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

基本信息

批准号：
10115123
负责人：
Anthony J Koleske
金额：
$ 39.37万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-06-01 至 2023-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10115123
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 organizational structure postnatal postsynaptic premature preservation protein function protein protein interaction recruit stoichiometry

项目摘要

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正确控制对于脊柱的长期稳定性至关重要，因为脊柱不稳定过早地出现精神和神经系统疾病。树突棘包含至少两个不同的池丝状- (F-) 肌动蛋白，一个小的稳定池，缓慢翻转并驻留在肌动蛋白的中央核心内脊柱，以及一个从中央核心延伸到脊柱外围的更大的动态池。什么机制控制这些不同的 F-肌动蛋白池，这些池如何与神经传递机制相互作用，以及如何它们有助于树突棘的可塑性和稳定性，是该领域尚未解决的基本问题。我们的实验室发现 Abl2/Arg 非受体酪氨酸激酶的破坏会导致广泛的产后树突棘不稳定和突触损失。 Arg 除了作为激酶外，还结合 F-肌动蛋白和皮质蛋白， Arg 和 cortactin 协同稳定 F-肌动蛋白并通过 Arp2/3 激活 F-肌动蛋白分支成核复杂的。我们假设 Arg 将 cortactin 招募到刺上，促进其与 F-肌动蛋白的结合，并且与 Arg 一起 Cortactin 维持稳定的 F-肌动蛋白池以稳定脊柱。我们将在三个目标中检验这一假设：我们的首要目标是确定 cortactin 与 F-肌动蛋白结合的分子基础。我们假设 cortactin 结合 F-肌动蛋白的能力对于调节 F-肌动蛋白动力学和介导树突棘稳定性至关重要，但我们完全缺乏对 cortactin 如何结合 F-肌动蛋白的高分辨率理解。我们发现皮质素重复序列（CR）结构域在溶液中自然展开，但我们可以获得适合高通量的 CR:F-肌动蛋白复合物。使用冷冻电镜分辨率结构测定。我们还将同时使用 CR 域截断氢-氘交换质谱法绘制 cortactin:F-肌动蛋白结合界面处的残基图。我们的第二个目标是阐明精氨酸和皮质蛋白如何相互作用来控制肌动蛋白丝动力学。这 Arg 和 cortactin 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 Arg 和 cortactin 相互作用，在体外控制 F-肌动蛋白和新肌动蛋白分支成核的稳定性。我们将使用基于全内反射显微镜的蛋白质：蛋白质相互作用的测量肌动蛋白丝测定，以及通过冷冻电镜进行结构测定，以了解精氨酸和皮质蛋白如何相互作用彼此之间以及 Arp2/3 复合体调节 F-肌动蛋白稳定性和肌动蛋白分支成核。我们的第三个目标是阐明稳定肌动蛋白池在树突棘基础设施和稳定性中的作用。我们的初步数据表明，稳定的 F-肌动蛋白池可以通过充当中央组织者来稳定脊柱脊柱基础设施的破坏和减弱 NMDA 受体 (NMDAR) 活性。我们将在培养的神经元中使用 Arg 或 Cortactin 突变体的敲低/互补策略来揭示它们的肌动蛋白是否需要调节和/或其他功能来维持脊柱稳定的 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的破坏程度稳定的 F-肌动蛋白池会影响脊柱内关键肌动蛋白调节因子和子区室的组织。