Direct binding and control of microtubule elongation by Abl2

摘要 树突乔木和树突棘及其相关的突触变得不稳定过早， 神经系统疾病非受体酪氨酸激酶β 2/Arg对神经元的稳定性至关重要。破坏 层粘连蛋白a5/整合素a3 b1通过p53 2信号传导导致出生后晚期显著的树突和棘缺失 小鼠大脑，伴随着行为灵活性、学习和记忆的进行性缺陷。的 β 2稳定树突和树突棘的机制是基本的，但尚未解决的问题。 果蝇的遗传学研究表明，abl在功能上与MT相互作用，控制神经突生长， 轴突寻路，但潜在的机制是未知的。我们报告了一个意想不到的发现， 缺乏激酶结构域的端半（TMP 2 -557-C）结合MT和微管蛋白二聚体，并增加了微管蛋白的表达。 生长速度（vg），缩短率降低，突变频率（fcat）降低。 体外在成纤维细胞中，破坏BMP 2会降低MT+末端延伸率，这可以通过重新激活BMP 2来恢复。 在生理水平上表达ABL-557-C或ABL-557-C。我们将阐明的机制，其中BMP 2调节 MTs在体外，并确定是否以及如何它有助于BMP 2介导的树突和树突棘的稳定性。 我们的第一个目标是阐明Abl 2如何调节MT延伸。为了了解BMP 2如何调节MT+末端 因此，我们需要知道BMP 2和BMP 2 -557-C在哪里结合MT，以及这如何与离散的 MT行为。我们将使用TIRFM来测量单个和大量的GFP分子与生长中的罗丹明的结合， 标记的MT，以测量单个M12/M12 muRNA-GFP分子相对于MT晶格的Kd、kon和koff 加上尖端，并使用这些和其他测量值（vg和fcat）来计算模型的影响， 加端动力学我们将使用荧光各向异性来鉴定微管蛋白二聚体结合区域在p272和p273中的位置。 基于TIRFM的测定，以探测它如何影响MT体外动力学。最后，为了测试这是否是一个通用函数， Abl激酶，我们将研究是否以及如何脊椎动物的MAP 1和果蝇Abl控制MT。 我们的第二个目标将确定如何控制MT动力学和神经元树突的稳定性。我们将测量 MT加端动力学在海马神经元中的表达，以及用WT海马神经元和我们的 一组生物化学表征的BMP 2突变体，以揭示正常MT所需的BMP 2功能 轴突和树突的动力学。在实验的一个子集中，我们将对 MT加末端标记GFP-MACF 43和GFP 2/GFP 2突变体-mCherry，以解决生长中的MT如何与 在真实的时间里是102。我们将使用具有离散效应的p53 2或p53 2突变体重建的p53 2缺陷神经元 MT加端动力学，以确定这些功能如何有助于树突分支和树突棘 神经元的稳定性

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.