Feedback-Driven Assembly of the Axon Initial Segment

Amélie Fréal,Dipti Rai,Roderick P Tas,Xingxiu Pan,Eugene A Katrukha,Dieudonnée Van De Willige,Riccardo Stucchi,Amol Aher,Chao Yang,A.F Maarten Altelaar,Karin Vocking,Jan Andries Post,Martin Harterink,Lukas C Kapitein,Anna Akhmanova,Casper C Hoogenraad

doi:10.1016/j.neuron.2019.07.029

Amélie Fréal, Dipti Rai + Show 14 more

Open Access

https://doi.org/10.1016/j.neuron.2019.07.029

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Abstract

SummaryThe axon initial segment (AIS) is a unique neuronal compartment that plays a crucial role in the generation of action potential and neuronal polarity. The assembly of the AIS requires membrane, scaffolding, and cytoskeletal proteins, including Ankyrin-G and TRIM46. How these components cooperate in AIS formation is currently poorly understood. Here, we show that Ankyrin-G acts as a scaffold interacting with End-Binding (EB) proteins and membrane proteins such as Neurofascin-186 to recruit TRIM46-positive microtubules to the plasma membrane. Using in vitro reconstitution and cellular assays, we demonstrate that TRIM46 forms parallel microtubule bundles and stabilizes them by acting as a rescue factor. TRIM46-labeled microtubules drive retrograde transport of Neurofascin-186 to the proximal axon, where Ankyrin-G prevents its endocytosis, resulting in stable accumulation of Neurofascin-186 at the AIS. Neurofascin-186 enrichment in turn reinforces membrane anchoring of Ankyrin-G and subsequent recruitment of TRIM46-decorated microtubules. Our study reveals feedback-based mechanisms driving AIS assembly.

Highlights

The axon initial segment (AIS) is a specialized membrane-associated structure at the base of the axon that generates and shapes the action potential before it is propagated along the axon (Kole and Stuart, 2008)
When targeted to the plasma membrane by AIS membrane proteins, 480AnkGGFP formed long linear stretches that stained positive for tubulin (Figure 1G)
The ability of 480AnkG to organize MT structures at the plasma membrane relies on the interaction with both MTs and AIS membrane proteins

Summary

Introduction

The axon initial segment (AIS) is a specialized membrane-associated structure at the base of the axon that generates and shapes the action potential before it is propagated along the axon (Kole and Stuart, 2008). The AIS contains a concentration of voltage-gated ion channels and cell adhesion molecules that are anchored by a submembranous layer of scaffolds, extending from the plasma membrane to the underlying microtubule (MT) cytoskeleton. Most of the AIS membrane proteins are directly recruited and concentrated by AnkG, including voltage-gated sodium (Nav) and potassium (Kv) channels and adhesion molecules, such as the 186-kDa isoform of Neurofascin (NF186) (Leterrier, 2018). The interaction between EBs and AnkG is necessary for both AIS formation and maintenance (Freal et al, 2016; Leterrier et al, 2011) These data highlight the importance of the association between the AIS submembrane complex and the underlying MT cytoskeleton. The molecular mechanisms connecting axonal MT organization and AIS formation are incompletely understood

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