A pair of E3 ubiquitin ligases compete to regulate filopodial dynamics and axon guidance

Abstract

Appropriate axon guidance is necessary to form accurate neuronal connections. Axon guidance cues that stimulate cytoskeletal reorganization within the growth cone direct axon navigation. Filopodia at the growth cone periphery have long been considered sensors for axon guidance cues, yet how they respond to extracellular cues remains ill defined. Our previous work found that the filopodial actin polymerase VASP and consequently filopodial stability are negatively regulated via nondegradative TRIM9-dependent ubiquitination. Appropriate VASP ubiquitination and deubiquitination are required for axon turning in response to the guidance cue netrin-1. Here we show that the TRIM9-related protein TRIM67 outcompetes TRIM9 for interacting with VASP and antagonizes TRIM9-dependent VASP ubiquitination. The surprising antagonistic roles of two closely related E3 ubiquitin ligases are required for netrin-1–dependent filopodial responses, axon turning and branching, and fiber tract formation. We suggest a novel model in which coordinated regulation of VASP ubiquitination by a pair of interfering ligases is a critical element of VASP dynamics, filopodial stability, and axon guidance.

The TRIM9/TRIM67 neuronal interactome reveals novel activators of morphogenesis

“…We have previously shown that deletion of Trim9 or Trim67 independently disrupt axon branching (Winkle et al, 2014;Menon et al, 2015;Plooster et al, 2017;Boyer et al, 2020). In Trim67 -/neurons, basal levels of axon branching are normal, but do not increase in response to netrin-1 addition.…”

“…Since the interaction between TRIM67 and Myo16 appeared stronger, we hypothesized that TRIM67 may alter Myo16 ubiquitination. To test this, we performed denaturing immunoprecipitation-based ubiquitination assays of GFP-Myo16 from HEK293 cells in which TRIM67 had been removed via CRISPR Cas9 genome editing (Boyer et al, 2020). GFP-Myo16 immunoprecipitation contained high molecular weight ubiquitin reactivity in TRIM67 -/-HEK cells expressing Myc alone or Myc-TRIM67 ( Figure 5E).…”

“…We have found that TRIM9 and TRIM67 mediate morphological changes downstream of netrin and DCC by regulating cytoskeletal dynamics and exocytosis (Winkle et al, 2014;Menon et al, 2015;Plooster et al, 2017;Urbina et al, 2018;Boyer et al, 2020). TRIM9 and TRIM67 interact with and modulate ubiquitination of the actin polymerase VASP to regulate growth cone dynamics and netrin-dependent axonal turning (Menon et al, 2015;Boyer et al, 2020).…”

The ubiquitylome of developing cortical neurons

“…TRIM9 and TRIM67 are neuronally-enriched E3 ubiquitin ligases (Boyer et al, 2018a; Berti et al, 2002). Both genes are required for neuronal morphological responses to the axon guidance cue netrin-1 (Winkle et al, 2014; Boyer et al, 2018; Plooster et al, 2017; Boyer et al, 2020; Menon et al, 2015; Urbina et al, 2018; Winkle et al, 2016). For example, our previously published work demonstrated that the actin polymerase VASP and the netrin receptor DCC exhibit TRIM9 dependent ubiquitylation that is lost upon netrin stimulation (Menon et al, 2015; Plooster et al, 2017; Boyer et al, 2020).…”

“…Both genes are required for neuronal morphological responses to the axon guidance cue netrin-1 (Winkle et al, 2014; Boyer et al, 2018; Plooster et al, 2017; Boyer et al, 2020; Menon et al, 2015; Urbina et al, 2018; Winkle et al, 2016). For example, our previously published work demonstrated that the actin polymerase VASP and the netrin receptor DCC exhibit TRIM9 dependent ubiquitylation that is lost upon netrin stimulation (Menon et al, 2015; Plooster et al, 2017; Boyer et al, 2020). Deletion of either gene in the mouse results in subtle neuroanatomical anomalies yet overt deficits in spatial learning and memory (Menon et al, 2015; Winkle et al, 2016; Boyer et al, 2018).…”