Isolation and characterization of libraries of monoclonal antibodies directed against various forms of tubulin in Paramecium

Abstract

Ciliates are very good models for studying post-translationally generated tubulin heterogeneity because they exhibit highly differentiated microtubular networks in combination with reduced genetic diversity. We have approached the analysis of tubulin heterogeneity in Paramecium through extensive isolation and characterization of monoclonal antibodies using various antigens and several immunization protocols. Eight monoclonal antibodies and 10 hybridoma supernatants were characterized by: i) immunoblotting on ciliate and pig brain tubulins as well as on peptide maps of Paramecium axonemal tubulin; ii) immunoblotting on ciliate tubulin fusion peptides generated in E coli, a procedure which allows in principle to discriminate antibodies that are directed against tubulin sequence (reactive on fusion peptides) from those directed against a post-translational epitope (non-reactive); and iii) immunofluorescence on Paramecium, 3T3 and PtK2 cells. Twelve antibodies labeled all microtubules in Paramecium cells and were found to be directed against tubulin primary sequences (nine of them being located in the alpha N-terminal domain, one in the beta C-terminal one, and two in alpha and beta central stretches). The remaining ones decorated only a specific subset of microtubules within the cell and were presumably directed against post-translational modifications. Among these, three antibodies are directed against an N-terminal acetylated epitope of alpha-tubulin whereas the epitopes of three other ones (TAP 952 degrees, AXO 58 and AXO 49 degrees) apparently correspond to still unidentified post-translational modifications, located in the C-terminal domain of both alpha- and beta-tubulins. The AXO 49 degrees specificity is similar to that of a previously described polyclonal serum raised against Paramecium axonemal tubulin [2]. The results are discussed in terms of identification and accessibility of the epitopes and immunogenicity of ciliate tubulin with reference to mammalian and ciliate tubulin sequences.

Identification, Localization, and Functional Implications of the Microdomain-Forming Stomatin Family in the Ciliated Protozoan Paramecium tetraurelia

“…The authors noted that the responsible ␣-tubulin acyltransferase also occurs in Paramecium. This would comply with the old observation that in Paramecium, Abs against acetylated ␣-tubulin recognize microtubule subpopulations around the oral cavity and over the entire extension of the CVC (99). To sum up, in agreement with work with metazoans, cytoskeletal elements are available in the Paramecium cell at the sites where we localized Stomatin forms and where silencing has affected function.…”

FOR20, a conserved centrosomal protein, is required for assembly of the transition zone and basal body docking at the cell surface

“…The following antibodies were used: the monoclonal 17E2 directed against HsFOR20p (Sedjaï et al, 2010) diluted 1:1000; the monoclonal anti-glutamylated tubulin ID5 (Wehland and Weber, 1987) diluted 1:500 to label basal bodies; the monoclonal TAP952 directed against monoglycylated tubulin (Callen et al, 1994;Janke and Bulinski, 2011) diluted 1:10 to decorate the cilia; the monoclonal CTS32 directed against epiplasmins (Nahon et al, 1993) diluted 1:20, to decorate the epiplasm, and the polyclonal anti-GFP antibody (Interchim, Montluçon, France) diluted 1:500 to amplify the GFP signal. Appropriate secondary (mouse or rabbit) antibodies from Jackson ImmunoResearch labs (West Grove, PA) Alexa 488 or Alexa 568 were used at a dilution of 1:200.…”

Kinesin-II Is Preferentially Targeted to Assembling Cilia and Is Required for Ciliogenesis and Normal Cytokinesis inTetrahymena

“…Coverslips were processed for immunofluorescent labeling as described (Gaertig et al, 1995), using rabbit polyclonal SG serum raised against Tetrahymena total tubulin (Guttman and Gorovsky, 1979) at 1:100 dilution. Cells expressing the epitope-tagged Kin1p were double labeled using the TAP952 mouse monoclonal antibodies directed against the monoglycylated isoforms of tubulins (Callen et al, 1994) and the anti-GFP rabbit polyclonal antibodies (Clontech, Palo Alto, CA) at 1:100 and 1:400 dilution, respectively. Secondary antibodies were goat anti-mouse FITC (Sigma), goat anti-rabbit-Cy3, and goatanti-rabbit-FITC (Zymed, San Francisco, CA) conjugates, and all were used at 1:100 dilution.…”