Abstract
The cytoskeleton microtubule consists of polymerized αβ-tubulin dimers and plays essential roles in many cellular events. Reagents that inhibit microtubule behaviors have been developed as antifungal, antiparasitic, and anticancer drugs. Benzimidazole compounds, including thiabendazole (TBZ), carbendazim (MBC), and nocodazole, are prevailing microtubule poisons that target β-tubulin and inhibit microtubule polymerization. The molecular basis, however, as to how the drug acts on β-tubulin remains controversial. Here, we characterize the S. pombe β-tubulin mutant nda3-TB101, which was previously isolated as a mutant resistance to benzimidazole. The mutation site tyrosine at position 50 is located in the interface of two lateral β-tubulin proteins and at the gate of a putative binging pocket for benzimidazole. Our observation revealed two properties of the mutant tubulin. First, the dynamics of cellular microtubules comprising the mutant β-tubulin were stabilized in the absence of benzimidazole. Second, the mutant protein reduced the affinity to benzimidazole in vitro. We therefore conclude that the mutant β-tubulin Nda3-TB101 exerts a dual effect on microtubule behaviors: the mutant β-tubulin stabilizes microtubules and is insensitive to benzimidazole drugs. This notion fine-tunes the current elusive molecular model regarding binding of benzimidazole to β-tubulin.
Highlights
Microtubules are a cytoskeleton essential for a number of cellular events in eukaryotes and in prokaryotes
Similar results had been obtained with antitubulin immunofluorescence images in the previous study in order to demonstrate the performance of MBC in comparison to TBZ [48]. These results demonstrate that microtubules assembled in the nda3-TB101 mutation appeared normal in the absence of benzimidazole drugs and that microtubules in the mutant are insensitive to the drugs
C, microtubule dynamics were not altered by either TBZ, MBC, nor nocodazole: frequencies of catastrophe and rescue did not change in comparison to those without drugs. These results indicate that the Y50S mutation of β-tubulin per se moderately stabilized microtubule dynamics, and the property was not altered by the addition of benzimidazole compounds
Summary
Microtubules are a cytoskeleton essential for a number of cellular events in eukaryotes and in prokaryotes. Microtubules are organized in the cytoplasm to form a network array during interphase. As αβ-tubulin dimers are aligned in a unidirectional manner, a microtubule filament possesses the polarity in direction: the end that exposes α-tubulin is called the minus end, whilst the other end exposing β-tubulin is called the plus end. Tubulins are a GTP-binding protein (GTPase), and they are GTP-bound before and right after incorporation into a microtubule, whereas GTP is hydrolyzed into GDP gradually after incorporation. Microtubules are stable in the GTP-bound form but unstable when hydrolyzed into GDP. These are the properties of tubulin dimers in polarity, and in nucleotide binding conditions, a microtubule displays dynamic instability in which the plus end is more dynamic than the minus end
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