VASP ubiquitination regulates actin dynamics in dendritic spines

VASP 泛素化调节树突棘肌动蛋白动力学

• 批准号: 10240485
• 负责人: Laura E McCormick
• 金额: $ 3.75万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10240485
• 关键词: Actins; Affect; Axon; Binding; Biochemical; Biochemistry; Biological Assay; Bundling; Cells; Cellular biology; Chemicals; Collaborations; Complex; Cytoskeleton; Data; Defect; Dendrites; Dendritic Spines; Development; Environment; Family; Filament; Filopodia; Fluorescence Microscopy; Growth Factor; Hippocampus (Brain); Impairment; In Vitro; Individual; Label; Learning; Maintenance; Mediating; Microfilaments; Microscopy; Modification; Morphology; Mus; Neurites; Neurodevelopmental Disorder; Neurons; Neurosciences; Plus End of the Actin Filament; Polymerase; Polymers; Process; Prosencephalon; Proteins; Regulation; Role; Shapes; Structure; Synapses; Synaptic Transmission; Synaptic plasticity; Syndrome; Testing; Ubiquitination; Work; axon guidance; base; density; experimental study; genetic regulatory protein; in vitro activity; monomer; neuron development; polymerization; prevent; profilin; single molecule; synaptic function; ubiquitin-protein ligase; vasodilator-stimulated phosphoprotein

ABSTRACT Filopodia are dynamic, actin-rich structures that extend outward from the cell and explore the local environment. In neurons, filopodia are critical for numerous stages of development, including neuritogenesis, axon guidance, and dendritic spine formation. Defects in any of these stages of neuronal development can result in improper synaptic connectivity, neurodevelopmental disorders, and psychiatric syndromes. The Ena/VASP of actin polymerizes is well appreciated to localize to the filopodial tip complex and influence actin dynamics. Recently, the Gupton lab showed VASP transiently co-localizes with the E3 ubiquitin ligase TRIM9 at the filopodial tip. TRIM9 was required for the reversible, non-degradative ubiquitination of VASP and this modification was associated with decreases in filopodia stability and number. Appropriate control of filopodial dynamics is critical for several neuronal processes, yet we still do not understand how VASP is regulated at the filopodial tip. My central hypothesis that VASP-Ub negatively regulates actin dynamics in filopodia and dendritic spines. To test this hypothesis, I will chemically ubiquitinate and fluorescently label purified VASP protein. Using various biochemical assays, I will define the impact of ubiquitination on VASP tetramerization and VASP-mediated regulation of actin dynamics. Mechanistically understanding the impact of VASP ubiquitination in vitro is critical in understanding the cellular role of ubiquitinated VASP (VASP-Ub). Furthermore, I will examine the localization and function of VASP-Ub in dendritic spines. Previous work has shown that VASP is required for proper dendritic spine formation and my preliminary data shows that VASP-Ub localizes to the post-synaptic density. I will examine VASP and VASP-Ub abundance and localization during dendritic spine maturation and chemically induced plasticity will be analyzed in forebrains and cultured cortical and hippocampal neurons isolated from wild-type and Trim9-/- mice using both microscopy-based and biochemical assays. These experiments will determine how ubiquitination regulates VASP activity, and consequently actin polymerization and synaptic plasticity, in dendritic spines. Appropriate control of the cytoskeleton is critical for dendritic spine formation, and synaptic transmission, yet it is still unknown how actin dynamics are controlled in these specialized neuronal structures. Working at the interface of biochemistry, cell biology, and neuroscience, I will determine how non- degradative ubiquitination regulates the actin polymerase VASP and shapes neuronal morphology and function.

抽象的 丝状伪足是动态的、富含肌动蛋白的结构，从细胞向外延伸并探索局部环境。 在神经元中，丝状伪足对于许多发育阶段都至关重要，包括神经突发生、轴突引导、 和树突棘的形成。神经元发育的任何这些阶段的缺陷都可能导致不适当的 突触连接、神经发育障碍和精神综合征。肌动蛋白的 Ena/VASP 聚合被广泛认为定位于丝状尖端复合物并影响肌动蛋白动力学。最近， Gupton 实验室表明 VASP 与 E3 泛素连接酶 TRIM9 短暂地共定位于丝状足尖端。 VASP 的可逆、非降解泛素化需要 TRIM9，这种修饰是 与丝状伪足稳定性和数量的减少有关。适当控制丝状伪足动力学至关重要 对于多个神经元过程，但我们仍然不了解 VASP 在丝状伪足尖端是如何调节的。我的 中心假设是 VASP-Ub 负向调节丝状伪足和树突棘中的肌动蛋白动力学。测试 根据这个假设，我将对纯化的 VASP 蛋白进行化学泛素化和荧光标记。使用各种 生化检测，我将定义泛素化对 VASP 四聚化和 VASP 介导的影响 肌动蛋白动力学的调节。从机制上理解 VASP 泛素化的体外影响至关重要 了解泛素化 VASP (VASP-Ub) 的细胞作用。此外，我将检查本地化 以及 VASP-Ub 在树突棘中的功能。先前的工作表明，VASP 是适当树突所必需的 脊柱形成和我的初步数据表明 VASP-Ub 定位于突触后密度。我会 检查树突棘成熟和化学过程中 VASP 和 VASP-Ub 的丰度和定位 将在前脑和培养的皮质和海马神经元中分析诱导的可塑性 使用基于显微镜和生化检测的野生型和 Trim9-/- 小鼠。这些实验将 确定泛素化如何调节 VASP 活性，从而调节肌动蛋白聚合和突触 可塑性，在树突棘中。细胞骨架的适当控制对于树突棘的形成至关重要，并且 突触传递，但仍不清楚肌动蛋白动力学如何在这些特殊的神经元中受到控制 结构。在生物化学、细胞生物学和神经科学的交叉领域工作，我将确定如何非 降解泛素化调节肌动蛋白聚合酶 VASP 并塑造神经元形态和功能。