Tubulin Tyrosination Is Required for the Proper Organization and Pathfinding of the Growth Cone

Séverine Marcos,Stéphanie Backer,Evelyne Bloch-Gallego,Julie Moreau,Didier Job,Annie Andrieux,Michael Hendricks

doi:10.1371/journal.pone.0005405

Abstract

BackgroundDuring development, neuronal growth cones integrate diffusible and contact guidance cues that are conveyed to both actin and microtubule (MT) cytoskeletons and ensure axon outgrowth and pathfinding. Although several post-translational modifications of tubulin have been identified and despite their strong conservation among species, their physiological roles during development, especially in the nervous sytem, are still poorly understood.Methodology/FindingsHere, we have dissected the role of a post-translational modification of the last amino acid of the α-tubulin on axonal growth by analyzing the phenotype of precerebellar neurons in Tubulin tyrosin ligase knock-out mice (TTL −/−) through in vivo, ex vivo and in vitro analyses. TTL −/− neurons are devoid of tyrosinated tubulin. Their pathway shows defects in vivo, ex vivo, in hindbrains open-book preparations or in vitro, in a collagen matrix. Their axons still orient toward tropic cues, but they emit supernumerary branches and their growth cones are enlarged and exhibit an emission of mis-oriented filopodia. Further analysis of the TTL −/− growth cone intracellular organization also reveals that the respective localization of actin and MT filaments is disturbed, with a decrease in the distal accumulation of Myosin IIB, as well as a concomitant Rac1 over-activation in the hindbrain. Pharmacological inhibition of Rac1 over-activation in TTL −/− neurons can rescue Myosin IIB localization.Conclusions/SignificanceIn the growth cone, we propose that tubulin tyrosination takes part in the relative arrangement of actin and MT cytoskeletons, in the regulation of small GTPases activity, and consequently, in the proper morphogenesis, organization and pathfinding of the growth cone during development.

Highlights

During embryonic development, axons have to make choices in order to reach their target in the nervous system
The tyrosine residue is removed by a carboxypeptidase and tubulin tyrosine ligase (TTL) brings an additional tyrosine residue According to the level of transcription-translation in a cell, the amount in neosynthesized tyr-tubulin varies
We show that tubulin retyrosination plays a major role in the morphology and functionality of the growth cone

Summary

Introduction

Axons have to make choices in order to reach their target in the nervous system. The neuronal growth cone is a dynamic structure at the tip of the axon that provides the traction force necessary for axon outgrowth and acts as a sensor. It explores the environment by integrating guidance cues that influence its direction and remodeling. The actin cytoskeleton has been proposed as a major target of guidance cues due to its peripheral location in the growth cone and its crucial role in extension and retraction of filopodia and lamellipodia when they encounter tropic cues, the microtubule (MT) cytoskeleton has been involved in the guiding process through intracellular signaling pathways [3]. Neuronal growth cones integrate diffusible and contact guidance cues that are conveyed to both actin and microtubule (MT) cytoskeletons and ensure axon outgrowth and pathfinding. Several post-translational modifications of tubulin have been identified and despite their strong conservation among species, their physiological roles during development, especially in the nervous sytem, are still poorly understood

Methods

Results

Conclusion