Direct binding and control of microtubule elongation by Abl2

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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.