Three-dimensional organization of pKi-67: a comparative fluorescence and electron tomography study using FluoroNanogold. |
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Authors: | Thierry Cheutin Marie-Fran?oise O'Donohue Adrien Beorchia Christophe Klein Hervé Kaplan Dominique Ploton |
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Affiliation: | Unité MéDian, CNRS UMR 6142, UFR de Pharmacie, 51 rue Cognacq-Jay, 51096 Reims Cedex, France. |
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Abstract: | 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. |
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Keywords: | confocal microscopy electron tomography heterochromatin Ki-67 antigen nucleolus |
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