Visualization of Individual Cryptophycin-Tubulin Rings by Electron Microscopy Tomography

The antimitotic drug Cryptophycin-1 (Cr) arrests cell growth by destabilizing microtubules. When added in vitro to either dimeric tubulin or preformed microtubules, Cr induces the formation of single protofilament rings that are highly monodisperse, having a diameter of ∼28nm and thickness ∼4 nm, and composed of eight tubulin dimers. Here we apply negative stain tomography [Fera, et al, J. Comp. Neurol., 2012] to Cr-tubulin rings. This new technique, which utilizes a beam stable organo-tungsten negative stain, allows construction of tomograms resolving the individual components of the protofilament rings without averaging. Molecular structure of Cr-tubulin rings is revealed in series of en face virtual sections ∼4 A thick positioned parallel to the plane of Cr-tubulin rings. We repeated the observations with rings obtained from tubulin-S, which lack the C-terminal acidic tail filaments that face the center of the Cr-tubulin rings. These tail filaments, normally on the outside of the microtubule and important for MAP and motor protein binding, have a diameter of ∼1 nm and significant conformational freedom, making them impossible to detect by averaging methods. Thus, comparison of tomograms from rings of tubulin and tubulin-S will gather further information about these filaments. We also investigate ring-ring interactions in samples of higher concentration to probe their material properties.