Wilhelm bernhard workshop on the cell nucleus

EFFECT OF ACTINOMYCIN-D UPON UP-STREAM BINDING FACTOR LOCALIZATION: A (3D + TIME) STUDY

Elias E., Tchélidzé P., Benassarou A.¹, Sauvage C., Bobichon H., Ploton D. and O’Donohue M.F.

Unité MéDIAN, CNRS UMR 6142 and ¹LERI, Reims, France.
Upstream Binding Factor (UBF), is present within the nucleolus as two closely related isoforms, UBF1 and UBF2. The fixation of a dimer of UBF1 remodels the promoter region, which increases RNA polymerase I (RNAP 1) binding affinity. In addition, UBF2 plays an anti-repressor role by binding to enhancer elements. 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. In the present work, we addressed the localisation of UBF1 and UBF2 both by electron microscopy and by confocal microscopy within living KB cells (4D study) during the inhibition of RNAP 1 by actinomycin D (AMD; 0.05 µg/ ml). The most sensitive method for ultrastructural immunolocalisation of GFP molecules was performed before embedding by using nanogold labelling, silver enhancement and observation of 100 nm ultrathin sections. In control cells, both proteins are mainly localised within fibrillar centers (FC) either as coils, for UBF1 (as for RNAP1, see T. Cheutin et al. J Cell Sci. 115, 3297, 2002) or with a more diffused pattern, for UBF2. During AMD treatment, both proteins remain within FC and are frequently organised as coils. For study on living cells by confocal microscopy, transfected cells were mounted in a perfusion chamber equipped with a heat controller and 40 optical sections were performed every five minutes for 8 hours. By developing a new software, we were able to study the complex 3D trajectories of the FC, identified by UBF-GFP, during all the steps of nucleolar segregation. This approach allowed us to demonstrate that segregation occurs by successive fusions of FC.