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Molecular Mechanisms of Spectraplakins

Spectraplakins 的分子机制

基本信息

批准号：
9118250
负责人：
Kevin C Slep
金额：
$ 7.29万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-08-01 至 2018-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9118250
关键词：
Actins Affect Architecture Axon Behavior Binding Biological Assay Brain Bulla C-terminal Cell Culture Techniques Cells Cytoskeleton Development Disease EF Hand Motifs Epitopes GAS2 gene Goals Growth Cones Health Impaired wound healing In Vitro Investigation Knowledge Life Maps Mediating Microfilaments Microtubule Stabilization Microtubules Modeling Molecular Mus Mutagenesis Mutate Mutation Nerve Degeneration Neurons Phenotype Play Plus End of the Microtubule Process Proteins Public Health Research Resolution Role Sedimentation process Series Skin Solvents Structure Surface System Testing Time Tubulin Work Wound Healing X-Ray Crystallography Zinc Fingers base brain malformation cell fixing cell motility crosslink fluorescence imaging in vitro activity insight migration mutant neuronal growth novel polarized cell reconstitution research study response retinal rods scaffold treatment strategy wound

项目摘要

DESCRIPTION (provided by applicant): The eukaryotic cytoskeleton is critical for cell migration. How the dynamic microtubule and actin cytoskeletal networks interact to coordinate polarized cell migration is poorly understood. Spectraplakins are key cytoskeletal cross-linkers that track microtubule plus ends and engage the leading edge actin network in migrating cells. Mutations in spectraplakins result in a wide spectrum of diseases including defective neuronal migration, brain malformations, neurodegeneration, and delayed wound healing. While the mechanism underlying spectraplakin binding to actin filaments is well established, how spectraplakins engage the microtubule cytoskeleton is poorly understood. Spectraplakins have a C-terminal EF-Hand-GAS2 microtubule- binding module and a proximal EB1-binding SxIP motif that confers microtubule plus end tracking. The architecture of the EF-Hand-GAS2 module, the identification of its microtubule-binding determinants, and an understanding of how the EF-Hand-GAS2-SxIP modules synergistically confer localization to leading edge microtubules remains to be determined. We hypothesize that the spectraplakin EF-Hand-GAS2 module forms a composite microtubule-binding structure that works synergistically with the EB1-binding SxIP motif to promote microtubule binding at the leading edge of migrating cells. Three series of experiments examine the structure, function and mechanism of the spectraplakin microtubule-binding module in cell migration. The first objective is to determine the atomic structure of the EF-Hand-GAS2 module using X-ray crystallography to elucidate how the EF-Hand and GAS2 domains collectively form a composite microtubule- binding structure. The second objective is to functionally map residues in the EF-Hand-GAS2 module involved in microtubule-binding and determine how the proximal EB1-binding SxIP module affects microtubule binding in vitro using microtubule dynamics reconstitution assays. The third objective is to characterize the synergistic behavior of the EF-Hand-GAS2 and SxIP modules in migrating cells. This investigation will use live cell and fixed cell fluorescence imaging to assay spectraplakin localization and cytoskeletal dynamics in motile cells. These three independent aims work to develop a multi-resolution model for spectraplakin microtubule-binding activity in migrating cells. The investigation's long term objective is to determine how spectraplakins regulate and coordinate microtubule and actin dynamics in neuronal growth cones. A mechanistic understanding of spectraplakins will enhance our knowledge of the inter-cytoskeletal coordination processes that underlie polarized neuronal migration and inform how spectraplakin mutations yield aberrant brain structure and neuronal connectivity. The proposed research will impact public health by establishing a mechanistic framework from which defective cell migration can be investigated, providing molecular insight into mutant spectraplakin phenotypes including delayed wound response, aberrant neuronal migration, and defective brain architecture.

描述(申请人提供)：真核细胞骨架是细胞迁移的关键。动态微管和肌动蛋白细胞骨架网络如何相互作用以协调极化细胞迁移的机制还知之甚少。斑点蛋白是关键的细胞骨架交联物，它跟踪微管和末端，并在迁移细胞中参与前沿肌动蛋白网络。光谱蛋白的突变会导致一系列疾病，包括神经元迁移缺陷、脑畸形、神经变性和伤口愈合延迟。虽然镜斑蛋白与肌动蛋白细丝结合的机制已经确定，但镜斑蛋白如何与微管细胞骨架结合却知之甚少。斑点蛋白有一个C-末端的EF-Hand-Gas2微管结合模块和一个近端的EB1结合的Sxip基序，它提供微管+末端跟踪。EF-Hand-Gas2模块的结构，其微管结合决定因素的鉴定，以及对EF-Hand-Gas2-Sxip模块如何协同赋予前沿微管定位的理解仍有待确定。我们假设spectraplakin EF-Hand-Gas2模块形成了一个复合的微管结合结构，它与EB1结合的Sxip基序协同作用，促进迁移细胞前沿的微管结合。三个系列的实验研究了微管结合模块在细胞迁移中的结构、功能和机制。第一个目标是用X射线结晶学确定EF-Hand-Gas2模块的原子结构，以阐明EF-Hand和Gas2结构域是如何共同形成复合微管结合结构的。第二个目标是在功能上定位参与微管结合的EF-Hand-Gas2模块中的残基，并通过微管动力学重建分析确定近端EB1结合Sxip模块在体外如何影响微管结合。第三个目标是表征EF-Hand-Gas2和Sxip模块在迁移细胞中的协同行为。这项研究将使用活细胞和固定细胞的荧光成像来检测运动细胞中的蛋白定位和细胞骨架动力学。这三个独立的目标致力于开发一个多分辨率的模型，用于研究迁移细胞中的spectraplakin微管结合活性。这项研究的长期目标是确定spectraplakins如何调节和协调神经元生长锥体中的微管和肌动蛋白动力学。从机制上理解spectraplakin将增强我们对细胞骨架间协调过程的了解，这些过程是极化神经元迁移的基础，并揭示spectraplakin突变如何产生异常的脑结构和神经元连接。这项拟议的研究将通过建立一个可以研究缺陷细胞迁移的机制框架来影响公共健康，提供对突变的特殊蛋白表型的分子洞察，包括创伤反应延迟、神经元异常迁移和大脑结构缺陷。