Ndfip Proteins Target Robo Receptors for Degradation and Allow Commissural Axons to Cross the Midline in the Developing Spinal Cord

Abstract

SUMMARY Commissural axons initially respond to attractive signals at the midline, but once they cross, they become sensitive to repulsive cues. This switch prevents axons from re-entering the midline. In insects and mammals, negative regulation of Roundabout (Robo) receptors prevents premature response to the midline repellant Slit. In Drosophila, the endosomal protein Commissureless (Comm) prevents Robo1 surface expression before midline crossing by diverting Robo1 into late endosomes. Notably, Comm is not conserved in vertebrates. We identified two Nedd-4-interacting proteins, Ndfip1 and Ndfip2, that act analogously to Comm to localize Robo1 to endosomes. Ndfip proteins recruit Nedd4 E3 ubiquitin ligases to promote Robo1 ubiquitylation and degradation. Ndfip proteins are expressed in commissural axons in the developing spinal cord and removal of Ndfip proteins results in increased Robo1 expression and reduced midline crossing. Our results define a conserved Robo1 intracellular sorting mechanism between flies and mammals to avoid premature responsiveness to Slit.

A Spatiotemporal Sequence of Sensitization to Slits and Semaphorins Orchestrates Commissural Axon Navigation

“…It is unlikely responsible for specifying the timing of surface sorting because our expression vectors do not encode the regulatory sequences underlying microRNA regulation. Ndfip1/2 proteins recruiting Nedd4E3 ubiquitin ligases were discovered to target Robo1 in endosomes for degradation before FP crossing (Gorla et al, 2019).…”

Conserved and divergent aspects of Robo receptor signaling and regulation betweenDrosophilaRobo1 andC. elegansSAX-3

“…We and others have shown that the perimembrane region and Fn3 domain of Robo1 are each required for downregulation by Comm, but Robo1 variants lacking these regions are still subject to sorting-independent Comm antagonism (Gilestro 2008;Brown et al 2018). Comm appears to be conserved only within insects, and Comm orthologs have not been identified in nematodes or other animal groups (though Ndfip and PRRG4 proteins have been proposed as functional analogs of Comm in mammals (Justice et al 2017;Gorla et al 2019)). We therefore asked whether SAX-3 is sensitive to endosomal sorting or sorting-independent antagonism by Drosophila Comm when expressed in Drosophila neurons.…”

“…Drosophila Robo1 is also subject to negative regulation by the endosomal sorting receptor Commissureless (Comm) (Tear et al 1996;Kidd et al 1998b;Keleman et al 2002;) and a second Robo family member, Robo2, which interacts with Robo1 in trans to inhibit premature Slit response in pre-crossing commissural axons (Simpson et al 2000b;Spitzweck et al 2010;Evans et al 2015). As Comm and Robo2 appear to be conserved only within a subset of insect species, it is not yet clear how well conserved the signaling and regulatory mechanisms of Drosophila Robo1 are, although the mammalian Nedd4-family interacting proteins Ndfip1 and Ndfip2 can act analogously to Drosophila Comm to regulate surface levels of Robo1 on precrossing commissural axons in the spinal cord (Gorla et al 2019) and the divergent Robo3/Rig-1 receptor in mammals appears to be able to antagonize Slit-Robo repulsion via a mechanism that is distinct from Drosophila Robo2 (Sabatier et al 2004;Jaworski et al 2010;Zelina et al 2014). The mammalian PRRG4 protein can also regulate subcellular distribution of mammalian Robo1, similar to Comm's effect on Drosophila Robo1, though whether PRRG4 influences midline crossing in mammalian neurons is not yet known (Justice et al 2017).…”

Conserved and divergent aspects of Robo receptor signaling and regulation betweenDrosophilaRobo1 andC. elegansSAX-3

“…Drosophila Robo1 is also subject to negative regulation by the endosomal sorting receptor Commissureless (Comm) ( Tear et al 1996 ; Kidd et al 1998b ; Keleman et al 2002 , 2005 ) and a second Robo family member, Robo2, which interacts with Robo1 in trans to inhibit premature Slit response in precrossing commissural axons ( Simpson et al 2000b ; Spitzweck et al 2010 ; Evans et al 2015 ). As Comm and Robo2 appear to be conserved only within a subset of insect species, it is not yet clear how well conserved the signaling and regulatory mechanisms of Drosophila Robo1 are, although the mammalian Nedd4-family interacting proteins Ndfip1 and Ndfip2 can act analogously to Drosophila Comm to regulate surface levels of Robo1 on precrossing commissural axons in the spinal cord ( Gorla et al 2019 ) and the divergent Robo3/Rig-1 receptor in mammals appears to be able to antagonize Slit-Robo repulsion via a mechanism that is distinct from Drosophila Robo2 ( Sabatier et al 2004 ; Jaworski et al 2010 ; Zelina et al 2014 ). The mammalian PRRG4 protein can also regulate subcellular distribution of mammalian Robo1, similar to Comm’s effect on Drosophila Robo1, though whether PRRG4 influences midline crossing in mammalian neurons is not yet known ( Justice et al 2017 ).…”