Myosin Va regulates terminal translocation in the neocortex by controlling the trafficking of Neuropilin-1

Proper neocortex formation relies on the precisely regulated migration of neurons into distinct cortical layers. This process requires the timely surface expression of receptors that decode extracellular guidance cues. Yet, the molecular machinery governing receptor trafficking in migrating neurons remains largely unclear. Here, we identified the motor protein myosin Va (Myo5a) as a key regulator of Neuropilin-1 (Nrp1) trafficking in the early postnatal neocortex. Both male and female mice were used for this study. Myo5a localized to the apical dendrites of superficial layer neurons, with expression increasing during cortical maturation. Functional inhibition of Myo5a led to a terminal translocation defect in superficial layer neurons, thereby preventing their proper entry into NeuN-negative regions of the neocortex. Additionally, Myo5a inhibition led to Nrp1 accumulation within the Golgi apparatus and a significant reduction in its surface expression. Remarkably, overexpression of Nrp1 or VLDLR fully rescued the terminal translocation defects and dendritic abnormalities caused by Myo5a inhibition, demonstrating that Myo5a-dependent Nrp1 trafficking underlies proper Reelin receptor availability during this process. Overall, these results reveal a pivotal Myo5a–Nrp1 trafficking pathway that governs the final stage of neuronal migration, offering a molecular mechanism for the spatial and temporal regulation of receptor dynamics essential for precise cortical layering. Significance Statement Proper neocortex formation is essential for establishing functional brain circuits; however, the mechanisms by which migrating neurons interpret extracellular cues remain poorly understood. This study identified the motor protein Myo5a as a key regulator of the cell-surface expression of the Reelin coreceptor Nrp1 during the final phase of neuronal migration. Myo5a inhibition disrupts Nrp1 trafficking to the plasma membrane , leading to terminal translocation defects and abnormal dendritic development. Overexpression of Nrp1 or VLDLR fully rescues both phenotypes, indicating that Myo5a-dependent Nrp1 trafficking is essential for proper Reelin receptor availability at the cell surface. These findings reveal a previously unrecognized mechanism that enables neurons to respond properly to Reelin signaling, thus ensuring precise cortical layer formation during brain development.