Visualization of cell microtubules in their native state.
BACKGROUND
Over the past decades, cryo-electron microscopy of vitrified specimens has yielded a detailed understanding of the tubulin and microtubule structures of samples reassembled in vitro from purified components. However, our knowledge of microtubule structure in vivo remains limited by the chemical treatments commonly used to observe cellular architecture using electron microscopy.


RESULTS
We used cryo-electron microscopy and cryo-electron tomography of vitreous sections to investigate the ultrastructure of microtubules in their cellular context. Vitreous sections were obtained from organotypic slices of rat hippocampus and from Chinese-hamster ovary cells in culture. Microtubules revealed their protofilament ultrastructure, polarity and, in the most favourable cases, molecular details comparable with those visualized in three-dimensional reconstructions of microtubules reassembled in vitro from purified tubulin. The resolution of the tomograms was estimated to be approx. 4 nm, which enabled the detection of luminal particles of approx. 6 nm in diameter inside microtubules.


CONCLUSIONS
The present study provides a first step towards a description of microtubules, in addition to other macromolecular assemblies, in an unperturbed cellular context at the molecular level. As the resolution appears to be similar to that obtainable with plunge-frozen samples, it should allow for the in vivo identification of larger macromolecular assemblies in vitreous sections of whole cells and tissues.
Bouchet-Marquis
Cédric
C
Laboratory for Ultrastructural Analysis, Biophore, University of Lausanne, CH-1015 Lausanne, Switzerland.
Zuber
Benoît
B
Glynn
Anne-Marie
AM
Eltsov
Mikhail
M
Grabenbauer
Markus
M
Goldie
Kenneth N
KN
Thomas
Daniel
D
Frangakis
Achilleas S
AS
Dubochet
Jacques
J
Chrétien
Denis
D
D000818:Animals; D016466:CHO Cells; D016764:Cell Polarity; D006224:Cricetinae; D003412:Cricetulus; D020285:Cryoelectron Microscopy; D006624:Hippocampus / Q000166:cytology / Q000648:ultrastructure; D008870:Microtubules / Q000648:ultrastructure*; D009474:Neurons / Q000166:cytology* / Q000648:ultrastructure*; D051381:Rats