Directed membrane trafficking is believed to be crucial for axon development during neuronal morphogenesis. However, the underlying mechanisms are poorly understood. Here, we report a role of Lgl1, the mammalian homolog of Drosophila tumor suppressor Lethal giant larvae, in controlling membrane trafficking underlying axonal growth. We find that Lgl1 is associated with plasmalemmal precursor vesicles and enriched in developing axons. Lgl1 upregulation promoted axonal growth, whereas downregulation attenuated it as well as directional membrane insertion. Interestingly, Lgl1 interacted with and activated Rab10, a small GTPase that mediates membrane protein trafficking, by releasing GDP dissociation inhibitor (GDI) from Rab10. Furthermore, Rab10 lies downstream of Lgl1 in axon development and directional membrane insertion. Finally, both Lgl1 and Rab10 are required for neocortical neuronal polarization in vivo. Thus, the Lgl1 regulation of Rab10 stimulates the trafficking of membrane precursor vesicles, whose fusion with the plasmalemma is crucial for axonal growth.

Polarized membrane addition is crucial for axon development and elongation during neuronal morphogenesis. This process is believed to be regulated by directed membrane trafficking of Rab10-containing post-Golgi carriers. However, the mechanisms underlying the biogenesis of these carriers remain unclear. Here, we report that Rab10 interaction with myosin Vb (MYO5B) determines the formation of Rab10 carriers and is important for axon development. Rab10 interacts with the exon D-encoded domain of MYO5B. Downregulating the expression of MYO5B ( þ D) or blocking its interaction with Rab10 impairs the fission of Rab10 vesicles from trans-Golgi membranes, causes a decrease in the number of Rab10 transport carriers and inhibits axon development in cultured hippocampal neurons. Furthermore, the MYO5B-Rab10 system is required for axon development of vertebrate neocortical neurons or zebrafish retinal ganglion cells in vivo. Thus, specific interaction between Rab10 and MYO5B controls the formation of Rab10 vesicles, which is required for axon development.

Axon development requires membrane addition from the intracellular supply, which has been shown to be mediated by Rab10-positive plasmalemmal precursor vesicles (PPVs). However, the molecular mechanisms underlying the membrane trafficking processes of PPVs remain unclear. Here, we show that myristoylated alanine-rich C-kinase substrate (MARCKS) mediates membrane targeting of Rab10-positive PPVs, and this regulation is critical for axon development. We found that the GTP-locked active form of Rab10 binds to membrane-associated MARCKS, whose affinity depends on the phosphorylation status of the MARCKS effector domain. Either genetic silencing of MARCKS or disruption of its interaction with Rab10 inhibited axon growth of cortical neurons, impaired docking and fusion of Rab10 vesicles with the plasma membrane, and consequently caused a loss of membrane insertion of axonal receptors responsive to extracellular axon growth factors. Thus, this study has identified a novel function of MARCKS in mediating membrane targeting of PPVs during axon development.