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.
Highlights
Microtubules (MTs) are cytoskeletal filaments that are vital for cell division and vesicle transport in cells [1]
We have shown through systematic experiments that sulfo-SMCC inhibits end-to-end annealing of taxol-stablized MTs
We speculate that the maleimide group on sulfo-SMCC binds to thiol groups on MT ends, and thereby prevents annealing
Summary
Microtubules (MTs) are cytoskeletal filaments that are vital for cell division and vesicle transport in cells [1]. The polarity of MTs is crucial for their biological function, making it possible for molecular motors to travel along MTs in a specific direction. Polymerization and depolymerization kinetics at the ends of MTs are dependent on the state of the nucleotide (GTP/GDP) bound to each tubulin monomer, and GTP hydrolysis, coupled to polymerization, makes MTs non-equilibrium polymers. MTs undergo various post-translational modifications and are the substrate for a multitude of MT-binding proteins that control many functions [6]. Because in vivo experiments are difficult to interpret due to the complexity of cells, protocols for polymerizing microtubules (MTs) in vitro have been developed providing a more controlled system for the study of MT structure and function [7,8,9]
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