Molecular Mechanisms of Spectraplakins

 描述（由申请人提供）：真核细胞骨架对细胞迁移至关重要。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还知之甚少。Spectraplakins是关键的细胞骨架交联剂，其跟踪微管正末端并在迁移细胞中接合前缘肌动蛋白网络。spectraplakins的突变导致多种疾病，包括神经元迁移缺陷、脑畸形、神经变性和伤口愈合延迟。尽管spectraplakin与肌动蛋白丝结合的机制已经很好地建立，但spectraplakin如何参与微管细胞骨架却知之甚少。Spectraplakins具有C-末端EF-Hand-GAS 2微管结合模块和近端EB 1结合SxIP基序，其赋予微管加末端追踪。EF-Hand-GAS 2模块的结构、其微管结合决定簇的鉴定以及EF-Hand-GAS 2-SxIP模块如何协同地赋予前沿微管定位的理解仍有待确定。我们假设，spectraplakin EF-Hand-GAS 2模块形成了一个复合微管结合结构，与EB 1结合SxIP基序协同作用，以促进微管结合在迁移细胞的前沿。三个系列的实验研究的结构，功能和细胞迁移中的spectraplakin微管结合模块的机制。第一个目标是使用X射线晶体学确定EF-Hand-GAS 2模块的原子结构，以阐明EF-Hand和GAS 2结构域如何共同形成复合微管结合结构。第二个目标是功能地图中的EF-Hand-GAS 2模块参与微管结合的残基，并确定如何近端EB 1结合SxIP模块影响微管结合在体外使用微管动力学重建测定。第三个目的是表征EF-Hand-GAS 2和SxIP模块在迁移细胞中的协同行为。本研究将使用活细胞和固定细胞荧光成像来分析 运动细胞中斑点蛋白定位和细胞骨架动力学。这三个独立的目标的工作，以开发一个多分辨率模型的spectraplakin微管结合活动的迁移细胞。这项研究的长期目标是确定spectraplakins如何调节和协调神经生长锥中的微管和肌动蛋白动力学。对spectraplakins的机械理解将增强我们对细胞骨架间协调过程的认识，这些协调过程是极化神经元迁移的基础，并告知spectraplakin突变如何产生异常的脑结构和神经元连接。拟议的研究将通过建立一个机制框架来影响公共卫生，从该框架中可以研究缺陷细胞迁移，为突变体spectraplakin表型提供分子见解，包括延迟伤口反应，异常神经元迁移和有缺陷的大脑结构。

 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.