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

Abstract

S U M M A R YThe 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

Correlation of Ki-67 and MCM-2 proliferative marker expression with grade of histological malignancy (G) in ductal breast cancers.

“…During interphase Ki-67 co-localizes with perinuclear located DNA and DNA located near the nuclear envelope what suggests that it is involved in perinuclear chromatin organization [9]. Expression of Ki-67 may develop also in situations of an inhibited DNA synthesis or when the cell undergoes the process of apoptosis [10].…”

Prediction of Response to Aromatase Inhibitors in Breast Cancer

“…The exact intracellular function of Ki67 is poorly understood [88][89][90]. It is a large protein (>350 Kda) interacting with chromatin [91,92]. It can be phosphorylated [93].…”

Electron tomography of biological samples

“…However, all the sample preparation methods for electron tomography impose specimen fixation, which constitute a major problem to study any living cells. A possible solu tion to overcome this drawback is to combine electron and confocal microscopy by using, for instance, samples combining a fluorescent dye with a dense marker, like Fluoro Nanogold (FNG) [57,58].…”