Cytoskeletal Roles of APC in Synaptic Remodeling

神经可塑性是大脑在细胞水平上对刺激做出反应而改变结构的能力；这种神经元形态的动态重塑是学习和记忆的基础。调节神经元形态需要动态微管和肌动蛋白细胞骨架之间的紧密协调。在神经元中，微管的动态加端被证明可以侵入树突棘，并刺激依赖肌动蛋白的脊椎重塑，以响应突触活动。这表明微管加端传递和/或激活局部诱导脊椎肌动蛋白组装的因子。然而，这种微管-肌动蛋白串扰的机制仍然难以捉摸。肿瘤抑制蛋白腺瘤性息肉病结肠腺样息肉病(APC)与微管和肌动蛋白结合，Goode实验室最近的工作表明，APC有效地使肌动蛋白组装成核，并需要协调肌动蛋白和微管的动力学，允许细胞定向迁移。然而，APC在促进神经系统内肌动蛋白组装和微管-肌动蛋白协调中的作用尚未被研究。APC富含树突棘，并通过末端结合蛋白EB3在微管生长的正端运输神经元。我的初步数据显示，EB3通过与APC的“基本结构域”结合，直接抑制APC介导的肌动蛋白组装。其他研究表明，耗尽APC或EB3会减少成熟树突棘的数量，这与APC和EB3共同调节肌动蛋白依赖的脊柱重塑的情况是一致的。该建议的目的是阐明APC介导的肌动蛋白组装的作用，以及EB3对其在神经元分支和树突棘形态发生中的负调控作用。这将通过在培养的原代小鼠海马神经元中使用现有的取消APC介导的肌动蛋白组装的显性突变体(APCm4)和新产生的对EB3抑制折射的APC突变体来实现。神经元形态的变化，以及肌动蛋白和微管的水平和动态将与固定细胞显微镜和活细胞显微镜下的APC定位相关。这些努力将得到与Erik Dent教授(威斯康星大学麦迪逊分校)的大力合作，他的实验室将被访问，进行神经元细胞骨架动态实时成像的动手培训。同时，单分子生化将被用来确定EB3在体外抑制APC介导的肌动蛋白组装的机制。这些信息将被用来在体内测试两个模型，以了解EB3对APC介导的肌动蛋白组装的调节如何影响树突棘：(1)“海绵模型”，假设侵袭的微管吸收EB3，释放/激活APC以促进肌动蛋白组装和脊柱重塑；(2)“传递模型”，假设APC介导的肌动蛋白组装帮助将富含EB3的微管招募到脊柱，导致APC抑制，以及随之而来的可用的肌动蛋白单体池向作为脊柱重塑基础的分支-肌动蛋白核系统(Arp2/3复合体)的转移。这项工作将揭示神经细胞生物学的基本机制，这与我们对人类认知和神经疾病的机械理解高度相关，在这些疾病中，这些途径被改变。

Neuroplasticity is the ability of the brain to change structure at the cellular level in response to stimulus; this dynamic remodeling of neuronal morphology is the basis of learning and memory. Regulating neuronal morphology requires tight coordination between the dynamic microtubule and actin cytoskeletons. In neurons, the dynamic plus-ends of microtubules have been shown to invade dendritic spines and stimulate actin- dependent spine remodeling in response to synaptic activity. This indicates that microtubule plus-ends deliver and/or activate factors that locally induce actin assembly in the spine. However, the mechanisms underlying this microtubule-actin crosstalk has remained elusive. The tumor suppressor protein adenomatous polyposis coli (APC) binds to microtubules and actin, and recent work from the Goode lab has shown that APC potently nucleates actin assembly and is required to coordinates actin and microtubule dynamics and allow directional cell migration. However, the role of APC in promoting actin assembly and microtubule-actin coordination in the nervous system has not been investigated. APC is enriched in dendritic spines and is trafficked in neurons on the growing plus ends of microtubule by the end-binding protein EB3. My preliminary data show that EB3 directly inhibits APC-mediated actin assembly by binding to the APC `Basic domain'. Other studies have shown that depleting either APC or EB3 diminishes the number of mature dendritic spines, consistent with APC and EB3 working together to regulate actin-dependent spine remodeling. The goals of this proposal are to elucidate the role of APC-mediated actin assembly, and its negative regulation by EB3, in neuronal branching and dendritic spine morphogenesis. This will be achieved by live-imaging in cultured primary mouse hippocampal neurons using an existing dominant mutant (APCm4) that abolishes APC- mediated actin assembly, and newly generated APC mutants refractive to EB3 inhibition. Changes in neuronal morphology, as well as the levels and dynamics of actin and microtubules will be correlated with APC localization by fixed- and live-cell microscopy. These efforts will be facilitated by a strong collaboration with Prof. Erik Dent (University of Wisconsin Madison), whose lab will be visited for hands-on training in live imaging of cytoskeleton dynamics in neurons. In parallel, single-molecule biochemistry will be used to define the mechanism by which EB3 inhibits APC-mediated actin assembly in vitro. This information will be used to test two models in vivo for how EB3 regulation of APC-mediated actin assembly influences dendritic spines: (1) The `sponge model', which postulates that invading microtubules soak up EB3, releasing/activating APC to promote actin assembly and spine remodeling; (2) The `delivery model', which postulates that APC-mediated actin assembly helps to recruit EB3-rich microtubules to spines, leading to APC inhibition, and an accompanying shift in the available actin monomer pool to a branched-actin nucleator system (Arp2/3 complex) underlying spine remodeling. This work will uncover fundamental mechanisms of neuronal cell biology, which are highly relevant to our mechanistic understanding of human cognition and neurological diseases in which these pathways are altered.