Genetically encoded live-cell sensor for tyrosinated microtubules.

Prakash Lama,Balaji M Rao,Satish Bodakuntla,Carsten Janke,Shubham Kesarwani,Minhajuddin Sirajuddin,Anchal Chandra,A.S Jijumon,P Purushotam Reddy,Ranabir Das

doi:10.1083/jcb.201912107

Abstract

Microtubule cytoskeleton exists in various biochemical forms in different cells due to tubulin posttranslational modifications (PTMs). Tubulin PTMs are known to affect microtubule stability, dynamics, and interaction with MAPs and motors in a specific manner, widely known as tubulin code hypothesis. At present, there exists no tool that can specifically mark tubulin PTMs in living cells, thus severely limiting our understanding of their dynamics and cellular functions. Using a yeast display library, we identified a binder against terminal tyrosine of α-tubulin, a unique PTM site. Extensive characterization validates the robustness and nonperturbing nature of our binder as tyrosination sensor, a live-cell tubulin nanobody specific towards tyrosinated microtubules. Using this sensor, we followed nocodazole-, colchicine-, and vincristine-induced depolymerization events of tyrosinated microtubules in real time and found each distinctly perturbs the microtubule polymer. Together, our work describes a novel tyrosination sensor and its potential applications to study the dynamics of microtubule and their PTM processes in living cells.

Highlights

Microtubules are cytoskeleton tubular polymers that perform diverse cellular functions, including intracellular cargo transport, chromosome segregation, and cell motility
We screened a yeast display library (Gera et al, 2012) against α-tubulin CTT and identified a binder molecule. We demonstrate that this binder is specific toward the tyrosinated state of tubulin and does not interfere with the cellular or microtubule-based functions when expressed in living cells
Using structural illumination microscopy (SIM), here we demonstrate the application of the tyrosination sensor in superresolution microscopy that yields resolution similar to the SiRtubulin probe (Fig. 8)

Summary

Introduction

Microtubules are cytoskeleton tubular polymers that perform diverse cellular functions, including (but not limited to) intracellular cargo transport, chromosome segregation, and cell motility. These cellular processes are mediated by interactions between microtubules and a cohort of molecular motors and microtubule-associated proteins (MAPs). Several tubulin PTMs, such as acetylation (L’Hernault and Rosenbaum, 1985), glutamylation (Eddeet al., 1990), and glycylation (Redeker et al, 1994), and their respective enzymes have been identified across species These PTMs, with the exception of acetylation, occur at the C-terminal tails (CTTs) of either α- and/or β-tubulin gene products. This, in turn, has allowed for in vitro reconstitution studies that have highlighted how single PTMs can uniquely modulate molecular motors (Barisic et al, 2015; McKenney et al, 2016; Nirschl et al, 2016; Sirajuddin et al, 2014), MAPs

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