Protein kinase CK2 is a multifunctional enzyme which has long been described as a stable heterotetrameric complex resulting from the association of two catalytic (␣ or ␣) and two regulatory (␤) subunits. To track the spatiotemporal dynamics of CK2 in living cells, we fused its catalytic ␣ and regulatory ␤ subunits with green fluorescent protein (GFP). Both CK2 subunits contain nuclear localization domains that target them independently to the nucleus. Imaging of stable cell lines expressing low levels of GFP-CK2␣ or GFP-CK2␤ revealed the existence of CK2 subunit subpopulations exhibiting differential dynamics. Once in the nucleus, they diffuse randomly at different rates. Unlike CK2␤, CK2␣ can shuttle, showing the dynamic nature of the nucleocytoplasmic trafficking of the kinase. When microinjected in the cytoplasm, the isolated CK2 subunits are rapidly translocated into the nucleus, whereas the holoenzyme complex remains in this cell compartment, suggesting an intramolecular masking of the nuclear localization sequences that suppresses nuclear accumulation. However, binding of FGF-2 to the holoenzyme triggers its nuclear translocation. Since the substrate specificity of CK2␣ is dramatically changed by its association with CK2␤, the control of the nucleocytoplasmic distribution of each subunit may represent a unique potential regulatory mechanism for CK2 activity.Protein kinase CK2 is a ubiquitous serine/threonine protein kinase, generally described as a stable ␣ 2 ␤ 2 tetramer, where ␣ and ␤ are the catalytic and regulatory subunits, respectively (3). Although its signaling function has long remained obscure, the importance of CK2 is suggested by the evolutionary conservation of the enzyme and by the fact that the disruption of both Saccharomyces cerevisiae genes encoding CK2 catalytic subunits is a lethal event (29). In addition to its role in embryonic development and terminal differentiation, the enzyme is required for normal cell cycle progression (20,30). At last, a function of CK2 in cell survival has recently emerged (1).Many of the identified CK2 substrates that are critical for cell proliferation and viability are localized in different cellular compartments. However, there is controversy as to the localization of CK2 and where its substrates are phosphorylated. Although the current prevailing view of CK2 is a tetrameric enzyme, accumulating evidence also indicates that free populations of both CK2 subunits can exist and exert specific functions in the cell (18, 37). At least in vitro, CK2␤ exerts a central role in modulating the catalytic activity of CK2 (26). Consequently, it is suspected that in vivo, the substrate specificity of the enzyme is likely to be determined both by subcellular localization and by affinity for its regulatory subunit that brings the kinase in proximity to the substrate.In a previous study, the behavior of CK2 subunits fused to GFP was characterized in living cells (25). The expressed fusion proteins were functional and interacted with endogenous CK2. Both subunits were mostl...

Histone macroH2A, which is a subtype of histone H2A, possesses a histone H2A-like portion fused to a relatively long non-histone portion. MacroH2A has been shown to associate preferentially with the inactive X chromosome [1]. To investigate the specificity of this association, the nuclear distribution of macroH2A was compared with that of regular core histones. In normal human female fibroblasts, all anti-histone antibodies that were tested (including anti-macroH2A antibody) preferentially labeled the inactive X chromosome. Moreover, when expressed as green fluorescent protein (GFP) fusions, both histone H2A and macroH2A were concentrated in the Barr body. These data clearly show the presence of a higher density of nucleosomes in the inactive X chromosome. Accordingly, the specificity of the macroH2A association with the inactive X chromosome should be reconsidered. While investigating the role of macroH2A, we found that the proximity of the non-histone region of macroH2A to a promoter could lead to a specific repression of transcription, suggesting that the incorporation of macroH2A into chromatin might help to establish the stable pattern of gene expression in differentiated cells.

To ascertain the ability of commercial and home-made anti-fading media to reduce the decrease of fluorescein isothiocyanate (FITC) fluorescence, we studied the bleaching characteristics of FITC-stained Reh 6 cells mounted in buffered glycerol and in anti-fading media. We measured the intensity of fluorescence over time with a confocal laser scanning microscope and a standard epifluorescence microscope coupled to an image analysis system. Most of the anti-fading media effectively retard fading but each has drawbacks. Better results were obtained with media containing p-phenylenediamine (solutions in buffered glycerol, Vectashield, Fluorstop). However, Mowiol, Slowfade, n-propyl gallate (20 g/liter) were also effective in retarding fading. Most of them, except Mowiol, reduced fluorescence intensity. We concluded that the choice of anti-fading medium would depend on the desired results: a slower decay of fluorescence despite an initial quenching of fluorescence or a lower retardant effect with no decrease in initial fluorescence intensity. Moreover, the combination of Mowiol with another anti-fading medium may be a useful compromise when a strong retardant effect is required without marked quenching of the initial fluorescence.