Direct binding and control of microtubule elongation by Abl2

Abl2 直接结合并控制微管伸长

基本信息

批准号：
9978453
负责人：
Anthony J Koleske
金额：
$ 45.87万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-04-01 至 2022-09-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9978453
关键词：
Address Affect Affinity Amino Acids Axon Behavior Behavioral Binding Binding Sites Biochemical Biological Assay Brain C-terminal COS-7 Cell Cells Color Computer Models Cytoskeletal Modeling Cytoskeleton Data Defect Dendrites Dendritic Spines Drosophila genus Family Fibroblasts Fluorescence Fluorescence Anisotropy Frequencies Genetic study Growth Guanosine Triphosphate Hippocampus (Brain)Hydrolysis Image Impairment In Vitro Insecta Integrins Invertebrates Kinetics Label Laminin Lead Learning Location Measurement Measures Mediating Memory Microtubules Modeling Mus Neurites Neurodegenerative Disorders Neurodevelopmental Disorder Neuroglia Neurons Pathologic Phenotype Phosphotransferases Physiological Plus End of the Microtubule Process Protein Tyrosine Kinase Regulation Reporting Rhodamine Role Sedimentation process Shapes Signal Transduction Skeleton Structure Synapses Testing Time Total Internal Reflection Fluorescent Tubulin Vertebral column Work axonal pathfinding base dimer experimental study flexibility imaging approach insight knock-down mutant nervous system disorder novel postnatal premature reconstitution recruit tool

项目摘要

ABSTRACT Dendritic arbors and dendritic spines and their associated synapses become destabilized prematurely in neurological disorders. The Abl2/Arg nonreceptor tyrosine kinase is essential for neuronal stability. Disruption of laminin a5/integrin a3b1 signaling through Abl2 causes significant dendrite and spine loss in the late postnatal mouse brain, accompanied by progressive defects in behavioral flexibility, learning, and memory. The mechanisms by which Abl2 stabilizes dendrites and dendritic spines are fundamental, yet unresolved questions. Genetic studies in Drosophila show that abl interacts functionally with MTs to control neurite outgrowth and axon pathfinding, but the underlying mechanism is unknown. We report the unexpected finding that the Abl2 C- terminal half (Abl2-557-C), which lacks the kinase domain, binds MTs and tubulin dimers and increases the growth velocity (vg), reduces shortening rate, and decreases catastrophe frequency (fcat) of MT plus ends in vitro. Disruption of Abl2 reduces MT plus end elongation rates in fibroblast cells, which can be restored by re- expression of Abl2 or Abl-557-C at physiological levels. We will elucidate the mechanism by which Abl2 regulates MTs in vitro and determine whether and how it contributes to Abl2-mediated dendrite and dendritic spine stability. Our first aim will elucidate how Abl2 regulates MT elongation. To understand how Abl2 regulates MT plus-end dynamics, we need to know where Abl2 and Abl2-557-C bind MTs and how this relates to regulation of discrete MT behaviors. We will use TIRFM to measure single and bulk Abl2-GFP molecule binding to growing rhodamine- labeled MTs to measure the Kd, kon, and koff of single Abl2/Abl2 mutant-GFP molecules to the MT lattice vs. MT plus tip, and use these and other measurements (vg and fcat) to computationally model the effects of Abl2 on MT plus-end dynamics. We will use fluorescence anisotropy to identify the tubulin dimer binding region in Abl2 and TIRFM-based assays to probe how it impacts MT dynamics in vitro. Finally, to test if this is a general function of Abl kinases, we will study whether and how vertebrate Abl1 and Drosophila Abl control MTs. Our second aim will determine how Abl2 controls MT dynamics and dendrite stability in neurons. We will measure MT plus-end dynamics in Abl2-deficient cultured hippocampal neurons and rescue them with WT Abl2 and our set of biochemically-characterized Abl2 mutants to reveal which Abl2 functions are required for normal MT dynamics in axons and dendrites. In a subset of experiments, we will perform two-color TIRFM imaging of the MT plus-end marker GFP-MACF43 and Abl2/Abl2 mutant-mCherry to address how growing MTs interact with Abl2 in real time. We will use Abl2-deficient neurons reconstituted with Abl2 or Abl2 mutants with discrete effects on MT plus-end dynamics to determine how these functions contribute to dendritic branch and dendritic spine stability in neurons.

抽象的树突乔木和树突棘及其相关突触在神经系统疾病。 Abl2/Arg 非受体酪氨酸激酶对于神经元稳定性至关重要。扰乱通过 Abl2 的层粘连蛋白 a5/整合素 a3b1 信号传导导致产后后期显着的树突和脊柱损失小鼠大脑，伴有行为灵活性、学习和记忆的渐进性缺陷。这 Abl2 稳定树突和树突棘的机制是基本但尚未解决的问题。果蝇的遗传学研究表明 abl 与 MT 功能性相互作用以控制神经突生长和轴突寻路，但其潜在机制尚不清楚。我们报告了一个意外的发现，即 Abl2 C- 末端半部 (Abl2-557-C) 缺乏激酶结构域，可结合 MT 和微管蛋白二聚体并增加 MT plus 的生长速度 (vg)、缩短率和灾难频率 (fcat) 降低体外。 Abl2 的破坏降低了成纤维细胞的 MT 和末端伸长率，这可以通过重新修复来恢复。 Abl2 或 Abl-557-C 在生理水平的表达。我们将阐明Abl2的调节机制体外 MT 并确定它是否以及如何促进 Abl2 介导的树突和树突棘稳定性。我们的首要目标是阐明 Abl2 如何调节 MT 伸长。了解Abl2如何调控MT加端动力学，我们需要知道 Abl2 和 Abl2-557-C 结合 MT 的位置以及这与离散调节的关系 MT 行为。我们将使用 TIRFM 来测量与生长的罗丹明结合的单个和整体 Abl2-GFP 分子标记 MT 以测量单个 Abl2/Abl2 突变体-GFP 分子与 MT 晶格与 MT 的 Kd、kon 和 koff 加提示，并使用这些和其他测量（vg 和 fcat）来计算模拟 Abl2 对 MT 的影响正端动态。我们将使用荧光各向异性来识别 Abl2 和中的微管蛋白二聚体结合区域基于 TIRFM 的检测，探讨其如何影响体外 MT 动态。最后，测试这是否是一个通用函数 Abl 激酶，我们将研究脊椎动物 Abl1 和果蝇 Abl 是否以及如何控制 MT。我们的第二个目标是确定 Abl2 如何控制神经元中的 MT 动力学和树突稳定性。我们将测量 Abl2 缺陷培养海马神经元中的 MT 加端动力学，并用 WT Abl2 和我们的一组具有生化特征的 Abl2 突变体，以揭示正常 MT 所需的 Abl2 功能轴突和树突的动力学。在实验的子集中，我们将执行双色 TIRFM 成像 MT 加端标记 GFP-MACF43 和 Abl2/Abl2 突变体 -mCherry，用于解决生长中的 MT 如何与 Abl2 实时。我们将使用具有离散效应的 Abl2 或 Abl2 突变体重建的 Abl2 缺陷神经元 MT 加端动力学，以确定这些功能如何促进树突分支和树突棘神经元的稳定性。