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

描述(由申请人提供)：在精神和神经退行性疾病中，树突棘及其相关突触过早地变得不稳定。对肌动蛋白细胞骨架的适当控制对于树突棘的长期结构稳定性至关重要，但目前人们对赋予树突棘长期结构稳定性的分子和机制知之甚少，该领域仍未得到充分研究。我们发现整合素�-3-�-1信号的缺失通过ABL2/Arg非受体酪氨酸激酶导致广泛的树突状突起丢失和树突棘失稳。尽管Arg抑制RhoA GTP酶稳定树突状分枝，但这一机制并不影响脊柱的稳定性，这提出了Arg如何稳定脊柱的根本问题。我们提供的证据表明，Arg直接结合和稳定肌动蛋白细丝，也调节肌动蛋白调节因子Cortactin和Arp2/3复合体对肌动蛋白细丝的结合和作用。我们还发现，Arg介导的皮质蛋白向树突棘的募集对脊柱的稳定性至关重要。我们的提案将检验高度创新的假设，即Arg与肌动蛋白细丝和肌动蛋白调节蛋白在物理和功能上相互作用 蛋白质直接调节肌动蛋白的动态，从而稳定树突棘。我们的第一个目标将阐明Arg：Cortactin相互作用如何控制肌动蛋白动力学。我们发现，Arg与肌动蛋白细丝的结合使它们稳定在解聚状态。Arg结合还招募肌动蛋白结合蛋白Cortactin，该蛋白稳定肌动蛋白细丝，并通过Arp2/3复合体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，并测量Arg和Cortactin如何影响肌动蛋白细丝的稳定性、Arp2/3复合体介导的分支形成以及粘连蛋白介导的肌动蛋白细丝断裂。我们将使用这些彼此不相互作用或不与肌动蛋白细丝相互作用的蛋白质的突变体来确定哪些蛋白质：蛋白质相互作用界面对肌动蛋白动力学的影响至关重要。这些研究将揭示AR和Cortactin如何影响肌动蛋白细丝的稳定性、分支和周转。我们的第二个目标将确定Arg和Cortactin如何通过对肌动蛋白动力学的影响来调节脊柱稳定性。我们发现，神经元中Arg的敲除会导致脊椎中皮质蛋白的丢失，并引发它们的不稳定。我们假设这种不稳定是由于脊柱中正常的肌动蛋白动力学的破坏。在已建立的海马神经元培养中敲除Arg或Cortactin会损害树突棘的稳定性。这些缺陷可以通过分别重新表达具有shRNA抗性的Arg或Cortactin版本来定量挽救。利用我们收集的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的形状和稳定性。我们将使用脊椎中GFP-肌动蛋白的光漂白后荧光恢复(FRAP)来揭示Arg和Cortactin功能的操纵如何影响脊柱中的肌动蛋白动态，并确定这与这些蛋白质对肌动蛋白生化和脊柱稳定性的影响之间的关系。

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.