Electron tomography and immuno-labeling of Tetrahymena thermophila basal bodies.
Basal bodies and centrioles are highly ordered, microtubule-based organelles involved in the organization of the mitotic spindle and the formation of cilia and flagella. The ciliate Tetrahymena thermophila has more than 700 basal bodies per cell, making it an excellent choice for the study of the structure, function, and assembly of basal bodies. Here, we describe methods for cryofixation of Tetrahymena by high-pressure freezing and freeze-substitution (HPF/FS) for the analysis of basal body structure with advanced electron microscopy techniques. Electron tomography of semi-thick HPF/FS sections was used to generate high-resolution three-dimensional images and models that reveal the intricate structure of basal bodies and associated structures. Immuno-labeling of thin sections from the same HPF/FS samples was used to localize proteins to specific domains within the basal body. To further optimize this model system, we used cell cycle synchronization to increase the abundance of assembling basal bodies. The Tetrahymena genome has been sequenced and techniques for genetic manipulations, such as construction of gene deletion strains, inducible expression and epitope tagging of proteins are now available. These advances have helped to make Tetrahymena a tractable experimental model system. Collectively, these methods facilitate studies of the mechanism of basal body assembly, the functions of basal body constituents and the cytological role of the basal body as a whole.
Giddings
Thomas H
TH
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309, USA.
Meehl
Janet B
JB
Pearson
Chad G
CG
Winey
Mark
M
D002502:Centrioles / Q000648:ultrastructure*; D055032:Electron Microscope Tomography / Q000295:instrumentation / Q000379:methods*; D017110:Freeze Substitution / Q000379:methods; D021621:Imaging, Three-Dimensional / Q000379:methods; D007150:Immunohistochemistry / Q000379:methods*; D008854:Microscopy, Electron / Q000295:instrumentation / Q000379:methods; D016808:Tetrahymena thermophila / Q000648:ultrastructure*