Localized plasma membrane expansion during axon branching mediated by Netrin-1 occurs via TRIM9-dependent regulation of SNARE-mediated vesicle fusion.

Summary Neuronal growth cone filopodia contain guidance receptors and contribute to axon guidance, however the mechanism by which the guidance cue netrin increases filopodia density is unknown. Here we demonstrate that TRIM9, an E3 ubiquitin ligase that localizes to filopodia tips and binds the netrin receptor DCC, interacts with and ubiquitinates the barbed-end polymerase VASP to modulate filopodial stability during netrin-dependent axon guidance. Studies with murine TRIM9+/+ and TRIM9−/− cortical neurons, along with a non-ubiquitinatable VASP mutant, demonstrate that TRIM9-mediated ubiquitination of VASP reduces VASP filopodial tip localization, VASP dynamics at tips, and filopodial stability. Upon netrin treatment, VASP is deubiquitinated, which promotes VASP tip localization and filopodial stability. TRIM9 deletion induces axon guidance defects in vitro and in vivo, whereas a gradient of deubiquitinase inhibition promotes axon turning in vitro. We conclude that a gradient of TRIM9-mediated ubiquitination of VASP creates a filopodial stability gradient during axon turning.

In the presence of netrin, tripartite motif protein 9 (TRIM9) promotes deleted in colorectal cancer (DCC) clustering, but TRIM9-dependent ubiquitination of DCC is reduced. Loss of ubiquitination promotes an interaction between DCC and FAK and FAK activation. FAK activation is required for the progression from SNARE assembly to exocytic vesicle fusion, which supplies membrane material for axon branching.

During hippocampal development, newly born neurons migrate to appropriate destinations, extend axons, and ramify dendritic arbors to establish functional circuitry. These developmental stages are recapitulated in the dentate gyrus of the adult hippocampus, where neurons are continuously generated and subsequently incorporate into existing, local circuitry. Here we demonstrate that the E3 ubiquitin ligase TRIM9 regulates these developmental stages in embryonic and adult-born mouse hippocampal neurons in vitro and in vivo. Embryonic hippocampal and adult-born dentate granule neurons lacking Trim9 exhibit several morphological defects, including excessive dendritic arborization. Although gross anatomy of the hippocampus was not detectably altered by Trim9 deletion, a significant number of Trim9 Ϫ/Ϫ adult-born dentate neurons localized inappropriately. These morphological and localization defects of hippocampal neurons in Trim9 Ϫ/Ϫ mice were associated with extreme deficits in spatial learning and memory, suggesting that TRIM9-directed neuronal morphogenesis may be involved in hippocampal-dependent behaviors.

The generation of axon collateral branches is a fundamental aspect of the development of the nervous system and the response of axons to injury. Although much has been discovered about the signaling pathways and cytoskeletal dynamics underlying branching, additional aspects of the cell biology of axon branching have received less attention. This review summarizes recent advances in our understanding of key factors involved in axon branching. Here we focus on how cytoskeletal mechanisms, intracellular organelles, such as mitochondria and the endoplasmic reticulum, and membrane remodeling (exocytosis and endocytosis) contribute to branch initiation and formation. Together this growing literature provides valuable insight as well as a platform for continued investigation into how multiple aspects of axonal cell biology are spatially and temporally orchestrated to give rise to axon branches.