The antifungal agent benomyl [methyl-1-(butylcarbamoyl)-2-benzimidazolecarbamate] is used throughout the world against a wide range of agricultural fungal diseases. In this paper, we investigated the interaction of benomyl with mammalian brain tubulin and microtubules. Using the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulfonic acid, benomyl was found to bind to brain tubulin with a dissociation constant of 11.9 +/- 1.2 microM. Further, benomyl bound to at a novel site, distinct from the well-characterized colchicine and vinblastine binding sites. Benomyl altered the far-UV circular dichroism spectrum of tubulin and reduced the accessibility of its cysteine residues to modification by 5,5'-dithiobis-2-nitrobenzoic acid, indicating that benomyl binding to tubulin induces a conformational change in the tubulin. Benomyl inhibited the polymerization of brain tubulin into microtubules, with 50% inhibition occurring at a concentration of 70-75 microM. Furthermore, it strongly suppressed the dynamic instability behavior of individual brain microtubules in vitro as determined by video microscopy. It reduced the growing and shortening rates of the microtubules but did not alter the catastrophe or rescue frequencies. The unexpected potency of benomyl against mammalian microtubule polymerization and dynamics prompted us to investigate the effects of benomyl on HeLa cell proliferation and mitosis. Benomyl inhibited proliferation of the cells with an IC(50) of 5 microM, and it blocked mitotic spindle function by perturbing microtubule and chromosome organization. The greater than expected actions of benomyl on mammalian microtubules and mitosis together with its relatively low toxicity suggest that it might be useful as an adjuvant in cancer chemotherapy.
We have utilized tau-assembled and tau-stabilized microtubules (MTs), in the absence of taxol, to investigate the effects of tau isoforms with three and four MT binding repeats upon kinesin-driven MT gliding. MTs were assembled in the presence of either 3-repeat tau (3R tau) or 4-repeat tau (4R tau) at tau:tubulin dimer molar ratios that approximate those found in neurons. MTs assembled with 3R tau glided at 31.1 lm/min versus 25.8 lm/min for 4R tau, a statistically significant 17% difference. Importantly, the gliding rates for either isoform did not change over a fourfold range of tau concentrations. Further, tau-assembled MTs underwent minimal dynamic instability behavior while gliding and moved with linear trajectories. In contrast, MTs assembled with taxol in the absence of tau displayed curved gliding trajectories. Interestingly, addition of 4R tau to taxol-stabilized MTs restored linear gliding, while addition of 3R tau did not. The data are consistent with the ideas that (i) 3R and 4R tau-assembled MTs possess at least some isoform-specific features that impact upon kinesin translocation, (ii) tau-assembled MTs possess different structural features than do taxol-assembled MTs, and (iii) some features of tau-assembled MTs can be masked by prior assembly by taxol. The differences in kinesin-driven gliding between 3R and 4R tau suggest important features of tau function related to the normal shift in tau isoform composition that occurs during neural development as well as in neurodegeneration caused by altered expression ratios of otherwise normal tau isoforms. V C 2010 Wiley-Liss, Inc.Key Words: neurodegeneration, axonal transport, frontotemporal dementia, motor protein Introduction T au is a neural microtubule (MT)-associated protein (MAP) that binds to MTs, promotes MT assembly, and regulates MT dynamics in the developing and adult nervous systems . Although there is only a single tau gene, alternative RNA splicing produces six tau isoforms, each possessing either three (3R tau) or four (4R tau) imperfect MT binding repeats located in the C-terminal half of the protein, and either zero, one, or two inserts located in the N-terminal portion of the protein (projection domain, Fig. 1), which is believed to extend outward from the MT surface [Hirokawa et al., 1988;Lee et al., 1988;Himmler et al., 1989;Himmler, 1989;Butner and Kirschner, 1991;Chen et al., 1992;Goode and Feinstein, 1994]. Whereas fetal brain expresses only the shortest 3R tau isoform, adult human brain expresses approximately equal amounts of the 3R tau and 4R tau isoforms [Kosik et al., 1989]. 4R tau generally exhibits higher levels of MT assembly promoting and dynamics regulatory activities than 3R tau [Goedert and Jakes, 1990;Trinczek et al., 1995;Goode et al., 2000;Panda et al., 2003;Bunker et al., 2004;Levy et al., 2005]. Qualitative mechanistic differences between 3R and 4R tau action have also been described [Levy et al., 2005]. The fact that 3R and 4R tau differentially regulate MT dynamics has given rise to a functional shift model in ...
The method of microtubule tracking and dynamics analysis, presented here, improves upon the current means of manual and automated quantification of microtubule behavior. Key contributions are increasing accuracy and data volume, eliminating user bias and providing advanced analysis tools for the discovery of temporal patterns in cellular processes. By tracking the entire length of each resolvable microtubule, as opposed to only the tip, it is possible to boost dynamics studies with positional information that is virtually impossible to collect manually. We demonstrate the method on the analysis of a microtubule dataset, which was manually tracked and analyzed in the study of βIII-tubulin isoform. Our results show that automated recognition of temporal patterns in cellular processes offers a highly promising potential.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.