Molecular Mechanisms of Cytoskeletal Regulators

描述（由申请人提供）：微管细胞骨架是一种动态支架，用于促进细胞内极化运输、细胞迁移和有丝分裂纺锤体的形成。微管是a -微管蛋白异二聚体的聚合物。微管具有内在的动力学，受GTP水解在微管蛋白的可交换核苷酸位点的调节。微管相关蛋白的宿主调节聚合物的动态在空间和时间上。微管动力学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体。TOG结构域是在两个调节微管动力学的保守蛋白家族中发现的微管蛋白结合结构域，由成员XMAP215和CLASP定义。在两个家族中，TOG结构域都是排列的；然而，XMAP215和CLASP分别调节微管动力学，促进聚合和暂停。TOG结构域结合微管蛋白的机制以及排列的TOG结构域调节微管动力学的作用仍有待确定。该研究提出了一个假设，即排列的TOG结构域提供了多个微管蛋白/微管结合位点，这种多价结构与TOG类特异性决定因素相结合，是XMAP215和CLASP差异调节微管动力学的机制的核心。三个系列的实验考察了TOG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射线晶体学在原子分辨率下定义XMAP215和CLASP蛋白家族的TOG结构域类的结构和独特特征。第二个目标是确定单个和排列的TOG结构域的微管和微管结合能力，并绘制微管蛋白结合决定因素。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生成tog -微管蛋白复合物的模型。第三个目标是确定TOG结构域阵列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野生型TOG蛋白被耗尽并被荧光标记的截断、突变或嵌合结构所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TOG结构域的排列机制，以单独或与其他微管相关蛋白一起调节微管动力学。对TOG结构域机制及其如何在不同蛋白质家族中被利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（包括各种神经病、纤毛病、非整倍体和癌症）中所起作用的理解。

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.