Molecular Mechanisms of Cytoskeletal Regulators

描述(申请人提供)：微管细胞骨架是一种动态支架，用于促进极化的细胞内运输、细胞迁移和有丝分裂纺锤体的形成。微管是微管蛋白杂二聚体的聚合物。微管具有固有的动力学，受微管蛋白的可交换核苷酸位置的GTP水解性调节。许多微管相关蛋白在空间和时间上调节聚合物的动力学。微管动力学缺陷导致多种疾病，包括但不限于神经变性、痉挛截瘫和非整倍体。TOG结构域是在两个保守的蛋白质家族中发现的微管蛋白结合域，它们调节微管的动力学，由成员XMAP215和CLASP定义。在这两个家族中，TOG结构域都被发现排列；然而，XMAP215和CLAP不同地调节微管动力学，分别促进聚合和暂停。TOG结构域用于结合微管蛋白的机制以及排列的TOG结构域对微管动力学的调节作用仍有待确定。这一建议发展了这样的假设，即排列的TOG结构域提供了多个微管/微管结合部位，这种与TOG类特异性决定因素结合的多价结构是XMAP215和CLAP差异调节微管动力学机制的核心。三个系列的实验考察了TOG结构域的结构和功能，以确定阵列TOG机构的多分辨率模型。第一个目标是使用X射线结晶学在原子分辨率下定义XMAP215和CLASP蛋白家族中TOG结构域类的结构和独特功能。第二个目标是确定单个和排列的TOG结构域的微管蛋白和微管结合能力，并绘制微管蛋白结合决定因素图。这项检查将使用体外微管和微管结合试验以及F“rster共振能量转移试验来产生TOG-微管蛋白复合体的模型。第三个目标是确定排列的TOG结构域用于调节体内微管动力学的机制。这项研究将使用活细胞荧光成像来监测当野生型TOG蛋白被耗尽并被荧光标记的截断、突变或嵌合构建体取代时的微管动力学。这项研究的长期目标是在原子水平上确定排列的TOG结构域单独以及与其他微管相关蛋白一起调节微管动力学的机制。深入了解TOG结构域的机制及其在不同蛋白质家族中的应用，将有助于我们更好地理解微管动力学及其在人类健康和疾病中的作用，包括各种神经疾病、纤毛疾病、非整倍体和癌症。 与公共健康相关：微管细胞骨架是一种动态的、极化的细胞支架，促进细胞内运输、纤毛发生、细胞迁移和有丝分裂；所有过程都依赖于微管聚合物调节的动态不稳定性。微管动力学缺陷表现在一系列影响人类健康的疾病中，包括痉挛截瘫、无脑、纤毛疾病和异倍体；同时，微管动力学是化疗的关键靶点，因为抑制微管动力学，因此有丝分裂是防止与癌症相关的快速细胞分裂的一种手段。这项拟议的研究旨在研究TOG结构域蛋白的分子机制及其在调节微管动力学中所起的作用；该研究将通过建立一个基本的机制框架来影响公众健康，在这个框架下，健康的微管动力学可以被正确地检查异常的表现并制定治疗策略。

DESCRIPTION (provided by applicant): The microtubule cytoskeleton is a dynamic scaffold used to facilitate polarized intracellular transport, cell migration and formation of the mitotic spindle. Microtubules are polymers of a¿-tubulin heterodimers. The microtubule has inherent dynamics regulated by GTP hydrolysis in ¿-tubulin's exchangeable nucleotide site. A host of microtubule associated proteins regulate the polymer's dynamics both spatially and temporally. Defects in microtubule dynamics result in a wide spectrum of diseases including, but not limited to neurodegeneration, spastic paraplegia and aneuploidy. TOG domains are tubulin binding domains found in two conserved protein families that regulate microtubule dynamics, defined by members XMAP215 and CLASP. Across both families, TOG domains are found arrayed; however XMAP215 and CLASP differentially regulate microtubule dynamics, promoting polymerization and pause respectively. The mechanism TOG domains use to bind tubulin and the role arrayed TOG domains play to modulate microtubule dynamics remains to be determined. This proposal develops the hypothesis that arrayed TOG domains provide multiple tubulin/microtubule binding sites and this multivalent architecture coupled with TOG class-specific determinants is central to the mechanism by which XMAP215 and CLASP differentially regulate microtubule dynamics. Three series of experiments examine the structure and function of TOG domains to determine a multi- resolution model for arrayed TOG mechanism. The first objective is to define, at atomic resolution, the structure and unique features of TOG domain classes across the XMAP215 and CLASP protein families using X-ray crystallography. The second objective is to ascertain the tubulin and microtubule binding capacity of individual and arrayed TOG domains and map tubulin binding determinants. This examination will use in vitro tubulin and microtubule binding assays as well as a F"rster resonance energy transfer assay to generate a model of the TOG-tubulin complex. The third objective is to determine the mechanism arrayed TOG domains use to modulate microtubule dynamics in vivo. This study will use live cell fluorescence imaging to monitor microtubule dynamics when the wild-type TOG protein has been depleted and replaced with a fluorescently- labeled truncated, mutant or chimeric construct. The long term objectives of this investigation are to determine at the atomic level, the mechanism arrayed TOG domains employ to modulate microtubule dynamics individually and in concert with other microtubule associated proteins. A fundamental understanding of TOG domain mechanism and how this is utilized in different protein families will enhance our understanding of microtubule dynamics and the role it plays in human health and disease including various neuropathies, ciliopathies, aneuploidy and cancer. PUBLIC HEALTH RELEVANCE: The microtubule cytoskeleton is a dynamic, polarized cellular scaffold that facilitates intracellular transport, ciliogenesis, cell migration and mitosis; all processes that rely on the regulated dynamic instability of the microtubule polymer. Defects in microtubule dynamics manifest in a range of disorders that effect human health including spastic paraplegia, lissencephaly, ciliopathies and aneuploidy; at the same time, microtubule dynamics is a key target for chemotherapeutics as inhibition of microtubule dynamics, and thus mitosis, is a means to prevent the rapid cell division associated with cancer. The proposed research aims to examine the molecular mechanism of TOG domain-containing proteins and the role they play in modulating microtubule dynamics; the research will impact public health by establishing a fundamental mechanistic framework under which healthy microtubule dynamics operates, from which aberrant manifestations can be properly examined and therapeutic strategies developed.