Direct binding and control of microtubule elongation by Abl2

摘要 树枝和树突及其相关突触过早地变得不稳定 神经紊乱。ABL2/Arg非受体酪氨酸激酶对神经元的稳定是必不可少的。中断 Abl2介导的层粘连蛋白a5/整合素a3b1信号导致出生后晚期树突状细胞和棘突的显著丧失 小鼠大脑，伴随着行为灵活性、学习和记忆的进行性缺陷。这个 ABL2稳定树突和树突棘的机制是基本的，但尚未解决的问题。 对果蝇的遗传学研究表明，ABL与MTS在功能上相互作用，控制轴突生长和 轴突寻路，但潜在的机制尚不清楚。我们报告了一个意想不到的发现，ABL2C- 末端半部分(Abl2-557-C)缺乏激活域，与MTS和微管蛋白二聚体结合，增加了MTS和微管蛋白的活性。 增长速度(VG)，减少缩短率，并减少灾难频率(FCAT)的MT+结束于 体外培养。Abl2的破坏降低了成纤维细胞的MT+末端延长率，这可以通过重新修复而恢复。 Abl2或Abl-557-C在生理水平的表达。我们将阐明ABL2调节的机制 并确定其是否以及如何有助于Abl2介导的树突状细胞和树突状突起的稳定性。 我们的第一个目标是阐明Abl2是如何调节MT延长的。了解ABL2如何调节MT+-END 动力学，我们需要知道Abl2和Abl2-557-C在哪里结合MT，以及这与离散的调节有什么关系 MT行为。我们将使用TIRFM测量单个和整体ABL2-GFP分子与生长中的罗丹明的结合。 用标记MTS测量单个Abl2/Abl2突变体-GFP分子对MT晶格的Kd、Kon和Koff与MT的关系 加上TIP，并使用这些和其他测量(VG和FCAT)来计算模拟ABL2对MT的影响 加端动态。我们将使用荧光各向异性来确定Abl2和Abl2中的微管蛋白二聚体结合区 基于TIRFM的检测以探讨其对体外MT动力学的影响。最后，测试这是否是的常规函数 我们将研究脊椎动物ABL1和果蝇ABL是否以及如何控制MTS。 我们的第二个目标将确定Abl2如何控制神经元中MT的动力学和树突的稳定性。我们将衡量 海马神经元MT+端动力学变化及WT-ABL2和OUR对其的挽救作用 一组生化表征的Abl2突变体，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+末端标记GFP-MACF43和ABL2/ABL2突变体-mCherry解决生长MT如何与 实时的ABL2。我们将使用具有离散效应的Abl2或Abl2突变体重组的Abl2缺陷神经元 MT加端动力学研究这些功能对树突棘和树突的作用 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.