Nanomechanics of biologically-relevant microtubule systems

描述（由申请人提供）：微管是真核细胞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新的或更有效的方法用于治疗各种常见疾病，如癌症，阿尔茨海默氏症或帕金森病。为此，将使用原子力显微镜（AFM）对微管进行研究，目的是将微管描述为非均相结构。AFM将用于高分辨率结构表征和局部力学表征的结合，并将研究分子尺度异质性与生物功能之间的关系。重点将放在两个不同的、与生物学相关的微管系统上：在核苷酸配体状态方面是异质的微管，以及具有相关蛋白tau的微管。将使用GTP的缓慢水解类似物GMPCPP来研究含有GDP和GTP离散区域的非均相微管，以创建分段微管。将对GDP和GMPCPP地区之间的差异进行详细研究。将特别关注GMPCPP和GDP区域之间的界面，以研究结构变化和晶格应变如何在界面上适应。将要研究的第二个微管系统是微管与相关蛋白tau，一种重要的微管相关蛋白，可促进微管生长并防止微管解聚。将检验Tau结合作为装配条件的函数，以评估两个假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，这些数据将与tau在每个结合位点的生物学功能有关。该项目将为评价微管的功能提供许多新的机会，因为它结合了分子水平的结构和力学表征，将微管作为异质和复杂结构进行检查，并研究与生理相关的微管系统。

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.