Regulation of the actin cytoskeleton by Abl2 and its role in dendritic spine stability

Abl2 对肌动蛋白细胞骨架的调节及其在树突棘稳定性中的作用

• 批准号: 10241272
• 负责人: Josie Bircher
• 金额: $ 2.15万
• 依托单位: YALE UNIVERSITY
• 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 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10241272
• 关键词: Actins; Behavioral; Binding; Biochemical; Biological Assay; Biophysics; Brain; C-terminal; Collection; Color; Complex; Cytoskeleton; Data; Dendritic Spines; Dependence; EMS1 gene; Excitatory Synapse; F-Actin; Filament; Fluorescence Recovery After Photobleaching; Immunofluorescence Immunologic; Individual; Learning; Measures; Mediating; Memory; Mental disorders; Microfilaments; Modeling; Molecular Conformation; Morphology; Mus; Neurodegenerative Disorders; Neurons; Nucleotides; Pathology; Patients; Phosphotransferases; Population; Protein Tyrosine Kinase; Regulation; Rhodamine; Role; Sedimentation process; Shapes; Site; Structure; Synapses; Testing; Time; Total Internal Reflection Fluorescent; Vertebral column; Visualization; Work; base; burden of illness; depolymerization; emerging adult; experimental study; flexibility; knock-down; mutant; nervous system disorder; postnatal; stoichiometry

Project Summary Disruption of dendritic spine stability is a hallmark of neurological and neurodegenerative disorders. Dendritic spines are supported by an underlying actin framework consisting of at least two distinct actin pools, with stable filaments concentrated in the spine core and dynamic branched filaments in the outer shell. How these distinct populations are regulated to maintain spine stability while allowing ongoing structural plasticity is unclear. The Abl2 nonreceptor tyrosine kinase is essential for dendritic spine stability. Previous work and my preliminary data show that Abl2 binding to actin both regulates actin filament stability and promotes Arp2/3 complex-mediated actin branching. These functions to not appear to require Abl2 kinase activity, suggesting that Abl2 modulates the cytoskeleton via direct interactions with actin. In this proposal, I will test the hypothesis that direct interactions of Abl2 with actin filaments, to control filament stability and actin branching, regulate dendritic spine morphology and stability. My first aim is to determine how Abl2 stabilizes filaments. In this aim, I will use single filament TIRF microscopy (TIRFm) assays to determine the minimal fragment of Abl2 capable of stabilizing filaments. I will then determine key features of Abl2 decoration of actin filaments required for stabilization using 2-color TIRFm with Abl2-GFP and Rhodamine-actin. This will reveal if filament stabilization requires a threshold of global Abl2 decoration or local Abl2 binding at the site of depolymerization. My second aim is to elucidate how Abl2 activates the Arp2/3 complex. It is not known what parts of Abl2 are required for Arp2/3 activation or which step of the Arp2/3 complex branching mechanism is impacted by Abl2. I will use TIRFm actin-branching assays to determine the minimal fragment of Abl2 capable of promoting actin branching. I will also use 2-color TIRFm to study the effects of Abl2 actin decoration on actin branching, testing if branches form preferentially on Abl2 decorated regions of filament. My third aim is to determine how Abl2 cytoskeletal regulation impacts dendritic spine stability and morphology. Knockdown (KD) of Abl2 in primary cultured neurons destabilizes dendritic spines, and alters spine shape, actin dynamics, and filamentous actin levels within the spines that remain. To test the functions of Abl2 required to restore these disruptions, I will rescue Abl2KD neurons with different mutants of Abl2 known to possess specific actin-regulating functions, including any found in Aims 1 and 2. I will use fluorescence recovery after photobleaching (FRAP) of GFP-actin and immunofluorescence to determine Abl2 functions sufficient to restore proper spine actin dynamics and filamentous actin levels in spines. I will then identify which functions of Abl2 are sufficient to support normal spine shape and long-term stability.

项目摘要 树突状脊柱稳定性的破坏是神经和神经退行性疾病的标志。树突状 刺由至少两个不同的肌动蛋白池组成的基础肌动蛋白框架支持，稳定 细丝集中在脊柱核心和外壳中的动态分支细丝中。这些如何与众不同 调节种群以维持脊柱稳定性，同时允许持续的结构可塑性尚不清楚。这 ABL2非受体酪氨酸激酶对于树突状脊柱稳定性至关重要。以前的工作和我的初步数据 表明ABL2与肌动蛋白结合既调节肌动蛋白丝稳定性并促进ARP2/3复合介导 肌动蛋白分支。这些功能似乎不需要ABL2激酶活动，表明ABL2调节 通过与肌动蛋白直接相互作用的细胞骨架。在此提案中，我将测试直接互动的假设 带有肌动蛋白丝的ABL2，以控制细丝稳定性和肌动蛋白分支，调节树突状脊柱形态 和稳定性。 我的第一个目的是确定ABL2如何稳定细丝。在此目标中，我将使用单细丝tirf 显微镜（TIRFM）测定法，以确定能够稳定细丝的ABL2的最小片段。然后我会 确定使用2色TIRFM与 ABL2-GFP和Rhodamine-Actin。这将揭示灯丝稳定是否需要全球ABL2的阈值 在去聚合部位的装饰或局部ABL2结合。 我的第二个目标是阐明ABL2如何激活ARP2/3复合物。不知道ABL2的哪些部分 ARP2/3激活或ARP2/3复合分支机构的哪个步骤受到所需 ABL2。我将使用TIRFM肌动蛋白分支测定法来确定能够促进的ABL2的最小片段 肌动蛋白分支。我还将使用2色TIRFM研究ABL2肌动蛋白装饰对肌动蛋白分支的影响， 测试是否优先在ABL2装饰的细丝区域上形成分支。 我的第三个目的是确定ABL2细胞骨架调节如何影响树突状脊柱稳定性和 形态学。原代培养神经元中ABL2的敲低（KD）破坏了树突状刺，并改变了脊柱 刺内的形状，肌动蛋白动力学和丝状肌动蛋白水平。测试ABL2的功能 需要恢复这些干扰，我将挽救具有不同ABL2突变体已知的ABL2KD神经元 具有特定的肌动蛋白调节功能，包括AIMS 1和2中发现的任何功能。我将使用荧光回收 GFP-肌动蛋白和免疫荧光的光漂白（FRAP）后，确定ABL2的功能足以使 恢复刺中适当的脊柱肌动蛋白动力学和丝状肌动蛋白水平。然后，我将确定哪些功能 ABL2足以支持正常的脊柱形状和长期稳定性。