Cytoskeletal Roles of APC in Synaptic Remodeling

APC 在突触重塑中的细胞骨架作用

• 批准号: 10394599
• 负责人: Colby P Fees
• 金额: $ 2.42万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2022-08-26
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10394599
• 关键词: APC gene; Actins; Affect; Binding; Binding Proteins; Biochemistry; Brain; COX7A2L Protein; Calcium; Cellular biology; Cognition; Cognition Disorders; Collaborations; Complex; Cytoskeleton; Data; Defect; Dendrites; Dendritic Spines; Epilepsy; Event; Exhibits; Feedback; Gene Proteins; Goals; Growth; Hippocampus (Brain); Human; Image; Impairment; In Vitro; Infantile spasms; Invaded; Learning; Length; Letters; Mediating; Memory; Microtubules; Mitochondria; Modeling; Molecular; Morphogenesis; Morphology; Mus; Mutagenesis; Mutation; Nervous System Physiology; Nervous system structure; Neuroglia; Neurologic; Neuronal Plasticity; Neurons; Pathway interactions; Phenotype; Play; Plus End of the Microtubule; Point Mutation; Porifera; Proteins; Regulation; Research; Role; Seizures; Signal Transduction; Spasm; Stimulus; Structure; Synapses; System; Testing; Time; Training; Tumor Suppressor Proteins; Universities; Vertebral column; Visit; Wisconsin; Work; cell motility; conditional knockout; density; directional cell; in vitro activity; in vivo Model; in vivo evaluation; live cell microscopy; monomer; mutant; nervous system disorder; recruit; relating to nervous system; response; single molecule

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.

神经可塑性是大脑在细胞水平上响应刺激而改变结构的能力;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侵入树突棘并刺激肌动蛋白依赖性棘重塑以响应突触活动。这表明微管正末端递送和/或激活局部诱导肌动蛋白在脊柱中组装的因子。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结肠息肉病（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-肌动蛋白协调的作用尚未研究。APC在树突棘中富集，并通过末端结合蛋白EB 3在微管的生长端运输到神经元中。我的初步数据表明，EB 3直接抑制APC介导的肌动蛋白装配结合APC的“基本结构域”。其他研究表明，耗尽APC或EB 3减少了成熟树突棘的数量，这与APC和EB 3共同调节肌动蛋白依赖性棘重塑一致。这个建议的目标是阐明APC介导的肌动蛋白组装的作用，其负调控EB 3，在神经元分支和树突棘形态发生。这将通过在培养的原代小鼠海马神经元中使用消除APC介导的肌动蛋白组装的现有显性突变体（APCm 4）和新产生的对EB 3抑制无效的APC突变体进行活体成像来实现。神经元形态的变化，以及肌动蛋白和微管的水平和动力学将与APC定位固定和活细胞显微镜。这些工作将通过与Erik Dent教授（威斯康星州大学麦迪逊分校）的密切合作来促进，他的实验室将被访问，以进行神经元细胞骨架动态实时成像的实践培训。同时，单分子生物化学将被用来定义EB 3抑制APC介导的肌动蛋白组装在体外的机制。这一信息将用于测试两种体内模型，以了解EB 3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”，该模型假设入侵的微管吸收EB 3，释放/激活APC以促进肌动蛋白组装和棘重塑;（2）“递送模型”，其假设APC介导的肌动蛋白组装有助于将富含EB 3的微管募集到棘，导致APC抑制，以及伴随的可用肌动蛋白单体库向分支肌动蛋白成核剂系统（Arp 2/3复合物）的转变，从而导致脊柱重塑。这项工作将揭示神经元细胞生物学的基本机制，这与我们对人类认知和神经系统疾病的机械理解高度相关，其中这些通路被改变。