The Nucleolus Exhibits an Osmotically Regulated Gatekeeping Activity That Controls the Spatial Dynamics and Functions of Nucleolin

Abstract

We demonstrate that physiologically relevant perturbations in the osmotic environment rheostatically regulate a gatekeeping function for the nucleolus that controls the spatial dynamics and functions of nucleolin. HeLa cells and U2-OS osteosarcoma cells were osmotically challenged with 100 -200 mM sorbitol, and the intranuclear distribution of nucleolin was monitored by confocal microscopy. Nucleolin that normally resides in the innermost fibrillar core of the nucleolus, where it assists rDNA transcription and replication, was expelled within 30 min of sorbitol addition. The nucleolin was transferred into the nucleoplasm, but it distributed there non-uniformly; locally high levels accumulated in 4,6-diamidino-2-phenylindole-negative zones containing euchromatic (transcriptionally active) DNA. Inositol pyrophosphates also responded within 30 min of hyperosmotic stress: levels of bisdiphosphoinositol tetrakisphosphate increased 6-fold, and this was matched by decreased levels of its precursor, diphosphoinositol pentakisphosphate. Such fluctuations in inositol pyrophosphate levels are of considerable interest, because, according to previously published in vitro data, they regulate the degree of phosphorylation of nucleolin through a novel kinase-independent phosphotransferase reaction (Saiardi, A., Bhandari, A., Resnick, R., Cain, A., Snowman, A. M., and Snyder, S. H. (2004) Science 306, 2101-2105). However, by pharmacologically intervening in inositol pyrophosphate metabolism, we found that it did not supervise the osmotically driven switch in the biological activities of nucleolin in vivo.

Diphosphoinositol Polyphosphates: Metabolic Messengers?

“…We therefore manipulated cellular levels of diphosphoinositol polyphosphates in an osteosarcoma cell line using a combination of hyperosmotic stress and some pharmacological tricks (Yang et al, 2008). We found that a hyperosmotic challenge indeed caused nucleolin to accumulate in the nucleoplasm-suggesting its degree of phosphorylation was increased-but this response occurred independently of changes in levels of diphosphoinositol polyphosphates (Yang et al, 2008). Nevertheless, our experiments with nucleolin can only be considered an indirect test of the hypothesis put forward by Saiardi et al (2004) and Bhandari et al (2007).…”

“…Thus, the extent to which nucleolin accumulates in the nucleoplasm can be anticipated to provide a measure of its degree of phosphorylation. We therefore manipulated cellular levels of diphosphoinositol polyphosphates in an osteosarcoma cell line using a combination of hyperosmotic stress and some pharmacological tricks (Yang et al, 2008). We found that a hyperosmotic challenge indeed caused nucleolin to accumulate in the nucleoplasm-suggesting its degree of phosphorylation was increased-but this response occurred independently of changes in levels of diphosphoinositol polyphosphates (Yang et al, 2008).…”

“…We have previously argued that the degree of osmotic stress that activates (PP) 2 -InsP 4 synthesis lies within the biologically relevant range of conditions to which cells are normally exposed (Yang et al, 2008).…”

“…Using the human homolog of Nsr1-nucleolin-as a model, we (Yang et al, 2008) have searched for evidence that its phosphorylation by diphosphoinositol polyphosphates might be physiologically relevant. We made the assumption that, if Saiardi et al (2004) and Bhandari et al (2007) are correct, the degree of nucleolin phosphorylation should increase as the cellular levels of (PP) 2 -InsP 4 and/or PP-InsP 5 are elevated.…”

Inositol Pyrophosphates Modulate S Phase Progression after Pheromone-induced Arrest in Saccharomyces cerevisiae

“…For Kcs1 mutants, the analysis revealed a decrease in the phosphorylation of some nucleolar proteins, including nucleolin (39). However, studies in mammalian cells ruled out the possibility that nucleolin serves as a molecular target for inositol pyrophosphate response to osmotic stress in vivo (40).…”