将（i）开辟新领域 通过引入新功能来进行研究，以及（ii）使单分子和纳米级大众化 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做出发现。我们将继续 开发诸如离心力显微镜之类的仪器，这是一种安装在 台式离心机即使非专业人员也能进行高通量单分子力分析 测量和纳米级设备，例如可编程 DNA 纳米开关。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 实现单分子蛋白质识别和形状确定。我们还将开发功能性 基于交互的纳米开关发现（FIND），一种不基于传统方法的高通量筛选方法 机器人技术，但在分子设备上将分子成分聚集在一起进行分析和筛选 兴趣互动。 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。 我们将应用纳米级方法来回答机械生物学中的关键开放问题。例如，要 为了揭示止血的机械调节，我们将使用单分子方法来研究力- 冯维勒布兰德因子的调节酶促裂解，及其流诱导的延伸和激活 粘合功能。我们还将通过使用单项研究来研究听力和耳聋的分子基础。 分子力光谱法探测毛细胞尖端连接的特性，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基础的分子运动。总体而言，这些 努力应该牢固地将力量确立为理解生命基本过程的关键参数，并且 为理解和治疗疾病提供了新的思路。