Project Summary/Abstract This proposal focuses on the fundamental question of how neuronal growth cones are guided to their targets. It has long been established that growth cone navigation depends on regulated changes in both F-actin and microtubule (MT) dynamics in response to guidance cues. However, the mechanisms by which these cues bring about specific changes in growth cone MT dynamics are unresolved. This proposal takes aim at that void, by investigating the function of the MT polymerase and ‘plus-end tracking protein’ (+TIP), XMAP215, along with its regulatory mechanisms. Our recently published data identified an ability for XMAP215 to bind directly to F-actin and promote MT extension into the growth cone. This is complemented by unpublished data that kinase signaling modulates the ability of XMAP215 to promote MT-F-actin interaction. Our data support a model in which XMAP215 mediates MT-F-actin coupling through structural domains distinct from those regulating MT polymerization, and that XMAP215 converts signals from upstream guidance cues into changes in cytoskeletal coordination, ultimately directing growth cone motility. We will test this in Xenopus laevis using an array of complementary cell-based and biochemical approaches. The specific aims are: Aim 1 – Identify the mechanism by which XMAP215 interacts with F-actin in vitro. We will determine the specific domain of XMAP215 that binds to F-actin as well as identify how the binding is regulated. We will produce a variety of deletion and phosphomutant XMAP215 proteins, and we will use a series of biochemical techniques including F-actin/MT co-sedimentation binding assays, multi-wavelength TIRF microscopy with in vitro cytoskeletal reconstitution assays, mass spectrometry, as well as EM experiments, to dissect the mechanism by which XMAP215 binds to F-actin in vitro. Aim 2 - Determine how regulation of XMAP215 interaction with F-actin modulates cytoskeletal dynamics and growth cone behaviors in cultured neurons. We will use structured illumination microscopy (SIM), spinning disk confocal microscopy, and quantitative imaging analysis of +TIP dynamics in cultured neurons, after expressing various XMAP215 mutants, to uncover new mechanistic insights into how XMAP215 regulates MT-F-actin interactions in growth cones during axon outgrowth. Aim 3 - Define how XMAP215 interaction with F-actin contributes to accurate axon guidance. We have discovered that normal XMAP215 levels are required for cultured growth cones to be repelled from the axon guidance cue, Slit2. We will use a combination of ex vivo and in vivo axon guidance assays to determine how growth cone steering is impacted by the ability of XMAP215 to facilitate MT-F-actin interactions. The results of these Aims will reveal the direct links between guidance cue signaling, kinase regulation of the only well-characterized MT polymerase (XMAP215), and cytoskeletal coordination, within the context of growth cone steering. As such, this pr...