Control of actin dynamics and dendritic spine stability by Arg and cortactin

Arg 和 cortactin 控制肌动蛋白动力学和树突棘稳定性

• 批准号: 8791215
• 负责人: Anthony J Koleske
• 金额: $ 20.81万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8791215
• 关键词: Actin-Binding Protein; Actins; Address; Affect; Alzheimer' s Disease; Area; Binding; Biochemistry; Biological Assay; Brain; Brain-Derived Neurotrophic Factor; C-terminal; Collection; Complex; Cytoskeleton; Data; Dendrites; Dendritic Spines; F-Actin; Filament; Fluorescence Recovery After Photobleaching; Guanosine Triphosphate Phosphohydrolases; Hippocampus (Brain); In Vitro; Integrins; Laboratories; Left; Major Depressive Disorder; Measures; Mediating; Mental disorders; Microfilaments; Microscopy; N-Methyl-D-Aspartate Receptors; Neurodegenerative Disorders; Neurons; Neurosciences; Protein Tyrosine Kinase; Proteins; Recruitment Activity; Resistance; Role; Schizophrenia; Sedimentation process; Shapes; Signal Transduction; Structure; Synapses; Testing; Vertebral column; Work; base; cofilin; density; depolymerization; genetic regulatory protein; human EMS1 protein; innovation; mutant; neural circuit; polymerization; protein protein interaction; public health relevance; small hairpin RNA

DESCRIPTION (provided by applicant): Dendritic spines and their associated synapses become prematurely destabilized in psychiatric and neurodegenerative diseases. Proper control of the actin cytoskeleton is critical for the long-term structural stability of dendritic spines, bt currently little is known about the molecules and mechanisms that confer long-term structural stability on spines and the field remains understudied. We discovered that loss of integrin ¿3¿1 signaling through the Abl2/Arg nonreceptor tyrosine kinase causes widespread dendrite arbor loss and dendritic spine destabilization. Even though Arg inhibits the RhoA GTPase to stabilize dendrite arbors, this mechanism does not impact spine stability, raising the fundamental question of how Arg stabilizes spines. We provide evidence that Arg directly binds and stabilizes actin filaments and also regulates the binding and actions of the actin regulators cortactin and Arp2/3 complex on actin filaments. We also find that Arg-mediated recruitment of cortactin to dendritic spines is crucial for spine stability. Our proposal will test the highly innovative hypothesis that Arg interacts physically and functionally with actin filaments and actin regulatory proteins to directly regulate actin dynamics and thereby stabilize dendritic spines. Our first aim will elucidate how Arg:cortactin interactions control actin dynamics. We find that Arg binding to actin filaments stabilizes them from depolymerization. Arg binding also recruits the actin-binding protein cortactin, which stabilizes actin filaments and increases actin branch formation by Arp2/3 complex. We will use total internal reflection microscopy to observe single filaments and to measure how Arg and cortactin affect actin filament stability, Arp2/3 complex-mediated branch formation, and cofilin-mediated actin filament severing. We will use mutants of these proteins that do not interact with each other or with actin filaments to identify which protein:protein interaction interfaces are critical for effects on actin dynamics. These studies will reveal how Ar and cortactin affect actin filament stability, branching, and turnover. Our second aim will determine how Arg and cortactin modulate spine stability via effects on actin dynamics. We find that knockdown of Arg in neurons results in the loss of cortactin from spines and triggers their destabilization. We hypothesize this destabilization is due to the disruption of normal actin dynamics in spines. Knockdown of Arg or cortactin in established hippocampal neuron cultures compromises dendritic spine stability. These deficits can be quantitatively rescued by re-expression of shRNA-resistant versions of Arg or cortactin, respectively. Employing our collection of Arg and cortactin mutants, we will test how mutational disruption of key interaction interfaces in these proteins affects dendritic spine shape and stability. We will use fluorescence recovery after photobleaching (FRAP) of GFP-actin in spines to reveal how manipulations of Arg and cortactin function affect actin dynamics in spines and determine how this relates to the effects of these proteins on actin biochemistry and spine stability.

描述（由申请人提供）：树突棘及其相关突触在精神病和神经退行性疾病中过早失稳。肌动蛋白细胞骨架的正确调控对树突棘的长期结构稳定性至关重要，但目前对树突棘长期结构稳定性的分子和机制知之甚少，这一领域仍处于研究阶段。我们发现整合素<$3 <$1信号通过β 2/Arg非受体酪氨酸激酶的丢失导致广泛的树突乔木丢失和树突棘不稳定。尽管Arg抑制RhoA GT3来稳定树突，但这种机制并不影响棘的稳定性，这就提出了Arg如何稳定棘的根本问题。我们提供的证据表明，精氨酸直接结合和稳定肌动蛋白丝，也调节肌动蛋白调节剂corp 2/3复合物对肌动蛋白丝的结合和行动。我们还发现，精氨酸介导的招聘corneum树突棘是至关重要的脊柱的稳定性。我们的建议将测试高度创新的假设，精氨酸相互作用的物理和功能与肌动蛋白丝和肌动蛋白调节 蛋白质直接调节肌动蛋白动力学，从而稳定树突棘。我们的第一个目标将阐明如何精氨酸：corneum相互作用控制肌动蛋白动力学。我们发现，精氨酸结合到肌动蛋白丝稳定他们从解聚。精氨酸结合也募集肌动蛋白结合蛋白cornein，其稳定肌动蛋白丝并通过Arp 2/3复合物增加肌动蛋白分支形成。我们将使用全内反射显微镜来观察单纤维，并测量精氨酸和cornein如何影响肌动蛋白丝的稳定性，Arp 2/3复合物介导的分支形成，和cofilin介导的肌动蛋白丝切断。我们将使用这些蛋白质的突变体，不相互作用，或与肌动蛋白丝，以确定蛋白质：蛋白质相互作用界面是关键的肌动蛋白动力学的影响。这些研究将揭示Ar和coronin如何影响肌动蛋白丝的稳定性，分支和营业额。我们的第二个目标将确定精氨酸和coronin调节脊柱的稳定性，通过对肌动蛋白动力学的影响。我们发现，敲低精氨酸在神经元中的结果在损失的corneum从棘，并触发其不稳定。我们假设这种不稳定是由于正常的肌动蛋白动力学的破坏脊柱。在已建立的海马神经元培养物中敲低Arg或coronin会损害树突棘的稳定性。这些缺陷可以通过分别重新表达Arg或coronin的shRNA抗性版本来定量地挽救。利用我们收集的精氨酸和coronin突变体，我们将测试这些蛋白质中关键相互作用界面的突变破坏如何影响树突棘的形状和稳定性。我们将使用荧光恢复后的GFP-肌动蛋白在脊柱的光漂白（FRAP），以揭示如何操纵的精氨酸和corneum的功能影响肌动蛋白的动态脊柱，并确定这与这些蛋白质的肌动蛋白的生物化学和脊柱的稳定性的影响。