Abstract
BackgroundThe growth of new synapses shapes the initial formation and subsequent rearrangement of neural circuitry. Genetic studies have demonstrated that the ubiquitin ligase Highwire restrains synaptic terminal growth by down-regulating the MAP kinase kinase kinase Wallenda/dual leucine zipper kinase (DLK). To investigate the mechanism of Highwire action, we have identified DFsn as a binding partner of Highwire and characterized the roles of DFsn in synapse development, synaptic transmission, and the regulation of Wallenda/DLK kinase abundance.ResultsWe identified DFsn as an F-box protein that binds to the RING-domain ubiquitin ligase Highwire and that can localize to the Drosophila neuromuscular junction. Loss-of-function mutants for DFsn have a phenotype that is very similar to highwire mutants – there is a dramatic overgrowth of synaptic termini, with a large increase in the number of synaptic boutons and branches. In addition, synaptic transmission is impaired in DFsn mutants. Genetic interactions between DFsn and highwire mutants indicate that DFsn and Highwire collaborate to restrain synaptic terminal growth. Finally, DFsn regulates the levels of the Wallenda/DLK kinase, and wallenda is necessary for DFsn-dependent synaptic terminal overgrowth.ConclusionThe F-box protein DFsn binds the ubiquitin ligase Highwire and is required to down-regulate the levels of the Wallenda/DLK kinase and restrain synaptic terminal growth. We propose that DFsn and Highwire participate in an evolutionarily conserved ubiquitin ligase complex whose substrates regulate the structure and function of synapses.
Highlights
The growth of new synapses shapes the initial formation and subsequent rearrangement of neural circuitry
We generated a transgene encoding Tandem affinity purification (TAP)-tagged HighwireΔRING (HiwΔRING), which carries two Cys-to-Ser mutations in the conserved RING domain, and demonstrated that HiwΔRING acts as a potent dominant negative when expressed in neurons [11]
We found that HiwΔRING shows a much stronger enrichment in the final complex than Highwire, possibly because of a higher stability of HiwΔRING
Summary
The growth of new synapses shapes the initial formation and subsequent rearrangement of neural circuitry. In Drosophila, genetic studies have identified a number of signaling pathways that regulate the morphology of the presynaptic arbor made by motoneurons onto muscles (reviewed in [2]). One such pathway requires highwire – in highwire mutants there is a dramatic increase in the complexity of the synaptic terminal, with a large increase in the number of synaptic boutons and branches [3]. This function for highwire is evolutionarily conserved: studies in worms, fish, and mammals all suggest that highwire homologs are required for normal synaptic development. In Caenorhabditis elegans, the homolog rpm-1 regulates the number, spacing, and morphology of presynaptic active zones [4,5], mutations in the zebrafish homolog esrom disrupt retinotectal projections [6], while mice carrying large deletions that remove the murine homolog phr and adjacent genes disrupt NMJ morphology [7]
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