The establishment of synaptic connectivity in the visual system relies on the precise spatiotemporal localization and function of guidance receptors that interpret attractive or repulsive guidance cues. However, little is known about the intracellular trafficking mechanisms that underlie the sorting and activity of these receptors. This gap in knowledge contrasts with the intensely studied critical roles of the endocytic regulation of receptor function in numerous other processes, including cell differentiation, tissue patterning and synaptic plasticity. The proposed work is designed to elucidate the role of endocytic regulation of receptor localization and function in neuronal growth cones in a developing brain. We will test the hypothesis that neuron-specific membrane trafficking controls guidance receptor dynamics and function to establish neuronal connectivity in the Drosophila visual system. Towards this goal, we have established non-invasive intravital imaging methods to quantitatively measure guidance receptor sorting and endolysosomal degradation at subcellular resolution in identified growth cones during visual system development in Drosophila. In addition, we have discovered a conserved neuron-specific branch of the endolysosomal system that regulates the sorting and degradation of guidance receptors in these growth cones. This synaptic endolysosomal system represents, to our knowledge, the first discovery of a neuron-specific endomembrane sorting and degradation pathway. Its mechanism is currently defined by two neuron-specific vesicle proteins, the vesicle SNARE neuronal Synaptobrevin (n-Syb) and the vesicle ATPase component V100. Mutations in n-syb and v100 provide genetic inroads to measure and manipulate guidance receptor dynamics during visual system development. In addition, in a systematic profiling effort of all Drosophila rab GTPases we have discovered that the novel rab GTPase Rab26 regulates receptor trafficking in concert with V100 and n-Syb. We will characterize the roles of these neuron-specific sorting mechanisms with a focus on the guidance receptor Flamingo, a protocadherin with a key role in visual map formation. Flamingo exhibits high turnover in photoreceptor growth cones and is deregulated in the absence of n-Syb, V100 or Rab26. An understanding of the regulation of receptor localization and function through intracellular trafficking will elucidate how guidance receptor dynamics and growth cone dynamics are integrated to specify a complicated neural connectivity pattern in vivo.
The proposed project has direct and substantial relevance for public health, because we present new insights into the cell biological mechanisms that underlie the regulation of neuronal connectivity in health and disease. The proposed experiments will elucidate our understanding of the intracellular trafficking mechanisms that control the function of synaptic receptors and orchestrate the specificity of synaptic contacts in the developing eye and brain. Deranged receptor signaling and synaptic connectivity are the cause of many pathological features in several neurological and psychiatric disorders.
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