Nanomechanics of biologically-relevant microtubule systems

描述（由申请人提供）：微管是真核细胞中结构和组织机制的重要组成部分，对其功能的全面了解可能会导致治疗各种常见疾病（如癌症、阿尔茨海默病或帕金森病）的新方法或更有效的方法。为此，微管将使用原子力显微镜（AFM）进行研究，其目的是将微管描述为异质结构。AFM将用于高分辨率结构表征和局部机械表征的组合，并将检查分子尺度异质性和生物功能之间的关系。重点将放在两个不同的，生物学相关的微管系统：微管是异质性的核苷酸配体状态，和微管与相关的蛋白质tau。含有GDP和GTP的离散区域的异质微管将使用GTP的可缓慢水解的类似物GMPCPP来研究，以产生分段的微管。将对国内生产总值和GMPCPP地区之间的差异进行详细研究。将特别注意GMPCPP和GDP区域之间的接口，以检查如何在整个接口的结构变化和晶格应变被容纳。将研究的第二个微管系统是具有相关蛋白tau的微管，tau是一种重要的微管相关蛋白，其促进微管的生长并防止微管的解聚。Tau结合将作为组装条件的函数进行检查，以评估两个推定结合位点的相对重要性。将检查不同的结合位点以确定tau如何影响微管力学，并且这些数据将与每个结合位点中tau的生物学功能相关。该项目将提供许多新的机会来评估微管的功能，因为它结合了分子水平的结构和机械特性，检查微管作为异质和复杂的结构，并研究微管系统，生理相关。

DESCRIPTION (provided by applicant): Microtubules are an essential part of the structural and organizing machinery in eukaryotic cells, and a complete understanding of their function could potentially lead to new or more effective approaches in the treatment of a variety of common diseases such as cancer, Alzheimer's or Parkinson's. Toward this end, microtubules will be studied using atomic force microscopy (AFM) with the objective of describing microtubules as heterogeneous structures. AFM will be used for a combination of high-resolution structural characterization and local mechanical characterization, and the relationships between molecular-scale heterogeneity and biological function will be examined. The focus will be on two distinct, biologically-relevant microtubule systems: microtubules that are heterogeneous with respect to nucleotide ligand state, and microtubules with the associated protein tau. Heterogeneous microtubules containing discrete regions of GDP and GTP will be studied using a slowly-hydrolyzable analog of GTP, GMPCPP, to create segmented microtubules. A detailed study of the differences between the GDP and GMPCPP regions will be undertaken. Particular attention will be paid to the interface between GMPCPP and GDP regions to examine how structural changes and lattice strain are accommodated across the interface. The second microtubule system that will be studied is microtubules with the associated protein tau, an important microtubule associated protein that promotes growth and prevents depolymerization of microtubules. Tau binding will be examined as a function of assembly conditions to assess the relative importance of two putative binding sites. The different binding sites will be examined to determine how tau affects microtubule mechanics and these data will be related to the biological function of tau in each binding site. This project will provide many new opportunities to evaluate the functions of microtubules because it combines molecular-level structural and mechanical characterization, examines microtubules as heterogeneous and complex structures, and studies microtubule systems that are physiologically relevant.