Shalini Menon

The E3 Ubiquitin Ligase TRIM9 Is a Filopodia Off Switch Required for Netrin-Dependent Axon Guidance

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.

Building Blocks of Functioning Brain: Cytoskeletal Dynamics in Neuronal Development

Neural connectivity requires proper polarization of neurons, guidance to appropriate target locations, and establishment of synaptic connections. From when neurons are born to when they finally reach their synaptic partners, neurons undergo constant rearrangment of the cytoskeleton to achieve appropriate shape and polarity. Of particular importance to neuronal guidance to target locations is the growth cone at the tip of the axon. Growth-cone steering is also dictated by the underlying cytoskeleton. All these changes require spatiotemporal control of the cytoskeletal machinery. This review summarizes the proteins that are involved in modulating the actin and microtubule cytoskeleton during the various stages of neuronal development.

TRIM9-dependent ubiquitination of DCC constrains kinase signaling, exocytosis, and axon branching

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.

Mammalian TRIM67 Functions in Brain Development and Behavior

Abstract Class I members of the tripartite motif (TRIM) family of E3 ubiquitin ligases evolutionarily appeared just prior to the advent of neuronal like cells and have been implicated in neuronal development from invertebrates to mammals. The single Class I TRIM in Drosophila melanogaster and Caenorhabditis elegans and the mammalian Class I TRIM9 regulate axon branching and guidance in response to the guidance cue netrin, whereas mammalian TRIM46 establishes the axon initial segment. In humans, mutations in TRIM1 and TRIM18 are implicated in Opitz Syndrome, characterized by midline defects and often intellectual disability. We find that although TRIM67 is the least studied vertebrate Class I TRIM, it is the most evolutionarily conserved. Here we show that mammalian TRIM67 interacts with both its closest paralog TRIM9 and the netrin receptor DCC and is differentially enriched in specific brain regions during development and adulthood. We describe the anatomical and behavioral consequences of deletion of murine Trim67. While viable, mice lacking Trim67 exhibit abnormal anatomy of specific brain regions, including hypotrophy of the hippocampus, striatum, amygdala, and thalamus, and thinning of forebrain commissures. Additionally, Trim67−/− mice display impairments in spatial memory, cognitive flexibility, social novelty preference, muscle function, and sensorimotor gating, whereas several other behaviors remain intact. This study demonstrates the necessity for TRIM67 in appropriate brain development and behavior.