The TRIM9/TRIM67 neuronal interactome reveals novel activators of morphogenesis

Abstract

TRIM9 and TRIM67 are neuronally-enriched E3 ubiquitin ligases essential for appropriate morphogenesis of cortical and hippocampal neurons and fidelitous responses to the axon guidance cue netrin-1. Deletion of murine Trim9 or Trim67 results in neuroanatomical defects and striking behavioral deficits, particularly in spatial learning and memory. TRIM9 and TRIM67 interact with cytoskeletal and exocytic proteins, but the full interactome is not known. Here we performed the unbiased proximity-dependent biotin identification (BioID) approach to define TRIM9 and TRIM67 protein-protein proximity network in developing cortical neurons and identified neuronal putative TRIM interaction partners. Candidates included cytoskeletal regulators, cytosolic protein transporters, exocytosis and endocytosis regulators, and proteins necessary for synaptic regulation. A subset of high priority candidates was validated, including Myo16, Coro1A, SNAP47, ExoC1, GRIP1, PRG-1, and KIF1A. For a subset of validated candidates, we utilized TIRF microscopy to demonstrate dynamic colocalization with TRIM proteins at the axonal periphery, including at the tips of filopodia. Further analysis demonstrated the RNAi-based knockdown of the unconventional myosin Myo16 in cortical neurons altered axonal branching patterns in a TRIM9 and netrin-1 dependent manner. Future analysis of other validated candidates will likely identify novel proteins and mechanisms by which TRIM9 and TRIM67 regulate neuronal form and function.

TRIM67 regulates exocytic mode and neuronal morphogenesis via SNAP47

“…However, simulations in Trim9 −/− neurons overestimated expansion, suggesting possibly that endocytosis was also elevated in these neurons. Consistent with this idea, we recently identified several candidate TRIM9 interactors associated with clathrin coats ( Menon et al, 2020 ). We conclude that VAMP2-mediated fusion is the primary source of membrane addition in developing neurons and that manipulating exocytic frequency or mode alters neuronal growth.…”

“…Because few interaction partners of TRIM67 are known, we used the BioID method ( Roux et al, 2012 ) to identify candidate TRIM67-interacting partners that may regulate exocytosis ( Menon et al, 2020 ). One interesting candidate and the only SNARE protein significantly enriched (~4-fold) over the negative control (Myc-BirA*; Table S1 ) was SNAP47, a t-SNARE in the SNAP25 family.…”

“…TRIM67 interacts with the actin-polymerase VASP at filopodia tips in the growth cone, a highly dynamic region with local changes in membrane tension and actin remodeling ( Boyer et al, 2020 ; Staykova et al, 2011 ; Wen et al, 2016 ), but whether VASP alters exocytosis is not known. Proximity biotinylation approaches revealed several candidate TRIM67 interactors that are poised to regulate exocytosis, including the exocyst complex, tomosyn, and Munc18 ( Menon et al, 2020 ). Separate pools of vesicles with distinct fusion characteristics and cargo ( Hua et al, 2011 ; Rizzoli and Jahn, 2007 ) may fuse with different modes of fusion.…”

“…Interactions were sorted by SAINT’s interaction probability and a false discovery rate threshold of 25% was employed. A full list of identified proteins are reported elsewhere ( Menon et al, 2020 ).…”

The ubiquitylome of developing cortical neurons

“…In a previous study, we identified a putative TRIM9 and TRIM67 interactome using proximitydependent labeling with the promiscuous biotin ligase (BirA*) attached to either TRIM9 or TRIM67 (Menon et al, 2020). A comparison of the ubiquitylated proteome from developing brains with the top 0.25% of the interaction candidates identified peptides from 55 proteins that showed an increase or decrease (albeit insignificant) in ubiquitylation levels in the absence of Trim9, Trim67 , or both relative to wildtype (Figure 1B-D).…”