E3 Ubiquitin Ligase Highwire/Phr1 Phase Separation Mediates Endocytic Control of JNK Signaling in Drosophila Neurons

Synaptic growth and organization are orchestrated by pre – and post-synaptic signaling, neuronal activity, and environmental cues. Although endocytosis is known to attenuate synaptic growth, the underlying signaling mechanisms have remained elusive. Here, we uncover a previously unrecognized mechanism by which endocytosis constrains synaptic growth through regulation of the neuronal E3 ubiquitin ligase Highwire (Hiw/Phr1). We show that loss of endocytosis causes Hiw to accumulate in neuronal cell bodies, leading to elevated MAP3K Wallenda (Wnd)/DLK levels and hyperactivation of JNK signaling. The accumulated Hiw assembles into dynamic liquid–liquid phase-separated condensates, as revealed by their rapid and reversible dissolution with 1,6-hexanediol. Acute blockade of endocytosis using a temperature-sensitive dynamin mutant Shibire ^ts similarly triggered robust Hiw phase separation. We further demonstrate that Rab11-positive recycling endosomes are essential for proper Hiw localization and turnover, directly linking endosomal trafficking to the control of JNK signaling. Finally, we show that both BMP and JNK signaling are necessary and sufficient to guide synaptic morphogenesis at the Drosophila NMJ, thereby integrating endocytic trafficking with synaptic growth signaling. Our findings establish endocytosis as a critical regulator of Hiw/Phr1-dependent JNK signaling via liquid–liquid phase separation, with implications that extend beyond synaptic morphogenesis to axon injury and degeneration pathways.