Abstract
Understanding how axon guidance receptors are activated by their extracellular ligands to regulate growth cone motility is critical to learning how proper wiring is established during development. Roundabout (Robo) is one such guidance receptor that mediates repulsion from its ligand Slit in both invertebrates and vertebrates. Here we show that endocytic trafficking of the Robo receptor in response to Slit-binding is necessary for its repulsive signaling output. Dose-dependent genetic interactions and in vitro Robo activation assays support a role for Clathrin-dependent endocytosis, and entry into both the early and late endosomes as positive regulators of Slit-Robo signaling. We identify two conserved motifs in Robo’s cytoplasmic domain that are required for its Clathrin-dependent endocytosis and activation in vitro; gain of function and genetic rescue experiments provide strong evidence that these trafficking events are required for Robo repulsive guidance activity in vivo. Our data support a model in which Robo’s ligand-dependent internalization from the cell surface to the late endosome is essential for receptor activation and proper repulsive guidance at the midline by allowing recruitment of the downstream effector Son of Sevenless in a spatially constrained endocytic trafficking compartment.
Highlights
The complex wiring patterns of the adult central nervous system are established by the stepwise navigation of growth cones and migrating cells through a series of choice points during development
We identify a novel mechanism that is required for Robo receptor activation and PLOS Genetics | DOI:10.1371/journal.pgen
If endocytosis modulates the amount of Robo receptor on the surface of the growth cone, a reduction in receptor endocytosis would be predicted to lead to increased levels of surface receptor and more robust repulsive signaling
Summary
The complex wiring patterns of the adult central nervous system are established by the stepwise navigation of growth cones and migrating cells through a series of choice points during development. Robo receptors have been implicated in genome-wide association studies with the pathogenesis of several human diseases including autism and schizophrenia [5,6], and they are thought to be causatively linked to dyslexia and periventricular nodular heterotopia [7], suggesting roles in guidance of more diverse axonal projections in the human cortex that are yet to be characterized. In both invertebrates and vertebrates, Slits serve as repulsive cues to their Robo receptors by demarcating regions into which axons cannot maintain their exploratory projections. There exists a relative valley in Slit expression in medio-lateral axis of the Drosophila VNC through which a sizeable set of longitudinal fascicles project [11]
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