Radial Carboxyterminal Peptides of Tubulin Directly Imaged in Microtubules by Electron Microscopy Tomography Exploiting Emplitude Contrast

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Abstract Tubulin carboxyterminal tails (CTT) are peptides of 10-20 amino acids, unstructured and acidic, that vary in sequence between tubulin isotypes and are exposed on the outer surface of microtubules (MTs). These peptides have, so far, eluded direct visualization. In this report, electron microscopy tomography was applied to isolated MTs stained with Uranyl and tungstate salts demonstrated to resist sustained electron beam irradiation. Such resistance of high electron doses allows each electron microscopy image to be recorded with a high signal-to-noise ratio. Corresponding tomograms reconstructed from tilt series at high magnification show exceptional resolution of details, revealing features of average dimension ~ 1 nm without the need of averaging multiple samples. The known three-dimensional structure of the MT wall is apparent. But now images also reveal small stalks on the outer surface of MTs. Inspection of virtual sections demonstrates that the stalks are up to ~2.5 nm long and ~1 nm wide (at half length), protruding every 4 ± 0.8 (22) nm along the microtubule. This spacing corresponds to one stalk per tubulin monomer. The grafting point on each monomer is not random but is positioned at one end of each monomer, identifying that end as toward the (-) end of the MT. The stalks are not observed following CTT removal with subtilisin. We conclude that these stalks are the CTT peptides of tubulin.

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