Nanomechanics of Tubulin Extraction from Microtubules and Adhesin Catch-Bond Rupture

细胞的形状和结构是由微管组成的丝状网络支持的，微管是被称为微管蛋白二聚体的构建块的动态组合，根据细胞的需要不断重塑。这些组件在细胞分裂和组织中起着重要作用，并作为货物运输的“轨道”。细胞机制可以从微管中提取微管蛋白二聚体来分解这些组件，但是这样做所需的力和微管的整体稳定性是未知的。了解这一过程将有助于了解突变如何导致具有生理后果的微管缺陷，以及了解紫杉醇等抗癌药物如何发挥作用。该项目的技术开发将通过在单个微管蛋白二聚体的水平上直接测量稳定程度，从而有助于更好地表征紫杉醇等化合物。这个项目的跨学科性质将为高中生、本科生和研究生提供一个极好的培训机会，并将整合到课程中，其中包括动手实验部分。本项目将建立一种单分子力谱分析方法，通过拉动β微管蛋白的c端尾部，从微管（MT）中提取微管蛋白。先进的原子力显微镜（AFM）研究将表征微管蛋白提取的纳米力学特性，使用各种拉伸协议和改进的AFM悬臂，以提高力精度和时间分辨率。对所得数据的分析将得出使微管蛋白稳定在MT晶格内的能量学和力。它还将深入了解切断酶的机制，这些酶对提取微管蛋白所需的力起作用。切断酶是提取微管蛋白二聚体的分子马达，是调控微管蛋白动力学的重要手段。切断酶的机制尚不清楚。相关的六聚体分子马达的最大作用力为~ 20-30 pN。然而，粗粒度模拟预测提取微管蛋白二聚体需要更高的力（~400 pN）。该项目将通过直接测量从MT中提取单个微管蛋白二聚体所需的力来解决这种二分法，这是一个长期追求的目标。为了促进这一分析的发展，几种粘附配体对的机械特性将被表征为优化其作为短基因编码手柄的使用并评估其捕获键行为的手段。因此，该项目将加速基于afm的各种生物系统的力谱研究，包括膜蛋白和核酸。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Cell shape and structure are supported by a filamentous network composed in part by microtubules, dynamic assemblies of building blocks known as tubulin dimers, that are constantly remodeled according to the cell’s needs. These assemblies play important roles in cell division and organization and serve as “tracks” for the transport of cargo. The assemblies can be disassembled by cellular machinery that extracts a tubulin dimer from the microtubule, but the force required to do so and the overall stability of the microtubule is unknown. Understanding this process will provide insight in how mutations lead to microtubule defects that have physiological consequences, and to understanding how cancer drugs like Taxol exert their effects. This project’s technology development will facilitate better characterization of compounds such as Taxol by enabling direct measurement of the degree of stabilization at the level of a single tubulin dimer. The interdisciplinary nature of this project will provide an excellent training opportunity for high-school, undergraduate, and graduate students and will be integrated into a course, with a hands-on lab component.This project will establish a single-molecule force-spectroscopy assay to extract tubulin from a microtubule (MT) by pulling on the C-terminal tail of β tubulin. Advanced atomic force microscopy (AFM) studies will characterize the nanomechanical properties of tubulin extraction using a variety of pulling protocols and modified AFM cantilevers for improved force precision and temporal resolution. Analysis of the resulting data will yield the energetics and forces that stabilize tubulin within the MT lattice. It will also yield insight into the mechanism of severing enzymes, which act against the force needed to extract tubulin. Severing enzymes are molecular motors that extract tubulin dimers from MTs and are an important means to modulate MT dynamics. The mechanism of severing enzymes remains unknown. Related hexameric molecular motors exert a maximum force of ~20–30 pN. Yet, coarse-grained simulations predict a much higher force (~400 pN) needed to extract a tubulin dimer. This project will resolve this dichotomy by directly measuring the force needed to extract an individual tubulin dimer from a MT, a long-sought goal. To facilitate this assay development, the mechanical properties of several adhesin-ligand pairs will be characterized as a means to optimize their use as short, genetically encoded handles and evaluate their catch bond behavior. This project will thereby accelerate AFM-based force-spectroscopy studies of diverse biological systems including membrane proteins and nucleic acids.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.