Effect of actinomycin D upon upstream binding factor localization: a (3D + time) study

Marie-Françoise O'Donohue, Emmanuel Elias, Pavel Tchelidze, Hélène Bobichon, Laurent Lucas, Éric Bittar et Dominique Ploton

The nucleolus is the site where synthesis and maturation of ribosomal RNAs occur. The tandemly repeated rDNA genes are transcribed by RNA polymerase I associated to several transcription factors. Among them, the Upstream Binding Factor (UBF) is expressed from a unique gene by alternative splicing as two closely related isoforms, UBF1 and UBF2. The fixation of a dimer of UBF1 remodels the promoter region, which increases RNA polymerase I binding affinity. In addition, UBF2 plays an anti-repressor role by binding to enhancer elements. Actinomycin D is a selective inhibitor of RNA polymerase I when used at low concentrations. Previous studies have shown that its action leads to nucleolar segregation and that UBF is located in perinucleolar caps, as observed after fixation followed by immunolabelling. In order to visualize both variants, which are not discriminated by specific anti-UBF antibodies, we previously developed chimeric proteins between UBF1/UBF2 and GFP, an auto-fluorescent protein. This allowed us to follow their threedimensional distribution for several hours into living cells by using confocal microscopy.
The aim of the present study was to follow the action of actinomycin D upon the threedimensional distribution of UBF1 and UBF2 within living cells. Human cancerous cells were grown on glass coverslips and transfected with the corresponding vectors. Twenty-four hours post-transfection, a coverslip was mounted in a perfusion chamber equipped with a heat controller. Acquisition conditions were optimised for time-lapse studies in order to collect zseries every five minutes for several hours. Then, projections and three-dimensional reconstructions were obtained from each time-point. Several tomographic and visualization tools were applied to these reconstructions and films were mounted in order to visualize the effect of the inhibitor as a function of time. This study revealed that both UBF1 and UBF2 were reorganized upon treatment. Movements as well as fusion of neighbouring spots were clearly observed. These data were analysed by new visualization tools using (2D + time) and (3D + time) modes, and compared to the results obtained in fixed cells. Finally, the sites containing the GFP-tagged proteins were clearly identified at the ultrastructural level by studying ultrathin sections of the same cells in which GFP molecules were immunolocalized before embedding by using fluoronanogold and silver amplification. Taken together, these different approaches allowed us to obtain very important new data which will greatly increase our understanding of the action of anticancerous drugs at the cellular level.