Spatiotemporal manipulation of ciliary glutamylation reveals its roles in intraciliary trafficking and Hedgehog signaling

Shi-Rong Hong,Ning Hsu,Cuei-Ling Wang,Rajat Rohatgi,Wei-En Huang,Takanari Inoue,Yu-Chun Lin,Yueh-Chen Chiang,Nathan C Shaner,Yao-Shen Huang,Hsiao-Chi Cheng,Chun-Yu Lin,Ganesh V Pusapati,Ya-Chu Chang,Yu-Chen Chang

doi:10.1038/s41467-018-03952-z

Abstract

Tubulin post-translational modifications (PTMs) occur spatiotemporally throughout cells and are suggested to be involved in a wide range of cellular activities. However, the complexity and dynamic distribution of tubulin PTMs within cells have hindered the understanding of their physiological roles in specific subcellular compartments. Here, we develop a method to rapidly deplete tubulin glutamylation inside the primary cilia, a microtubule-based sensory organelle protruding on the cell surface, by targeting an engineered deglutamylase to the cilia in minutes. This rapid deglutamylation quickly leads to altered ciliary functions such as kinesin-2-mediated anterograde intraflagellar transport and Hedgehog signaling, along with no apparent crosstalk to other PTMs such as acetylation and detyrosination. Our study offers a feasible approach to spatiotemporally manipulate tubulin PTMs in living cells. Future expansion of the repertoire of actuators that regulate PTMs may facilitate a comprehensive understanding of how diverse tubulin PTMs encode ciliary as well as cellular functions.

Highlights

Tubulin post-translational modifications (PTMs) occur spatiotemporally throughout cells and are suggested to be involved in a wide range of cellular activities
We assessed the potential effect of overexpression of Cerulean3–FKBP–rapamycin-binding domain (FRB)–MAP4m on the primary cilia
The rates of intraflagellar transport (IFT) in anterograde and retrograde directions in the cilia with Cerulean3–FRB–MAP4m were not significantly different from those under control conditions (Supplementary Fig. 1d,e). These results confirmed that Cerulean3–FRB– MAP4m is a valid fusion construct to be anchored at the ciliary axoneme

Summary

Introduction

Tubulin post-translational modifications (PTMs) occur spatiotemporally throughout cells and are suggested to be involved in a wide range of cellular activities. We develop a method to rapidly deplete tubulin glutamylation inside the primary cilia, a microtubule-based sensory organelle protruding on the cell surface, by targeting an engineered deglutamylase to the cilia in minutes This rapid deglutamylation quickly leads to altered ciliary functions such as kinesin-2-mediated anterograde intraflagellar transport and Hedgehog signaling, along with no apparent crosstalk to other PTMs such as acetylation and detyrosination. The genetic or morpholino-mediated perturbation of polyglutamylases or deglutamylases across different model organisms results in morphological and/or functional defects in cilia and flagella[19,20,21,22,23,24,25,26,27,28,29,30,31,32,33] These studies strongly suggest the importance of tubulin polyglutamylation in the structural integrity and functionality of microtubules in cilia, as well as other subcellular compartments. Constitutive genetic perturbation often allows for compensation where cells adapt to their new genetic environment, likely leading to a missed detection of immediate consequences of loss-of-function, such as an effect on transient interactions between tubulins with specific

Methods

Results

Conclusion