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

基本信息

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
项目类别：
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&apos 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

项目摘要

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（由适用提供）：树突状刺及其相关的突触在精神病和神经退行性疾病中过早稳定。对肌动蛋白细胞骨架的适当控制对于树突状刺的长期结构稳定性至关重要，目前，BT对棘突上的长期结构稳定性的分子和机制知之甚少，并且该田仍然了解了。我们发现通过ABL2/ARG非受体酪氨酸激酶通过ABL2/ARG的信号传导的损失会导致宽度的树突植物植物植物损失和树突状脊柱不稳定。即使ARG抑制了RhoA GTPase稳定树突轴，但该机制也不会影响脊柱稳定性，从而提出了ARG如何稳定刺的基本问题。我们提供了ARG直接结合并稳定肌动蛋白丝的证据，并调节肌动蛋白调节剂Cortactin和Arp2/3复合物对肌动蛋白丝的结合和作用。我们还发现，ARG介导的皮质素募集到树突状棘对脊柱稳定性至关重要。我们的建议将检验ARG与肌动蛋白丝和肌动蛋白调节的物理和功能相互作用的高度创新假设蛋白质直接调节肌动蛋白动力学，从而稳定树突状刺。我们的第一个目标将阐明ARG：Cortactin相互作用控制肌动蛋白动力学。我们发现，与肌动蛋白丝结合的arg稳定在沉积中。 ARG结合还募集了肌动蛋白结合蛋白皮质素，该蛋白质可以稳定肌动蛋白丝并通过ARP2/3复合物增加肌动蛋白分支的形成。我们将使用总内反射显微镜观察单细丝，并测量ARG和皮质素如何影响肌动蛋白丝稳定性，ARP2/3复合物介导的分支形成以及Cofilin介导的肌动蛋白丝几种。我们将使用这些蛋白质不相互作用或与肌动蛋白丝的突变体来识别哪种蛋白质：蛋白质相互作用界面对于对肌动蛋白动力学的影响至关重要。这些研究将揭示AR和皮质素如何影响肌动蛋白丝稳定性，分支和周转率。我们的第二个目标将决定ARG和Cortactin如何通过对肌动蛋白动力学的影响调节脊柱稳定性。我们发现神经元中ARD的敲低导致棘突失去皮质素并触发其不稳定。我们假设这种不稳定是由于刺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敲低会损害树突状脊柱稳定性。这些缺陷可以分别通过重新表达ARG或Cortactin的抗shRNA版本的重新表达来定量响应。我们将采用我们的ARG和Cortactin突变体的收集，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状的脊柱形状和稳定性。我们将在刺中使用GFP-肌动蛋白的光漂白（FRAP）后使用荧光恢复，以揭示ARG和皮质素功能的操纵如何影响刺中的肌动蛋白动力学，并确定这与这些蛋白质对肌动蛋白对肌动蛋白生物化学和棘突稳定性的影响如何相关。