Chapter 24 - Cryo-Electron Tomography of Cellular Microtubules

Abstract

Microtubules are intrinsically dynamic structures. In the cellular environment many proteins and protein complexes are associated with microtubules that influence or functionalize microtubule dynamics. Therefore, investigation of the structure and dynamics of microtubules with their associated complexes inside the cellular environment lies at the heart of fully understanding their function. Cryo electron microscopy has been essential in structural microtubule research since the atomic structure of tubulin and the structure of microtubules were unraveled using this technique. Furthermore, the specific structures at the microtubule ends linked to the growing or shrinking states were also detected by cryo electron microscopy. Electron microscopy studies on microtubules were mainly performed in vitro but microtubules can also be investigated inside cells, using cryo electron tomography. Cryo electron tomography is an important tool in structural biology research because it enables visualization of single and unique protein complexes in a cellular environment and at a molecular resolution. Cryo electron tomography is a three-dimensional (3D) imaging technique in which electron microscopy tomographic imaging is performed on cryogenically cooled, vitrified specimens after which the object is computationally reconstructed. Here, I describe the materials and methods for cryo electron tomography of microtubules and in whole cells, describing cell growth, specimen vitrification, localization of microtubules, cryo electron tomography recording, tomographic image reconstruction, and 3D visualization techniques.