Demonstration of Detyrosinated alpha-Tubulin in the Feeding Apparatus (Siphon) of Entosiphon sulcatum Depends on Incubation Time in a Lysis Buffer

Witold Mütze, Ralf N. Breuker, Manfred Hauser; Lehrstuhl für Zellmorphologie, Ruhr-Universität Bochum, 44780 Bochum, FRG

Recent work provides evidence that post-translational modifications of MTs by acetylation or detyrosination correlate with the stability of the MTs, and that this stability may be due to the binding of MAPs to the walls of the MTs. The siphon of E. sulcatum consists of three microtubular C-shaped arranged rods, which are embedded in an electron-dense material (cement) in the anterior cytostom region1. The MTs forming the single rods are arranged in exact rows spaced by short and long crossbridges and appear remarkably stable against depolymerizing treatments. Surprisingly, immunocytochemical staining with antibodies against Tyr- and Glu-tubulin after a 2 minute lysis in a microtubule-stabilizing PHEM-buffer, yielded only a positive reaction with the YL 1/2 antibody directed against the labile Tyr-tubulin, suggesting a complete absence of the more stabile Glu-tubulin fraction.
However, prolonged incubation after lysis in a PEM-medium (with or without 2 mM Ca2+) resulted in a strong positive reaction with the 1D5 antibody directed against Glu-tubulin. Obviously, the intensity of this Glu-positive reaction is time-dependent, since the reactivity of the 1D5 antibody increased steadily within 10 minutes. Treatment in a Ca2+-containing buffer did not influence the binding capacity of the Tyr-antibody, thus indicating that all MTs represent a Ca2+-insoluble fraction. On the ultrastructural level, we were able to demonstrate that a 10 minute incubation leads to the disintegration of the cement and of the crossbridges spacing the distance between the single MT-rows.
These results suggest two plausible explanations: Firstly, conformational changes of the tubulin itself make the Glu-epitopes accessible to the 1D5-antibody and/or secondly, the MAPs, representing the inter-row crossbridges, mask exactly the Glu-tubulin epitopes. Interestingly, this confirms the results of Sherwin et al. (cited in Greer and Rosenbaum2) working on trypanosomes, that detyrosination of a-tubulin may be correlated with the formation of cross-bridges between neighboring microtubules.
References: [1] Belhadri, A., G. Brugerolle: Morphogenesis of the feeding apparatus of Entosiphon sulcatum. An immunofluorescence and ultrastructural study. In: Protoplasma 168, 125-135 (1992). [2] Greer, K. and J.L. Rosenbaum: Post-Translational Modifications of Tubulin. In: Cell Movement, Vol. 2: Kinesin, Dynein, and Microtubule Dynamics, 47-66 (1989).

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