Date Published: June 01, 2018
Publisher: International Union of Crystallography
Author(s): Joseph Atherton, Melissa Stouffer, Fiona Francis, Carolyn A. Moores.
Electron microscopy is a key methodology for studying microtubule structure and organization. Here, the results of cryo-electron tomography experiments on in vitro-polymerized microtubules and comparisons with microtubule ultrastructure in cells are described.
The microtubule (MT) cytoskeleton is vital for many aspects of cell function, including division, migration and definition of cell architecture. MTs act as tracks for the cellular transport motors kinesin and dynein, but are also dynamic polymers, and both growing and shrinking MT ends are put to work in numerous contexts (Akhmanova & Steinmetz, 2015 ▸; McIntosh et al., 2012 ▸). MTs are built from αβ-tubulin heterodimers that polymerize longitudinally to form polar protofilaments (PFs) and laterally to form the walls of the hollow MTs (Nogales et al., 1998 ▸, 1999 ▸). β-Tubulin is exposed at the so-called plus ends of MTs, which are typically more dynamic, while α-tubulin is exposed at the minus ends. MT dynamics are driven by the tubulin GTPase cycle: GTP-bound tubulin favours MT nucleation and growth, but polymerization stimulates tubulin GTPase activity, yielding MTs built from GDP-bound tubulin which are intrinsically unstable (Desai & Mitchison, 1997 ▸; Mitchison & Kirschner, 1984 ▸).
The technologies leading to the ‘resolution revolution’ (Kühlbrandt, 2014 ▸) have dramatically increased the information content of all cryo-EM data, shedding light on numerous dynamic cellular processes. Here, we have highlighted the power of cryo-electron tomography in studying the diversity of MT ultrastructure, thereby shedding light on the molecular mechanisms of dynamic instability. MTs polymerized in vitro from solutions of pure tubulin, although experimentally extremely simple compared with cellular MTs, allow the heterogeneity of MT structures to be manipulated and probed in detail. Our data emphasize that several features of these in vitro-polymerized MTs, the relatively short sheet-like ends of stable GMPCPP-MTs and the presence of lattice defects, are also found in neuronal MTs. Obviously, the structures of cellular MTs are highly context-dependent, being governed by the specific proteome of a given cell, post-translational modifications of both tubulin and its regulators, and the developmental and physiological status of the cell. As more precise correlated light- and electron-microscopy imaging is achieved, regulators associated with different MT morphologies in cells will be defined (Kukulski et al., 2011 ▸; Wolff et al., 2016 ▸).