Direct Optical Microscopic Observation of the Microtubule Polymerization Intermediate Sheet Structure in the Presence of Gas7

Abstract

The process of microtubule elongation is thought to consist of two stages—formation of a tubulin sheet structure and its closure into a tube. However, real-time observation of this process has been difficult. Here, by utilizing phospho-tau binding protein Gas7 (growth-arrest-specific protein 7), we visualized the polymer transformation process by dark-field microscopy. Upon elongation, thin and flexible structures, often similar to a curved hook, appeared at the end of microtubules. Electron microscopic observations supported the idea that these flexible structures are tubulin sheets. They maintained their length until they gradually became thick and rigid beginning in the central portion, resulting in straight microtubules. In the absence of Gas7, the sheet-like structure was rarely observed; moreover, when observed, it was fragile and engaged in typical dynamic instability. With Gas7, no catastrophe was observed. These results suggest that Gas7 enhances microtubule polymerization by stabilizing sheet intermediates and is a useful tool for analyzing microtubule transformation.