Abstract
In vertebrates, nucleostemin (NS) is an important marker of proliferation in several types of stem and cancer cells, and it can also interact with the tumor-suppressing transcription factor p53. In the present study, the intra-nuclear diffusional dynamics of native NS tagged with GFP and two GFP-tagged NS mutants with deleted guanosine triphosphate (GTP)-binding domains were analyzed by fluorescence correlation spectroscopy. Free and slow binding diffusion coefficients were evaluated, either under normal culture conditions or under treatment with specific cellular proliferation inhibitors actinomycin D (ActD), 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), or trichostatin A (TSA). When treated with ActD, the fractional ratio of the slow diffusion was significantly decreased in the nucleoplasm. The decrease was proportional to ActD treatment duration. In contrast, DRB or TSA treatment did not affect NS diffusion. Interestingly, it was also found that the rate of diffusion of two NS mutants increased significantly even under normal conditions. These results suggest that the mobility of NS in the nucleoplasm is related to the initiation of DNA or RNA replication, and that the GTP-binding motif is also related to the large change of mobility.
Highlights
Nucleostemin (NS) is a protein that is preferentially expressed in certain vertebrate stem cells and tumor cells, where it is thought to play an essential role in the regulation of stem cell proliferation, self-renewal, and differentiation
NS-GFP was mainly located in the nucleolus of HeLa cells, which is in line with previous studies, as shown in Figure 1B [2]
These results suggest that actinomycin D (ActD) induces both less2 transient bonding treatment, while the untreated NS-GFP equivalent was 0.34 ± 0.08 μm s−1
Summary
Nucleostemin (NS) is a protein that is preferentially expressed in certain vertebrate stem cells and tumor cells, where it is thought to play an essential role in the regulation of stem cell proliferation, self-renewal, and differentiation. While nucleostemin was initially identified as a nucleolar protein, it has long been known to shuttle between the nucleolus and nucleoplasm [1,2], and is apparently involved in cell cycle regulation rather than ribosome production [3]. Recent studies have advanced the hypothesis that, instead of promoting cellular growth via nucleolar interactions, the primary role of nucleostemin seems to be tied to DNA replication in the nucleoplasm during S-phase. There, it may coordinate the correction of deleterious mutations that would limit the efficiency of stem (and tumor) cell proliferation [4]. Other studies have suggested that NS fulfills three distinct roles in the nucleoplasm [4]
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