By controlling the localization of an actin-binding protein, PI(4,5)P2 controls synapse growth in Drosophila.
During the larval stages of Drosophila melanogaster development, there is a neuromuscular junction (NMJ) growth spurt to accommodate large increases in body wall muscle area. Using a genetic approach, Patrik Verstreken and colleagues report in Proceedings of the National Academy of Sciences of the USA that the phosphoinositide lipidPI(4,5)P2 helps to maintain NMJ size and morphology by regulating the actin-binding protein WASP.
Phosphoinositide (PI) lipids play numerous roles in the regulation of synaptic transmission, including the formation of new synaptic vesicles at neuronal membranes. By employing a PI(4,5)P2-binding protein domain (PH) from phospholipase C, which causes a dominant negative effect of reduced PI(4,5)P2 availability, the authors were able to test the effect of PI(4,5)P2 depletion in a Drosophila larval system of NMJ growth. Indeed, they saw decreased stimulation-induced synaptic vesicle cycling, consistent with PI(4,5)P2depletion. This depletion, as well as knockdown of the kinases immediately upstream of PI(4,5)P2, increased the total branch length and the number of satellite boutons — knob-like projections of neurons. Increasing PI(4,5)P2 levels by mutating a PI phosphatase restored the NMJ overgrowth caused by PH domain overexpression.
Because mutations in the WASP protein also show enlarged NMJs, the authors wanted to know if expressing the PH domain to deplete PI(4,5)P2 in mutants lacking the WASP protein (wsp) would have an additive effect on NMJ overgrowth. This was not the case, suggesting that PI(4,5)P2 controls NMJ growth in a WASP-dependent manner. This regulation was dependent on the known PI(4,5)P2-binding domain of WASP. The WASP- and PI(4,5)P2-mediated effects on NMJ function are likely not completely overlapping, as the authors saw differences in vesicle cycling and neurotransmitter release between the two mutant systems.
Monitoring the localization of WASP, the authors determined that PI(4,5)P2 is involved in localizing or stabilizing WASP. Consistent with a role for WASP in regulating actin branching and for the actin cytoskeleton in NMJ development, the authors found that PI(4,5)P2 depletion, like wsp mutants, accumulates actin in patches in NMJs. The actin patches, labelled with the actin-binding protein Moesin tagged with GFP, correlate with NMJ growth. The results suggest that unbranched actin, induced during WASP inhibition by PI(4,5)P2-depletion, supports new bouton sprouting.
It is thought that WASP restricts NMJ growth by cooperating with its binding partner, Nervous Wreck (NWK). Testing various single and double mutant combinations of wsp, nwk, and wit, the gene encoding a bone morphogenetic protein receptor that NWK inhibits, the authors determined that PI(4,5)P2 regulates NMJ morphology by acting in parallel to NWK and WIT. A similar epistasis analysis using tweek mutants, which show reduced boutonic PI(4,5)P2 availability, suggests that NWK, WSP and PI(4,5)P2 act in a Tweek-dependent pathway to regulate NMJ growth.
These results suggest that Tweek regulates NMJ growth by signaling through the two parallel pathways: the NWK/WIT system of synaptic BMP signaling and the WSP/PI(4,5)P2 pathway that regulates the actin cytoskeleton.
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Mirella Bucci
Lipidomics Gateway (27 October 2010) [doi:10.1038/lipidmaps.2010.33]
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