Nanoimages show disruption of tubulin polymerization by chlorpyrifos oxon: implications for neurotoxicity

Abstract

Organophosphorus agents cause cognitive deficits and depression in some people. We hypothesize that the mechanism by which organophosphorus agents cause these disorders is by modification of proteins in the brain. One such protein could be tubulin. Tubulin polymerizes to make the microtubules that transport cell components to nerve axons. The goal of the present work was to measure the effect of the organophosphorus agent chlorpyrifos oxon on tubulin polymerization. An additional goal was to identify the amino acids covalently modified by chlorpyrifos oxon in microtubule polymers and to compare them to the amino acids modified in unpolymerized tubulin dimers. Purified bovine tubulin (0.1 mM) was treated with 0.005-0.1 mM chlorpyrifos oxon for 30 min at room temperature and then polymerized by addition of 1 mM GTP to generate microtubules. Microtubules were visualized by atomic force microscopy. Chlorpyrifos oxon-modified residues were identified by tandem ion trap electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry of tryptic peptides. Nanoimaging showed that low concentrations (0.005 and 0.01 mM) of chlorpyrifos oxon yielded short, thin microtubules. A concentration of 0.025 mM stimulated polymerization, while high concentrations (0.05 and 0.1 mM) caused aggregation. Of the 17 tyrosines covalently modified by chlorpyrifos oxon in unpolymerized tubulin dimers, only 2 tyrosines were labeled in polymerized microtubules. The two labeled tyrosines in polymerized tubulin were Tyr 103 in EDAANNYR of alpha tubulin, and Tyr 281 in GSQQYR of beta tubulin. In conclusion, chlorpyrifos oxon binding to tubulin disrupts tubulin polymerization. These results may lead to an understanding of the neurotoxicity of organophosphorus agents.

Figure 1

Effect of chlorpyrifos oxon on tubulin polymerization visualized by atomic force microscopy. A) 0.1 mM bovine tubulin in PEM buffer, no GTP; B) 0.1 mM tubulin polymerized with 1 mM GTP, no chlorpyrifos oxon; C) 0.1 mM tubulin pretreated with 0.005 mM chlorpyrifos oxon before polymerization with GTP; D) pretreated with 0.010 mM chlorpyrifos oxon before polymerization; E) pretreated with 0.025 mM chlorpyrifos oxon before polymerization; F) pretreated with 0.050 mM chlorpyrifos oxon before polymerization; G) 0.1 mM tubulin pretreated with 0.1 mM chlorpyrifos oxon before polymerization; H) polymerized in the presence of 0.025 mM colchicine. The dimensions of each panel are 20 × 20 μm. Images are presented in height trace mode.

Figure 2

Ten times magnified view of polymerized tubulin. Samples are the same as in Figure 1. A) no GTP, no chlorpyrifos oxon; B) control with GTP, no chlorpyrifos oxon; C) 0.005 mM chlorpyrifos oxon; D) 0.010 mM chlorpyrifos oxon; E) 0.025 mM chlorpyrifos oxon; F) 0.050 mM chlorpyrifos oxon; G) 0.100 mM chlorpyrifos oxon; H) 0.025 mM colchicine. The dimensions of each panel are 2 × 2 μm. Images are presented in phase trace mode.

Figure 3

MS spectra showing chlorpyrifos oxon-labeled and unlabeled tubulin tryptic peptides. A) control tubulin dimer without chlorpyrifos oxon (CPO); B) tubulin dimer treated with 0.5 mM CPO; C) polymerized microtubules treated with 0.5 mM CPO. Peaks at 1023.5 and 1039.6 amu correspond to unlabeled EDAANNYAR and YLTVAAVFR peptides from alpha and beta tubulin, respectively. Peaks at 1059.6 and 1075.6 amu correspond to CPO labeled (+136 amu added mass from CPO) EDAANNY*AR and Y*LTVAAVFR peptides. The labeled peak at 1059.5 amu is present in panels B and C. The labeled peak at 1075.6 amu is present only in panel B. The letter X in panels A and C indicates absent peaks.