HighwireFunction at theDrosophilaNeuromuscular Junction: Spatial, Structural, and Temporal Requirements

Abstract

Highwire is a huge, evolutionarily conserved protein that is required to restrain synaptic growth and promote synaptic transmission at the Drosophila neuromuscular junction. Current models of highwire function suggest that it may act as a ubiquitin ligase to regulate synaptic development. However, it is not known in which cells highwire functions, whether its putative ligase domain is required for function, or whether highwire regulates the synapse during development or alternatively sets cell fate in the embryo. We performed a series of transgenic rescue experiments to test the spatial, structural, and temporal requirements for highwire function. We find that presynaptic activity of highwire is both necessary and sufficient to regulate both synapse morphology and physiology. The Highwire RING domain, which is postulated to function as an E3 ubiquitin ligase, is required for highwire function. In addition, highwire acts throughout larval development to regulate synaptic morphology and function. Finally, we show that the morphological and physiological phenotypes of highwire mutants have different dosage and temporal requirements for highwire, demonstrating that highwire may independently regulate the molecular pathways controlling synaptic growth and function.

Rapid in vivo forward genetic approach for identifying axon death genes in Drosophila

“…These data argue that specific loss of the Hiw Ring domain-which would lead to a protein predicted to bind its substrate but not transfer ubiquitin-can dominantly suppress axon degeneration. Consistent with this notion, Gal4/UAS-mediated overexpression of Highwire containing a nonfunctional RING domain (HiwΔRING) in wild-type animal phenocopies loss of hiw function both in NMJ morphological defects and axon degeneration (13,29). Our observation that both hiw 403d and hiw 275d fully suppress axon degeneration for 1 wk after axotomy argues for a key role for the Hiw RING domain in axon death signaling.…”

“…Thus, the Hiw RING domain is a key functional domain required to drive axonal degeneration. It remains to be determined how these dominant hiw alleles block axon degeneration (13,29).…”

“…Through this approach, we identified a premature stop in codon 2791 (CAG → TAG) of the highwire locus, which has recently been shown to be required for axon degeneration (13). We tested for allelism by performing complementation tests using a large N-terminal deletion allele of hiw termed hiw ΔN (29). At 1 dpa, severed wild-type and heterozygous Tables S2-S4.…”

The B′ Protein Phosphatase 2A Regulatory Subunitwell-roundedRegulates Synaptic Growth and Cytoskeletal Stability at theDrosophilaNeuromuscular Junction

“…Bouton analysis. Quantification of bouton number and size was performed as described previously (Wu et al, 2005) using confocal images analyzed with MetaMorph software (Universal Imaging Corporation). Wandering third-instar larvae were costained for antibody to HRP and DVGLUT.…”

Transmission, Development, and Plasticity of Synapses

“…Work in Drosophila and Caenorhabditis elegans showed that in the absence of the E3 ubiquitin ligase Highwire (Hiw; RPM-1 in C. elegans), Wnd levels increase, overactivating the Jnk pathway (Nakata et al 2005;Collins et al 2006). Loss of function of hiw gives rise to a distinct synaptic morphology characterized by a greatly expanded arbor and abnormally small boutons ( Figure 5B) (Wan et al 2000;Wu et al 2005). Despite the increase in bouton number, these animals exhibit synaptic dysfunction with decreased quantal size and quantal content.…”