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疾病和神经退行性疾病中过早不稳定，肌动蛋白细胞骨架的正确控制对于树突棘的长期结构稳定性至关重要，但目前人们对这些分子和突触知之甚少。赋予脊柱和领域长期结构稳定性的机制仍未得到充分研究。通过 Abl2/Arg 非受体酪氨酸激酶的 3¿1 信号传导导致广泛的树突轴损失和树突棘不稳定，尽管 Arg 抑制 RhoA GTPase 来稳定树突轴，但这种机制不会影响树突轴稳定性，从而提出了 Arg 如何稳定的基本问题。我们提供的证据表明精氨酸直接结合和稳定肌动蛋白丝，并且还调节肌动蛋白调节剂的结合和作用。我们还发现，Arg 介导的 cortactin 募集到树突棘对于树突棘的稳定性至关重要，我们的提议将测试 Arg 在物理和功能上与肌动蛋白丝和肌动蛋白调节相互作用的高度创新假设。 因此，我们的首要目标是阐明精氨酸：皮质蛋白相互作用如何控制肌动蛋白动力学。它通过 Arp2/3 复合物稳定肌动蛋白丝并增加肌动蛋白分支的形成我们将使用全内反射显微镜观察单丝和为了测量 Arg 和 cortactin 如何影响肌动蛋白丝稳定性、Arp2/3 复合物介导的分支形成和肌动蛋白丝切蛋白介导的肌动蛋白丝切断，我们将使用这些蛋白质的突变体，这些蛋白质彼此不相互作用，也不与肌动蛋白丝相互作用来鉴定哪种蛋白质。 ：蛋白质相互作用界面对于肌动蛋白动力学的影响至关重要。这些研究将揭示 Ar 和 cortactin 如何影响肌动蛋白丝的稳定性、分支和更新。我们发现，神经元中 Arg 的敲低会导致棘中皮质蛋白的丢失，并引发其不稳定，这是由于棘中正常肌动蛋白动力学的破坏所致。在已建立的海马神经元培养物中，Arg 或 Cortactin 的存在会损害树突棘的稳定性，这些缺陷可以通过 shRNA 抗性版本的重新表达来定量修复。分别使用我们收集的 Arg 和 cortactin 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形状和稳定性。揭示精氨酸和皮质蛋白功能的操纵如何影响脊柱中的肌动蛋白动力学，并确定这与这些蛋白质对肌动蛋白生物化学和脊柱稳定性的影响有何关系。