Using cryo-electron tomography and live-cell fluorescent imaging to study the role of cofilin in regulating neuronal filopodial structure and dynamics

利用冷冻电子断层扫描和活细胞荧光成像研究丝切蛋白在调节神经元丝状伪足结构和动力学中的作用

• 批准号: 10586225
• 负责人: MATTHEW SWULIUS
• 金额: $ 43.54万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-15 至 2028-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10586225
• 关键词: Actin-Binding Protein; Actins; Appearance; Architecture; Behavior; Binding; Binding Proteins; Brain; Cell membrane; Cells; Cellular Structures; Chemicals; Complex; Crosslinker; Cryo-electron tomography; Cytoskeletal Proteins; Cytoskeleton; Data; Development; Distal; Exclusion; F-Actin; Filament; Filopodia; Fluorescence; Goals; Growth Cones; Health; Hippocampus; Homologous Gene; Human; Immunofluorescence Immunologic; In Situ; Ischemia; LIM Domain Kinase 1; Length; Link; Mechanics; Microfilaments; Modification; Molecular; Molecular Structure; Natural regeneration; Nerve Tissue; Neurites; Neurons; Neuropil; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Play; Pliability; Process; Property; Protein Dephosphorylation; Proteins; Resolution; Role; Running; Signal Pathway; Stains; Structure; Testing; Time; Traction; Translating; base; cell motility; cofilin; crosslink; experimental study; extracellular; fascin; flexibility; fluorescence imaging; imaging modality; injury recovery; live cell imaging; macromolecular assembly; mechanical signal; nanoarchitecture; nanometer resolution; neuron regeneration; novel; pharmacologic; receptor

Project Summary/Abstract How neurons guide their processes to the correct binding partner is a complicated task, but is critical during development and recovery from injury. It involves the highly coordinated action of many cytoskeletal proteins and their binding partners within the growth cone at the tips of extending neurites, as they feel their way through the neuropil. There is a lot known about the signaling pathways that regulate neurite outgrowth and turning, but the details of how molecular structures come together to achieve growth cone behavior are still unclear. This project will initially focus on the structure of bundled cofilactin filaments (cofilin-decorated F-actin) in situ, and how this novel filament structure and fascin cross-linking determine filopodial dynamics. Here we propose that filopodial behavior is governed partially by the transition from the fascin cross-linked to a cofilin cross-linked filaments, that makes filopodial bundles more pliable. Experiments are focused around three aims: 1) to study the high-resolution structure of fascin- and cofilin-linked actin bundles to determine their impact on actin structure, 2) to determine how changes in fascin and cofilin concentration regulate filopodial dynamics and structure, and 3) determine how LIMKI and SSH1 form the core of a bidirectional regulatory mechanism for regulating actin architecture via tuning the phosphorylation state of Ser3 on cofilin.

项目总结/摘要 神经元如何将它们的过程引导到正确的结合伴侣是一项复杂的任务，但在神经元的发育过程中至关重要。 从受伤中恢复和发展。它涉及许多细胞骨架蛋白的高度协调作用 和它们的结合伙伴在生长锥内的尖端延伸的神经突，因为它们感觉到他们的方式通过 神经系统关于调节神经突生长和转向的信号通路，我们知道很多，但是， 分子结构如何结合在一起以实现生长锥行为的细节仍然不清楚。这 该项目最初将专注于原位成束的cofilactin细丝（cofilin修饰的F-肌动蛋白）的结构， 这种新的丝状结构和肌成束蛋白交联如何决定丝状伪足动力学。在此，我们建议， 丝状伪足的行为部分地受从肌束蛋白交联到cofilin交联的转变所控制 花丝，使丝状伪足束更柔韧。实验主要围绕三个目标：1）研究 fascin和cofilin连接的肌动蛋白束的高分辨率结构，以确定它们对肌动蛋白的影响 结构，2）确定肌成束蛋白和切丝蛋白浓度的变化如何调节丝状伪足动力学， 结构，以及3）确定LIMKI和SSH 1如何形成双向调节机制的核心， 通过调节cofilin上Ser 3的磷酸化状态来调节肌动蛋白结构。