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

对肌动蛋白细胞骨架的适当控制对脊柱的长期稳定至关重要，因为脊柱是不稳定的。 过早地出现精神和神经紊乱。树突棘至少包含两个不同的池 丝状-(F-)肌动蛋白，一个小的稳定的池，缓慢地翻转，驻留在 脊椎，以及从中央核心延伸到脊椎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交互，以及如何 它们促进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出生后 树突棘失稳和突触丢失。除了作为一种激酶，Arg还与F-肌动蛋白和皮质动蛋白结合， Arg和Cortactin通过Arp2/3协同稳定F-肌动蛋白并激活F-肌动蛋白分支成核 很复杂。我们假设Arg将皮质蛋白招募到脊柱上，促进其与F-肌动蛋白的结合，并共同作用于Arg 而Cortactin维持稳定的F-肌动蛋白池，以稳定脊柱。我们将从三个方面检验这一假设： 我们的第一个目标是确定皮质肌动蛋白与F-肌动蛋白结合的分子基础。我们假设Cortactin的 结合F-肌动蛋白的能力对于它调节F-肌动蛋白动力学和调节树突棘稳定性是必不可少的，但我们 完全缺乏对皮质肌动蛋白如何与F-肌动蛋白结合的高分辨率理解。我们发现Cortactin 重复序列(CR)结构域在溶液中自然展开，但我们可以得到适合于高密度脂蛋白的CR：F-肌动蛋白复合体 用低温电子显微镜测定拆分结构。我们还将结合使用CR域截断和 氢-重离子交换质谱仪绘制皮层肌动蛋白：F-肌动蛋白结合界面的残基图。 我们的第二个目标将阐明Arg和Cortactin如何相互作用来控制肌动蛋白细丝的动力学。这个 Arg和Cortactin维持树突棘中稳定的F-肌动蛋白池的机制尚不清楚。 我们发现Arg和Cortactin在体外相互作用来控制F-肌动蛋白的稳定性和新的肌动蛋白分支的成核。 我们将使用蛋白质的测量：蛋白质相互作用，基于全内反射显微镜的单一 肌动蛋白细丝分析和冷冻-EM结构测定，以了解Arg和Cortactin如何相互作用 与彼此和Arp2/3复合体一起调节F-肌动蛋白的稳定性和肌动蛋白分支的成核。 我们的第三个目标将阐明稳定的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-肌动蛋白池可能通过发挥中央组织者的作用来稳定脊柱 通过减弱NMDA受体(NMDAR)的活性，改善脊椎基础设施。我们将在培养的神经元中使用Arg或Cortactin突变体的击倒/互补策略来揭示它们的肌动蛋白 调节和/或其他功能是维持脊柱稳定的F-肌动蛋白池所必需的，调节NMDAR， 和脊椎的稳定性。我们还将使用活细胞和超分辨率显微镜来测量 稳定的F-肌动蛋白池影响脊柱内关键肌动蛋白调节器和亚室的组织。

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.