Molecular Mechanisms of Cytoskeletal Regulators

描述（由申请方提供）：微管细胞骨架是一种动态支架，用于促进极化细胞内转运、细胞迁移和有丝分裂纺锤体形成。微管是α-微管蛋白异源二聚体的聚合物。微管具有内在的动力学，受微管蛋白可交换核苷酸位点中GTP水解的调节。大量的微管相关蛋白在空间和时间上调节聚合物的动力学。微管动力学的缺陷导致广泛的疾病，包括但不限于神经变性、痉挛性截瘫和非整倍性。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结构域，其调节微管动力学，由成员XMAP 215和CLASP定义。在这两个家族中，TOG结构域被发现排列;然而XMAP 215和CLASP差异调节微管动力学，分别促进聚合和暂停。TOG结构域用于结合微管蛋白的机制和排列的TOG结构域在调节微管动力学中的作用仍有待确定。这一建议发展的假设，排列TOG结构域提供多个微管蛋白/微管结合位点，这种多价的架构加上TOG类特异性决定因素是中央的XMAP 215和CLASP差异调节微管动力学的机制。三个系列的实验研究TOG结构域的结构和功能，以确定阵列TOG机制的多分辨率模型。第一个目标是使用X射线晶体学以原子分辨率定义XMAP 215和CLASP蛋白家族中TOG结构域类的结构和独特特征。第二个目标是确定个别和排列的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簇。该检查将使用体外微管蛋白和微管结合测定以及F“rster共振能量转移测定来产生TOG-微管蛋白复合物的模型。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。本研究将使用活细胞荧光成像来监测当野生型TOG蛋白已被耗尽并被荧光标记的截短、突变或嵌合构建体取代时的微管动力学。本研究的长期目标是在原子水平上确定排列的TOG结构域用于单独调节微管动力学以及与其他微管相关蛋白协同调节微管动力学的机制。对TOG结构域机制以及如何在不同蛋白质家族中使用的基本理解将增强我们对微管动力学及其在人类健康和疾病中所起作用的理解，包括各种神经病，纤毛病，非整倍体和癌症。 公共卫生相关性：微管细胞骨架是一种动态的、极化的细胞支架，其促进细胞内运输、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的调节动态不稳定性。微管动力学的缺陷表现在一系列影响人类健康的疾病中，包括痉挛性截瘫、无脑畸形、纤毛病和非整倍性;同时，微管动力学是化疗的关键靶点，因为抑制微管动力学，因此抑制有丝分裂是防止与癌症相关的快速细胞分裂的手段。拟议的研究旨在研究含TOG结构域的蛋白质的分子机制及其在调节微管动力学中的作用;该研究将通过建立健康微管动力学运作的基本机制框架来影响公共卫生，从中可以正确检查异常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.