Control of Dendritic Spine Stability via Regulation of a Stable Actin Pool

通过稳定肌动蛋白库的调节控制树突棘稳定性

• 批准号: 10365989
• 负责人: Anthony J Koleske
• 金额: $ 39.38万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-06-01 至 2023-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10365989
• 关键词: Actins; Affinity; Attenuated; Binding; Biochemistry; Biological Assay; Brain; Cells; Collection; Complement; Complex; Cryoelectron Microscopy; Cytoskeleton; Data; Dendritic Spines; Deuterium; EMS1 gene; Excitatory Synapse; Exhibits; F-Actin; Filament; Hippocampus (Brain); Hydrogen; In Vitro; Infrastructure; Length; Maintenance; Major Depressive Disorder; Maps; Mass Spectrum Analysis; Measurement; Measures; Mediating; Mental disorders; Microfilaments; Microscopy; Molecular; Mus; Mutation; N-Methyl-D-Aspartate Receptors; Neurons; Phosphotransferases; Protein Tyrosine Kinase; Proteins; Proteolysis; Regulation; Resolution; Role; Schizophrenia; Stable Populations; Structure; Synapses; Testing; Vertebral column; Work; alpha Actin; base; cohort; density; experimental study; interdisciplinary approach; knock-down; monomer; mutant; nervous system disorder; neural circuit; neurotransmission; novel; postnatal; postsynaptic; premature; preservation; protein function; protein protein interaction; recruit; stoichiometry

Proper control of the actin cytoskeleton is critical for long-term stability of spines, which are destabilized prematurely in psychiatric and neurological disorders. Dendritic spines contain at least two distinct pools of filamentous- (F-) actin, a small stable pool that turns over slowly and resides within the central core of the spine, and a larger dynamic pool that extends from the central core to the spine periphery. What mechanisms control these distinct F-actin pools, how the pools interface with the neurotransmission machinery, and how they contribute to dendritic spine plasticity and stability are fundamental unresolved questions in the field. Our lab discovered that disruption of the Abl2/Arg nonreceptor tyrosine kinase causes widespread postnatal dendritic spine destabilization and synapse loss. In addition to being a kinase, Arg binds F-actin and cortactin, and Arg and cortactin synergize to stabilize F-actin and activate F-actin branch nucleation by the Arp2/3 complex. We hypothesize that Arg recruits cortactin to spines, promotes its binding to F-actin, and together Arg and cortactin maintain the stable F-actin pool to stabilize spines. We will test this hypothesis in three Aims: Our first aim will define the molecular basis for cortactin binding to F-actin. We hypothesize that cortactin's ability to bind F-actin is essential for it to regulate F-actin dynamics and mediate dendritic spine stability, but we completely lack a high-resolution understanding of how cortactin binds F-actin. We find that the cortactin repeats (CR) domain is natively unfolded in solution, but we can obtain CR:F-actin complexes suitable for high resolution structure determination using cryo-EM. We will also use CR domain truncations in tandem with hydrogen-deuterium exchange mass spectrometry to map residues at the cortactin:F-actin binding interface. Our second aim will elucidate how Arg and cortactin interact to control actin filament dynamics. The mechanisms by which Arg and cortactin maintain the stable pool of F-actin in dendritic spines are unknown. We find that Arg and cortactin interact to control the stability of F-actin and new actin branch nucleation in vitro. We will use measurements of protein:protein interactions, total internal reflection microscopy-based single actin filament assays, and structure determination via cryo-EM to understand how Arg and cortactin interact with each other and the Arp2/3 complex to regulate F-actin stability and actin branch nucleation. Our third aim will elucidate the role of the stable actin pool in dendritic spine infrastructure and stability. Our preliminary data suggest that the stable F-actin pool may stabilize spines both by acting as a central organizer of spine infrastructure and by attenuating NMDA receptor (NMDAR) activity. We will use a knockdown/complementation strategy with Arg or cortactin mutants in cultured neurons to reveal whether their actin regulatory and/or other functions are required to maintain the spine's stable F-actin pool, regulate NMDARs, and spine stability. We will also use live cell and super-resolution microscopy to measure how disruption of the stable F-actin pool impacts the organization of key actin regulators and subcompartments within the spine.

肌动蛋白细胞骨架的适当控制对于不稳定的棘的长期稳定性至关重要 过早地出现精神和神经疾病。树突棘含有至少两个不同的池 丝状-（F-）肌动蛋白，一个小的稳定的池，翻转缓慢，并驻留在中央核心的 脊柱，以及从中心核心延伸到脊柱外围的更大的动态池。哪些机制 控制这些不同的F-肌动蛋白池，池如何与神经传递机制接口，以及如何 它们有助于树突棘的可塑性和稳定性，这是本领域尚未解决的基本问题。 我们的实验室发现，在出生后， 树突棘不稳定和突触丧失。除了作为激酶外，Arg还结合F-肌动蛋白和cornein， Arg和coronin协同作用稳定F-肌动蛋白，并通过Arp 2/3激活F-肌动蛋白分支成核 复杂.我们推测，精氨酸招募coronin棘，促进其结合F-肌动蛋白，并一起精氨酸 和coronin维持稳定的F-肌动蛋白池以稳定棘。我们将在三个目标中检验这一假设： 我们的第一个目标是确定皮质素结合F-肌动蛋白的分子基础。我们假设coronann 结合F-肌动蛋白的能力对于它调节F-肌动蛋白动力学和介导树突棘稳定性是必不可少的，但是我们 完全缺乏对Cordiform如何结合F-肌动蛋白的高分辨率理解。我们发现， 重复序列（CR）结构域在溶液中天然未折叠，但我们可以获得适合于高浓度的CR：F-肌动蛋白复合物。 分辨率结构测定使用cryo-EM。我们还将使用CR结构域截短串联， 氢-氘交换质谱法以绘制core-binding：F-肌动蛋白结合界面处的残基。 我们的第二个目标将阐明精氨酸和coronin如何相互作用，以控制肌动蛋白丝动力学。的 Arg和coronin维持树突棘中F-肌动蛋白稳定池的机制尚不清楚。 我们发现，精氨酸和coronin相互作用，以控制稳定的F-肌动蛋白和新的肌动蛋白分支成核在体外。 我们将使用测量蛋白质：蛋白质相互作用，全内反射显微镜为基础的单 肌动蛋白丝测定，并通过冷冻电镜结构测定，以了解精氨酸和cornein如何相互作用 与Arp 2/3复合物相互作用以调节F-肌动蛋白稳定性和肌动蛋白分支成核。 我们的第三个目标将阐明树突棘的基础设施和稳定性的稳定肌动蛋白池的作用。我们 初步数据表明，稳定的F-肌动蛋白库可能通过充当中心组织者和细胞外基质， 脊髓基础设施和衰减NMDA受体（NMDAR）的活动。我们将在培养的神经元中使用敲低/互补策略，用Arg或coronin突变体来揭示它们的肌动蛋白 需要调节和/或其它功能来维持脊柱的稳定F-肌动蛋白库，调节NMDAR， 和脊柱稳定性。我们还将使用活细胞和超分辨率显微镜来测量细胞分裂是如何发生的。 稳定的F-肌动蛋白库影响脊柱内关键肌动蛋白调节子和亚区室的组织。