SummaryRegulated protein degradation via the ubiquitin-proteasome system (UPS) plays a central role in building synaptic connections, yet little is known about either which specific UPS components are involved or UPS targets in neurons. We report that inhibiting the UPS in developing Xenopus retinal ganglion cells (RGCs) with a dominant-negative ubiquitin mutant decreases terminal branching in the tectum but does not affect long-range navigation to the tectum. We identify Nedd4 as a prominently expressed E3 ligase in RGC axon growth cones and show that disrupting its function severely inhibits terminal branching. We further demonstrate that PTEN, a negative regulator of the PI3K pathway, is a key downstream target of Nedd4: not only does Nedd4 regulate PTEN levels in RGC growth cones, but also, the decrease of PTEN rescues the branching defect caused by Nedd4 inhibition. Together our data suggest that Nedd4-regulated PTEN is a key regulator of terminal arborization in vivo.

Slit is a large secreted protein that provides important guidance cues in the developing nervous system and in other organs. Signaling by Slit requires two receptors, Robo transmembrane proteins and heparan sulfate (HS) proteoglycans. How HS controls Slit-Robo signaling is unclear. Here we show that the second leucine-rich repeat domain (D2) of Slit, which mediates binding to Robo receptors, also contains a functionally important binding site for heparin, a highly sulfated variant of HS. Heparin markedly enhances the affinity of the Slit-Robo interaction in a solid-phase binding assay. Analytical gel filtration chromatography demonstrates that Slit D2 associates with a soluble Robo fragment and a heparin-derived oligosaccharide to form a ternary complex. Retinal growth cone collapse triggered by Slit D2 requires cell surface HS or exogenously added heparin. Mutation of conserved basic residues in the C-terminal cap region of Slit D2 reduces heparin binding and abolishes biological activity. We conclude that heparin/HS is an integral component of the minimal Slit-Robo signaling complex and serves to stabilize the relatively weak Slit-Robo interaction.Slits are large secreted leucine-rich repeat (LRR) 6 proteins with multiple roles in cell signaling and adhesion. They have well established and evolutionarily conserved functions as guidance cues in the developing nervous system (1, 2), but Slits are also important in the development of the vasculature (3) and other organs (4). The first class of Slit receptors to be identified were Robo family members, which are transmembrane proteins with an extracellular domain resembling cell adhesion molecules and a large cytosolic signaling domain (1, 2). Biochemical studies have defined the domains mediating the SlitRobo interaction (5, 6), as well as some of the components of the signaling cascade downstream of Robo activation (7, 8), but how binding of Slit to Robo receptors conveys a signal across the cell membrane remains unknown.The first indication that there might exist a second Slit receptor came from the observation that heparan sulfate (HS) was required for the repellent activity of Slit in vitro (9) and in vivo (10). The identity of this receptor was revealed by recent genetic studies in invertebrates, which showed that Slit signaling requires Robo to be co-expressed on the same cell with the HS proteoglycan syndecan (11-13). Syndecan is a membranespanning protein to which are covalently attached several HS chains, consisting of repeating sulfated disaccharide units (14). Heparin is a member of the HS family that is more highly and uniformly sulfated than other HS. Johnson et al. (12) showed that both Slit and Robo can be co-immunoprecipitated with syndecan, suggesting the presence of a ternary (or higher order) complex at the neuronal cell membrane. However, the composition and functional relevance of this putative ternary complex was not established. Because the Slit distribution was found to be altered in syndecan-deficient embryos, HS may also be required for...

At the neuromuscular junction, acetylcholinesterase (AChE) is mainly present as asymmetric forms in which tetramers of catalytic subunits are associated to a specific collagen, collagen Q (ColQ). The accumulation of the enzyme in the synaptic basal lamina strictly relies on ColQ. This has been shown to be mediated by interaction between ColQ and perlecan, which itself binds dystroglycan. Here, using transfected mutants of ColQ in a ColQ-deficient muscle cell line or COS-7 cells, we report that ColQ clusterizes through a more complex mechanism. This process requires two heparin-binding sites contained in the collagen domain as well as the COOH terminus of ColQ. Cross-linking and immunoprecipitation experiments in Torpedo postsynaptic membranes together with transfection experiments with muscle-specific kinase (MuSK) constructs in MuSK-deficient myotubes or COS-7 cells provide the first evidence that ColQ binds MuSK. Together, our data suggest that a ternary complex containing ColQ, perlecan, and MuSK is required for AChE clustering and support the notion that MuSK dictates AChE synaptic localization at the neuromuscular junction.