Project Summary/Abstract The goal of this program is to understand how informational signaling proteins that pattern tissues distribute in space and time during development. We study this process in Drosophila, investigating the Decapentaplegic, Hedgehog, FGF, EGF, Wg, and Notch-Delta signaling systems. The mechanisms that move these proteins from their sources and distribute them to their targets involve cellular machines and organelles whose actions precisely control their movement, and our work has identified novel structures we named “cytonemes” that mediate their dispersion. This proposal describes the approaches we will take to further characterize cytonemes and the machines and organelles that make them work. Cytonemes are specialized filopodia that extend between cells that produce signaling proteins and their signaling targets. Our work has now established that cytonemes are key elements of paracrine cell-cell signaling, and their properties led us to propose that signaling proteins move between non-neuronal cells in a manner similar to the way neurons exchange signals with post-synaptic target cells – by exchanging information at synaptic contacts that connect cell extensions such as axons that span the distance between signaling and target cells. Findings made during the current grant period show that both the composition and activities of cytonemes are remarkably similar to axons and chemical synapses. Cytonemes are constituted with proteins that have been shown to function and to be required at neuronal synapses, such as the cell adhesion proteins Capricious and Neuroglian, and are calcium dependent, excitable, and glutamatergic. They require the glutamate receptor, glutamate transporter, voltage-gated calcium channel, synaptobrevin, and synaptotagmin. We have also learned that cytonemes have alternating regions of thin and wide diameter akin to “beads on a string”, and borrow deep into invaginations of target cells. These unexpected properties have fascinating implications for mechanisms of pathfinding and signal transduction, and the work we propose both develops new tools for imaging cytonemes and builds upon our previous findings to determine the roles, composition and functions of these remarkable organelles and this mechanism of contact-based signaling.