Sulfo-SMCC Prevents Annealing of Taxol-Stabilized Microtubules In Vitro

TL;DR

This study demonstrates that the chemical crosslinker sulfo-SMCC inhibits the end-to-end annealing of taxol-stabilized microtubules in vitro by blocking cysteine sulfhydryl groups, affecting microtubule length distribution.

Contribution

It reveals that sulfo-SMCC prevents microtubule annealing by targeting cysteine sulfhydryl groups, a novel insight into microtubule stabilization mechanisms.

Findings

Sulfo-SMCC blocks microtubule annealing in vitro.

Maleimide dye alone does not prevent annealing.

Cysteine sulfhydryl groups are crucial for microtubule polymerization and annealing.

Abstract

Microtubule structure and functions have been widely studied in vitro and in cells. Research has shown that cysteines on tubulin play a crucial role in the polymerization of microtubules. Here, we show that blocking sulfhydryl groups of cysteines in taxol-stabilized polymerized microtubules with a commonly used chemical crosslinker prevents temporal end-to-end annealing of microtubules in vitro. This can dramatically affect the length distribution of the microtubules. The crosslinker sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, sulfo-SMCC, consists of a maleimide and an N-hydroxysuccinimide ester group to bind to sulfhydryl groups and primary amines, respectively. Interestingly, addition of a maleimide dye alone does not show the same interference with annealing in stabilized microtubules. This study shows that the sulfhydryl groups of cysteines of tubulin that are vital for the polymerization are also important for the subsequent annealing of microtubules.