(Supported by CNRS and E.C. CHRX-CT940642 to M. Bornens). Glutamylation represents the major post-translational modification of brain microtubule protein (tubulin). However, although this modification is also detected in non-neuronal cell lines, it appears to be largely restricted to centrioles-minute organelles located within the centrosome or cell center. Dr. Bornens's group has been loading vertebrate somatic cells with monolconal antibodies (GT335) to glutamylated tubulin, using micro-injection and electro-permeabilization methods, to determine the relationship between glutamylated tubulin and stability of the centrioles/centrosome. Twenty-four hours after loading HeLa cells centrosomes and/or centrioles appear absent by immunofluorescent assays, and short -term microtubule re-growth experiments confirmed the loss of a functional centrosome. Centrosome disappearance takes place within the first 12 hours of exposure to the antibody, and is maintained during 4 8 hours, but surprisingly cells regain functional centrosomes after 48 hrs. During the acentrosomal period, cells were still able to progress through mitosis. In order to confirm and extend these LM results the BMIRR was used to generate double-tilt IVEM tomographic reconstructions of the centrosome region in cells that had been loaded with anGTR335 antibody. In essence we examined the destruction of centrioles by tomography, and also confirmed by thick serial section analyses that they were absent from spindle poles and interphase cells. Our structural data directly demonstrate that antibodies to glutamylated microtubule protein disrupt centriolar structure, and therefore that this post-translational modification is important for normal centrosomal/centriolar function. This results of this study are currently in press. Bobinnec, Y., A. Khodjakov, L. M. Mir, C. L. Rieder, B. Edd? and M. Bornens. (1998) Centriole disassembly in vivo and its effect on centrosome structure and function in vertebrate cells. J. Cell Biol. (in press).
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