Extra-synaptic localization of Bruchpilot in association with neurite outgrowth and cell motility in dissociated Drosophila embryonic cell cultures

Neurite outgrowth and motility are essential for neuronal development and migration, and they play crucial roles in nervous system formation and axonal regeneration. Cytosolic scaffold proteins are key regulators of these processes, and their dysregulation can lead to neurodevelopmental disorders. In flies, Bruchpilot (Brp), an ortholog of the mammalian active zone scaffold protein ELKS/CAST, is a key factor in synapse formation and maintenance and in neurotransmitter release at chemical synapses. Although mammalian ELKS has been implicated in the growth and motility of both neuronal and non-neuronal cells, the roles of Brp beyond synaptic function remain unclear. Here, we show that Brp contributes to neurite outgrowth and cell motility in dissociated embryonic cell cultures of Drosophila. We detected Brp formed discrete cytoplasmic puncta at early developmental stages in dissociated giant neurons derived from cytokinesis-arrested neuroblasts. During neurite extension and branching, the density of Brp puncta increased over time prior to growth events. Consistently, Brp downregulation impeded cell growth. Brp accumulated in the growth cone, the most motile region of dissociated neurons. Brp was also expressed in non-neuronal cell types, such as highly motile hemocytes, raising the possibility that Brp may have broader context-dependent roles in motile cells. Our findings provide the first evidence of a novel functional link between the Brp scaffold and neuronal growth and cell motility, highlighting the conserved multifunctionality of this scaffold protein.