Three-dimensional organization of pKi-67: a comparative fluorescence and electron tomography study using FluoroNanogold.
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.
Cheutin
Thierry
T
Unité MéDian, CNRS UMR 6142, UFR de Pharmacie, 51 rue Cognacq-Jay, 51096 Reims Cedex, France.
O'Donohue
Marie-Françoise
MF
Beorchia
Adrien
A
Klein
Christophe
C
Kaplan
Hervé
H
Ploton
Dominique
D
D002460:Cell Line; D004247:DNA / Q000378:metabolism; D005456:Fluorescent Dyes; D017612:Gold Compounds; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D019394:Ki-67 Antigen / Q000737:chemistry* / Q000378:metabolism; D018613:Microscopy, Confocal; D017348:Microscopy, Electron, Scanning Transmission; D011487:Protein Conformation
D005456:Fluorescent Dyes; D017612:Gold Compounds; D019394:Ki-67 Antigen; D004247:DNA