Cytological indicators of overall suppression of protein synthesis revealed by staining with a new monoclonal antibody

Grigoryev, A. A.; Bulycheva, T. I.; Sheval, Eugene V.; Kalinina, Ilona; Зацепина, О. В.

doi:10.1134/s1990519x08020144

Cited by 3 publications

(4 citation statements)

References 28 publications

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“…No signals were found in the nucleoplasm and cytoplasm. Similar pattern of A3 antigen distribution was obser ved in other cell types except resting lymphocytes, whose nucleoli contained only one large granule corresponding to the location of RNA polymerase I and argentophilic proteins (data not shown [1]). Resting fi broblasts contained lower number of granules in the nuclei (<30) compared to HEp-2 and HeLa cells (up to 100).…”

Section: Resultssupporting

confidence: 63%

“…Changes in the localization of A3 antigen induced by anisomycin were previously described for HeLa cells [1] belonging similarly to HEp-2 cells to tumor cells. To evaluate the response of A3 antigen to anisomycin in other cell types, including non-tumor cells, we used skin fi broblasts and lymphocytes isolated from healthy donors.…”

Section: Resultsmentioning

confidence: 99%

“…Ramos cells and resting and activated lymphocytes were placed on poly-L-lysine-precoated coverslips. Anisomycin (Sigma) was added to the culture medium in a fi nal concentration of 100 μM for 2 h. We previously showed that changes in the localization of A3 antigen in HeLa cells were most pronounced under these conditions [1]. The cells were fi xed in 2% paraformaldehyde in phosphate buffered saline (140 mM NaCl, 2.7 mM KCl, 1.5 mM KH 2 PO 4 , 8.1 mM Na 2 HPO 4 , pH 7.2-7.4) for 20 min at room temperature, treated with 0.1% Triton X-100 for 10 min, and stained with A3 antibodies [1] and then with FITC-conjugated antibodies to murine immunoglobulins (Sigma).…”

Section: Methodsmentioning

confidence: 99%

“…The reaction of the nucleolus, the only place of 28S rRNA synthesis, is also poorly studied [6]. We previously showed that anisomycin changes the localization of A3 antigen, a nucleolar protein entering the composition of RNA polymerase I transcription complex, in HeLa cells [1]. However, the question whether the nucleolar response to anisomycin is universal for all types of cells was never studied.…”

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Enhanced Sensitivity of Nucleoli in Human Proliferating Cells to Inhibition of Protein Synthesis with Anisomycin

et al. 2010

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We describe the reaction of nuclei in cultured human cells from different tissues to inhibition of total protein synthesis with anisomycin - ribotoxin, which is now considered as a potential antitumor drug. It was shown that nucleoli in sensitive cells demonstrate typical reaction: under the action of the inhibitor, labile nucleolar protein, a component of RNA polymerase I transcription complex (previously called A3 antigen), rapidly migrates from the nucleolus to numerous discrete foci in the nucleoplasm. These changes are specific for translation suppression and are not induced by other influences on the cells. Migration of A3 antigen into the nucleoplasm manifests primarily in cells at the stage of DNA replication and is absent in resting cells. These results suggest that localization of A3 antigen can be a marker of artificial suppression of translation in proliferating human cells in vitro.

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Section: Resultssupporting

confidence: 63%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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Enhanced Sensitivity of Nucleoli in Human Proliferating Cells to Inhibition of Protein Synthesis with Anisomycin

et al. 2010

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Nucleolar localization/retention signal is responsible for transient accumulation of histone H2B in the nucleolus through electrostatic interactions

Musinova

Lisitsyna

Golyshev

et al. 2011

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

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The majority of known nuclear proteins are highly mobile. The molecular mechanisms by which they accumulate inside stable compartments that are not separated from the nucleoplasm by membranes are obscure. The compartmental retention of some proteins is associated with their biological function; however, some protein interactions within distinct nuclear structures may be non-specific. The non-specific retention may lead to the accumulation of proteins in distinct structural domains, even if the protein does not function inside this domain. In this study, we have shown that histone H2B-EGFP initially accumulated in the nucleolus after ectopic expression, and then gradually incorporated into the chromatin to leave only a small amount of nucleolus-bound histone that was revealed by removing chromatin-bound proteins with DNase I treatment. Nucleolar histone H2B had several characteristics: (i) it preferentially bound to granular component of the nucleolus and interacted with RNA or RNA-containing nucleolar components; (ii) it freely exchanged between the nucleolus and nucleoplasm; (iii) it associated with the nuclear matrix; and (iv) it bound to interphase prenuclear bodies that formed after hypotonic treatment. The region in histone H2B that acts as a nucleolar localization/retention signal (NoRS) was identified. This signal overlapped with a nuclear localization signal (NLS), which appears to be the primary function of this region. The NoRS activity of this region was non-specific, but the molecular mechanism was probably similar to the NoRSs of other nucleolar proteins. All known NoRSs are enriched with basic amino acids, and we demonstrated that positively charged motifs (nona-arginine (R9) and nona-lysine (K9)) were sufficient for the nucleolar accumulation of EGFP. Also, the correlation between measured NoRS activity and the predicted charge was observed. Thus, NoRSs appear to achieve their function through electrostatic interactions with the negatively charged components of the nucleolus. Though these interactions are non-specific, the functionally unrelated retention of a protein can increase the probability of its interaction with specific and functionally related binding sites.

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Construction of the plasmid for expression of ETA-EGFP fusion protein under control of the cytomegalovirus promoter and its effects in HeLa cells

Glinka

Andryushchenko

Sapozhnikov

et al. 2009

Plasmid

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Cytological indicators of overall suppression of protein synthesis revealed by staining with a new monoclonal antibody

Cited by 3 publications

References 28 publications

Enhanced Sensitivity of Nucleoli in Human Proliferating Cells to Inhibition of Protein Synthesis with Anisomycin

Enhanced Sensitivity of Nucleoli in Human Proliferating Cells to Inhibition of Protein Synthesis with Anisomycin

Nucleolar localization/retention signal is responsible for transient accumulation of histone H2B in the nucleolus through electrostatic interactions

Construction of the plasmid for expression of ETA-EGFP fusion protein under control of the cytomegalovirus promoter and its effects in HeLa cells

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