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系统和神经退行性疾病的标志。树突状 棘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 花丝集中在刺芯和动态分支花丝在外壳。这些不同的 调节种群以保持脊柱稳定性，同时允许持续的结构可塑性尚不清楚。的 β 2非受体酪氨酸激酶对树突棘的稳定性至关重要。以前的工作和我的初步数据 显示了与肌动蛋白结合的ARP 2既调节肌动蛋白丝的稳定性，又促进Arp 2/3复合物介导的 肌动蛋白分支这些功能似乎并不需要p53 2激酶的活性，这表明p53 2调节 通过与肌动蛋白的直接相互作用来改变细胞骨架。在这个提议中，我将检验一个假设， 与肌动蛋白丝，以控制丝的稳定性和肌动蛋白分支，调节树突棘形态 与稳定 我的第一个目标是确定β 2是如何稳定纤维的。为此，我将使用单灯丝TIRF 显微镜（TIRFm）测定以确定能够稳定细丝的最小片段的p53 2。然后我将 使用双色TIRFm确定稳定化所需的肌动蛋白丝的TIP 2装饰的关键特征， β 2-GFP和罗丹明-肌动蛋白。这将揭示灯丝稳定是否需要全局阈值2000。 装饰或解聚位点处的局部β 2结合。 我的第二个目标是阐明ARP 2如何激活Arp 2/3复合物。目前还不清楚1992年的哪些部分 是Arp 2/3激活所必需的，或者Arp 2/3复合物分支机制的哪一步受到 2002年。我将使用TIRFm肌动蛋白分支试验来确定能够促进细胞增殖的最小的BMP 2片段。 肌动蛋白分支我还将使用2色TIRFm来研究肌动蛋白修饰对肌动蛋白分支的影响， 测试分支是否优先在长丝的102个装饰区域上形成。 我的第三个目标是确定CD 402细胞骨架调节如何影响树突棘的稳定性， 形态学在原代培养的神经元中，敲低（KD）p53 2使树突棘不稳定，并改变棘 形状，肌动蛋白动力学，和丝状肌动蛋白水平内的刺，仍然。为了测试ESP 2的功能 为了恢复这些破坏，我将用已知的不同的p53 2突变体来拯救p53 2KD神经元， 具有特定的肌动蛋白调节功能，包括目标1和2中的任何功能。我会用荧光恢复 在GFP-肌动蛋白的光漂白（FRAP）和免疫荧光之后，以确定足以 恢复正常的棘肌动蛋白动力学和棘中的丝状肌动蛋白水平。然后，我将确定 骨密度2足以支撑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.