The membrane periodic skeleton is an actomyosin network that regulates axonal diameter and conduction.

Ana Rita Costa,Inês Mendes Pinto,Boris Y Rubinstein,Paulo Aguiar,José C Mateus,Xuewei Wang,Feng-Quan Zhou,Luis Pajuelo,Marko Lampe,Sara C Sousa,David Rosa,Paula Sampaio,António J Pereira,Diana Machado,Cátia Df Lopes,Ana Catarina Costa,Monica M Sousa,Rita Pinto-Costa

doi:10.7554/elife.55471

Abstract

Neurons have a membrane periodic skeleton (MPS) composed of actin rings interconnected by spectrin. Here, combining chemical and genetic gain- and loss-of-function assays, we show that in rat hippocampal neurons the MPS is an actomyosin network that controls axonal expansion and contraction. Using super-resolution microscopy, we analyzed the localization of axonal non-muscle myosin II (NMII). We show that active NMII light chains are colocalized with actin rings and organized in a circular periodic manner throughout the axon shaft. In contrast, NMII heavy chains are mostly positioned along the longitudinal axonal axis, being able to crosslink adjacent rings. NMII filaments can play contractile or scaffolding roles determined by their position relative to actin rings and activation state. We also show that MPS destabilization through NMII inactivation affects axonal electrophysiology, increasing action potential conduction velocity. In summary, our findings open new perspectives on axon diameter regulation, with important implications in neuronal biology.

Highlights

When considering an adult axon, its diameter can oscillate depending on organelle transport (Greenberg et al, 1990), neuronal activity (Fields, 2011), deformations generated by movement or degeneration
The function of non-muscle myosin II (NMII) is controlled by myosin light chain (MLC) kinase (MLCK) that phosphorylates the NMII regulatory light chains (RLC) leading to conformational changes and self-assembly in myosin filaments (Vicente-Manzanares et al, 2009; Figure 1A)
ML-7, a selective MLCK inhibitor that decreases pMLC levels in hippocampal neurons (Figure 1—figure supplement 1A, B), led to an increase in axonal diameter similar to that produced by blebbistatin (Figure 1D,E)

Summary

Introduction

When considering an adult axon, its diameter can oscillate depending on organelle transport (Greenberg et al, 1990), neuronal activity (Fields, 2011), deformations generated by movement or degeneration. In the initial MPS model, each ring was hypothesized to be composed of actin filaments capped by the actin-binding protein adducin (Xu et al, 2013). Combining platinum-replica electron and optical super-resolution microscopy, the MPS actin rings were shown to be made of two long, intertwined actin filaments (Vassilopoulos et al, 2019). According to this novel view, adducin might be responsible to enhance the lateral binding of spectrin to actin. Since reduction in axon diameter with time occurs both in WT and a-adducin knock-out (KO) neurons, MPS dynamics is probably regulated by additional actin-binding proteins. We demonstrate that the MPS affects signal propagation velocity, a property with important functional implications

Results and discussion

A Non-competent NMII

Materials and methods