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

项目摘要 树突棘稳定性的破坏是神经和神经退行性疾病的标志。树枝状 脊柱由一个基本的肌动蛋白框架支持，该框架由至少两个不同的肌动蛋白池组成，具有稳定的 细丝集中在脊骨核心，动态分枝细丝集中在外壳。如何区分这些不同 种群被调节以维持脊柱的稳定性，同时允许持续的结构可塑性尚不清楚。这个 ABL2非受体酪氨酸激酶对树突棘的稳定性是必不可少的。以前的工作和我的初步数据 结果表明，Abl2与肌动蛋白结合既调节肌动蛋白细丝稳定性，又促进Arp2/3复合体介导 肌动蛋白分支。这些功能似乎不需要Abl2激酶的活性，这表明Abl2调节 通过与肌动蛋白直接相互作用的细胞骨架。在这个提议中，我将检验这样一个假设，即直接交互 Abl2与肌动蛋白细丝结合，控制细丝稳定性和肌动蛋白分支，调节树突棘形态 和稳定性。 我的第一个目标是确定Abl2如何稳定细丝。在这个目标中，我将使用单丝TIRF 显微镜(TIRFM)分析确定能够稳定细丝的ABL2的最小片段。那我会的 用双色TIRFM确定稳定所需肌动蛋白细丝的ABL2修饰的关键特征 ABL2-GFP和罗丹明-肌动蛋白。这将揭示灯丝稳定是否需要全局Abl2的阈值 在解聚部位修饰或局部结合Abl2。 我的第二个目的是阐明Abl2如何激活Arp2/3复合体。目前尚不清楚Abl2的哪些部分 是Arp2/3激活所必需的，或者Arp2/3复杂分支机制的哪个步骤受到影响 ABL2.我将使用TIRFM肌动蛋白分支分析来确定ABL2能够促进 肌动蛋白分支。我还将使用双色TIRFM来研究Abl2肌动蛋白修饰对肌动蛋白分支的影响， 测试是否优先在花丝的Abl2装饰区域形成分支。 我的第三个目标是确定ABL2细胞骨架调节如何影响树突棘的稳定性和 形态学。原代培养神经元中Abl2基因的敲除(KD)使树突棘不稳定，并改变了脊椎 形状、肌动蛋白动态和残留脊椎内的丝状肌动蛋白水平。测试Abl2的功能 为了恢复这些干扰，我将用已知的不同的Abl2突变体来拯救Abl2KD神经元 具有特定的肌动蛋白调节功能，包括目标1和目标2中发现的任何功能。我将使用荧光回收 在光漂白(FRAP)GFP-肌动蛋白和免疫荧光后确定ABL2功能足以 恢复适当的脊椎肌动蛋白动力学和脊椎中的丝状肌动蛋白水平。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。

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.