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Journal of Histochemistry and Cytochemistry
Volume 51 (11): 1411-1423, 2003
Copyright ©The Histochemical Society, Inc.

Three-dimensional Organization of pKi-67 : A Comparative Fluorescence and Electron Tomography Study Using Fluoronanogold

Thierry Cheutin1, Marie-Françoise O'Donohue, Adrien Beorchia, Christophe Klein, Hervé Kaplan and Dominique Ploton

Unité MéDian, CNRS UMR 6142, UFR de Pharmacie (TC,M-FO,DP), Reims, France; DTI, UMR 6107, UFR de Sciences (AB), Reims, France; Service Commun d'Imagerie Cellulaire et de Cytométrie, INSERM IFR58, Institut Biomédical des Cordeliers, Paris, France (CK); and IFR53, Reims, France (HK)

Correspondence to: Dominique Ploton, Unité MéDian, CNRS UMR 6142, UFR de Pharmacie, 51 rue Cognacq-Jay, 51096 Reims Cedex, France. E-mail: dominique.ploton{at}univ-reims.fr

The monoclonal antibody (MAb) Ki-67 is routinely used in clinical studies to estimate the growth fraction of tumors. However, the role of pKi-67, the protein detected by the Ki-67 MAb, remains elusive, although some biochemical data strongly suggest that it might organize chromatin. To better understand the functional organization of pKi-67, we studied its three-dimensional distribution in interphase cells by confocal microscopy and electron tomography. FluoroNanogold, a single probe combining a dense marker with a fluorescent dye, was used to investigate pKi-67 organization at the optical and ultrastructural levels. Observation by confocal microscopy followed by 3D reconstruction showed that pKi-67 forms a shell around the nucleoli. Double labeling experiments revealed that pKi-67 co-localizes with perinucleolar heterochromatin. Electron microscopy studies confirmed this close association and demonstrated that pKi-67 is located neither in the fibrillar nor in the granular components of the nucleolus. Finally, spatial analyses by electron tomography showed that pKi-67 forms cords 250–300 nm in diameter, which are themselves composed of 30–50-nm-thick fibers. These detailed comparative in situ analyses strongly suggest the involvement of pKi-67 in the higher-order organization of perinucleolar chromatin. (J Histochem Cytochem 51:1411–1423, 2003)

Key Words: confocal microscopy • electron tomography • heterochromatin • Ki-67 antigen • nucleolus


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