Localization of γ-Tubulin in Tetrahymena

To localize γ-tubulin in vivo, log phase cells from strain tG2N-HA or cTTMG-HA were processed for immunofluorescent labeling as described (Gaertig et al., 1995 (link)). HA antibody (16B12) was used at a 1:1,000 dilution. Secondary antibodies were goat anti–mouse FITC (Sigma-Aldrich) at a 1:200 dilution or Alexa fluor 568 goat anti–mouse IgG (Molecular Probes) at a 1:500 dilution. DAPI (4′, 6′-diamidino-2-phenylindole) was used for DNA staining at 100 ng/ml. To study the effects of γ-tubulin depletion on BBs, GTU1 knockout heterokaryon progeny or cTTMG-HA cells in CdCl₂-free SPP medium were stained with anti-centrin monoclonal or polyclonal antibodies, provided by Dr. Jeffrey L. Salisbury (Mayo Clinic Foundation, Rochester, NY). Anti-glutamic acid polyclonal antibody (R-polyE), a rabbit antibody raised commercially (Alpha Diagnostic International) against a Cys (Glu)₉ peptide coupled to keyhole limpet hemocyanin, was used to stain BBs and cilia. MT structures were stained with anti–α-tubulin antibodies (DM1A; Sigma-Aldrich). All antibodies were used at a 1:1,000 dilution. Anti-TFKBP12 polyclonal antibody, provided by Dr. Osamu Numata (University of Tsukuba, Ibaraki, Japan) was used to stain the TFKBP12 (Tetrahymena thermophila FK506 binding protein of 12 kD) at a 1:250 dilution. Secondary antibodies were goat anti–mouse FITC (Zymed Laboratories, Inc.), goat anti–rabbit rhodamine (Zymed), and goat anti–rabbit FITC (Sigma) at 1:200 dilutions. To stain kinetodesmal fibers (KF, also known as striated rootlets), 2–5 × 10⁵ depleted cells were placed on ice for 5 min, fixed in 35% EtOH/0.12% Triton X-100 on ice for 30 min, washed with cold TBS twice (Nelsen et al., 1994 (link)), and stained with mAb FI-5D8 (Jerka-Dziadosz et al., 1995 (link)) provided by Dr. Joseph Frankel (University of Iowa, Iowa City, IA). Secondary antibodies, Alexa fluor 568 goat anti–mouse IgG (Molecular Probes) and goat anti–rabbit FITC (Sigma-Aldrich), were used at a 1:500 or 1:250 dilution, respectively. Four to eight stacked images of cell were obtained using a Leica TCS SP confocal microscope (PL Apo 100× lens, N.A. 1.4, and 1.8× zoom).