PHR regulates growth cone pausing at intermediate targets through microtubule disassembly.

Axonal growth cones use intermediate targets to navigate in the developing nervous system. Encountering these sites is correlated with growth cone pausing. PHR (Phr1, Esrom, Highwire, RPM-1) is a large neuronal ubiquitin ligase that interacts with multiple signaling pathways. Mouse and zebrafish phr mutants have highly penetrant axon pathfinding defects at intermediate targets. Mouse phr mutants contain excessive microtubules in the growth cone, which has been attributed to upregulation of DLK/p38 signaling. Here, we ask whether this pathway and microtubule misregulation are indeed linked to guidance errors in the vertebrate brain, using the zebrafish. By live imaging, we show that loops form when microtubules retract without depolymerizing. JNK, but not p38, phosphorylation is increased in mutant growth cones. However microtubule looping cannot be suppressed by inhibiting JNK. The phr microtubule defect can be phenocopied by taxol, while microtubule destabilization in vitro using nocodazole prevents loop formation. Acute disruption in vivo with nocodazole suppresses the intermediate target guidance defect. Given that microtubule looping is associated with growth cone pausing, we propose that microtubule disassembly, mediated by PHR, is essential for exiting the paused state at intermediate targets.