Cytoskeletal Roles of APC in Synaptic Remodeling

神经可塑性是指大脑在细胞水平上对刺激作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，微管的动态正端已被证明可以侵入树突棘，并刺激肌动蛋白依赖性脊柱重构以响应突触活动。这表明微管正端传递和/或激活局部诱导脊柱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是难以捉摸的。肿瘤抑制蛋白腺瘤性息肉大肠杆菌（APC）与微管和肌动蛋白结合，Goode实验室最近的研究表明，APC可以有效地形成肌动蛋白组装的核，并且需要协调肌动蛋白和微管动力学，并允许定向细胞迁移。然而，APC在神经系统中促进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在树突棘中富集，并通过末端结合蛋白EB3在微管生长正端的神经元中运输。我的初步数据显示，EB3通过结合APC“基本结构域”直接抑制APC介导的肌动蛋白组装。其他研究表明，消耗APC或EB3都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性脊柱重塑的观点一致。本研究的目的是阐明apc介导的肌动蛋白组装及其由EB3负调控在神经元分支和树突棘形态发生中的作用。这将通过使用现有的显性突变体（APCm4）在培养的原代小鼠海马神经元中进行活体成像来实现，该突变体可以消除APC介导的肌动蛋白组装，以及新产生的APC突变体对EB3的抑制。在固定细胞和活细胞显微镜下，神经元形态学的变化，以及肌动蛋白和微管的水平和动态变化将与APC定位相关。这些努力将通过与埃里克·登特教授（威斯康星大学麦迪逊分校）的强有力合作来促进，他的实验室将被访问，进行神经元细胞骨架动力学实时成像的实践培训。同时，单分子生物化学将用于确定EB3在体外抑制apc介导的肌动蛋白组装的机制。这些信息将用于在体内测试两种模型，以了解EB3调节APC介导的肌动蛋白组装如何影响树突棘：(1)“海绵模型”，假设入侵的微管吸收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有助于将富含eb3的微管招募到脊柱，导致APC抑制，并伴随可用的肌动蛋白单体池向分支肌动蛋白成核系统（Arp2/3复合物）转移，这是脊柱重塑的基础。这项工作将揭示神经细胞生物学的基本机制，这与我们对这些通路改变的人类认知和神经系统疾病的机制理解高度相关。

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.