Mechanisms that control neuronal microtubule polarity

PROJECT SUMMARY Tightly regulated microtubule dynamics and polarity are essential for long-term survival of neurons. Nucleation of new microtubules in axons and dendrites contributes to dynamics and polarity and is also critical for neuroprotection and dendrite regeneration. In a previous study, Wnt signaling proteins on endosomes were shown to control microtubule nucleation in dendrites. The current proposal expands on this finding to answer fundamental questions about neuronal microtubule organization and the relationship between cell shape and regional specialization. Proposed experiments analyze neurons in their normal environment and rely on the powerful genetic tools available in the Drosophila model system. Aim 1. Do surrounding cells control neuronal microtubule nucleation? The finding that Wnt signaling proteins, including four receptors, are required to position nucleation sites in dendrites raised the possibility that ligands from surrounding cells could control neuronal microtubule dynamics. Preliminary data suggests that epithelial cells that contact dendrites are the Wnt source. Aim 1A focuses on confirming that epithelial Wnts regulate neuronal microtubule nucleation. In Aim 1B the potential that Wnt signaling could tune nucleation to help neurons respond to injury will be explored. Aim 2. How are nucleation sites targeted to branch points? The distribution of Wnt signaling endosomes that control nucleation is non-random in dendrites: they are typically positioned at dendrite branch points. This localization links neuronal structure directly to regulation of microtubule dynamics. In Aim 2A several different hypotheses about how Wnt signaling endosomes are localized to branch points will be tested. Potential mechanisms that could contribute to targeting include local delivery of Wnt receptors or ligands and local endocytosis. Preliminary data indicates endocytic sites are enriched at dendrite branch points. In Aim 2B this preliminary data will be used to ask how branch points are recognized as different from the rest of the cell by the endocytic machinery. Two hypotheses that will be tested are: 1) membrane curvature-recognizing proteins recruit endocytic proteins to branch points, and 2) the tight curvature of the plasma membrane outside branch points mechanically restricts endocytosis to branch points. Aim 3. Axonal parallels: Is axonal microtubule nucleation the same as dendritic? While studies in invertebrate model systems have provided insights into how microtubules are nucleated in dendrites, these systems have not yet been used to probe axonal nucleation. In Aim 3A, a sensitized nucleation assay will be used to determine whether any dendritic nucleation regulators also function in the main axon shaft. In Aim 3B, nucleation at the neuromuscular junction will be investigated. Summary: Neuronal microtubule nucleation is still poorly understood, and the proposed experiments will substantially advance our understanding of this fundamental aspect of neuronal cell biology.