Centrosome organization and centriole architecture: their sensitivity to divalent cations.

The centrosome plays a major role in the spatial organization of the microtubular network and has a controlled cycle of duplication, the two duplicated centrosomes functioning as mitotic poles during subsequent cell division. However, a comprehensive description of the overall organization of the centrosome in animal cells is lacking. In order to integrate the various pieces contributing to the centrosome structure and to optimize the quality of the data, we have undertaken an extensive ultrastructural study of centrosomes isolated from human lymphoblasts, which involved (i) orientation of centrosomes by sedimentation before embedding and sectioning, (ii) ultrathin serial sectioning, (iii) digitalization of micrographs to obtain quantitative data, and finally, (iv) comparison between two methods of isolation, which differ by the presence or absence of EDTA. Using this strategy, we have unambiguously described the pericentriolar organization of two distinct sets of appendages (distal and subdistal) about the so-called parental centriole. New structures have been also observed in association with the microtubule sets in this study: (i) external columns, which are dense structures localized at the basis of the subdistal appendages and (ii) internal columns, which are made of globular subunits integrated in a more luminal and probably helical structure. We have also observed that removal of divalent cations by the EDTA during the isolation procedure could affect the centrosomal structure at different levels (subdistal appendages, internal and external columns, pericentriolar matrix), including a significant variation in centriole diameter. A scheme of the overall organization of the centrosome from animal cells and of its modulation by divalent cations can be drawn from this study. Our data gives a view of the centrosome as an organelle displaying a complex and possibly dynamic structural organization.