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Domain analysis of the Nematostella vectensis SNAIL ortholog reveals unique nucleolar localization that depends on the zinc-finger domains.

Dattoli AA, Hink MA, DuBuc TQ, Teunisse BJ, Goedhart J, Röttinger E, Postma M - Sci Rep (2015)

Bottom Line: Truncation of the N-terminal SNAG domain, reported to contain Nuclear Localization Signals, markedly reduces nucleolar levels, without effecting nuclear localization or mobility.Truncation of the C-terminal zinc-fingers, involved in DNA binding in higher organisms, significantly affects subcellular localization and mobility.Our findings implicate additional roles for SNAG and zinc-finger domains, suggesting a role for NvSNAILA in the nucleolus.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam Science Park 904, NL-1098 XH Amsterdam The Netherlands.

ABSTRACT
SNAIL transcriptional factors are key regulators during development and disease. They arose early during evolution, and in cnidarians such as Nematostella vectensis, NvSNAILA/B are detected in invaginating tissues during gastrulation. The function of SNAIL proteins is well established in bilaterians but their roles in cnidarians remain unknown. The structure of NvSNAILA and B is similar to the human SNAIL1 and 2, including SNAG and zinc-finger domains. Here, we performed a molecular analysis on localization and mobility of NvSNAILA/B using mammalian cells and Nematostella embryos. NvSNAILA/B display nuclear localization and mobility similar to HsSNAIL1/2. Strikingly, NvSNAILA is highly enriched in the nucleoli and shuttles between the nucleoli and the nucleoplasm. Truncation of the N-terminal SNAG domain, reported to contain Nuclear Localization Signals, markedly reduces nucleolar levels, without effecting nuclear localization or mobility. Truncation of the C-terminal zinc-fingers, involved in DNA binding in higher organisms, significantly affects subcellular localization and mobility. Specifically, the zinc-finger domains are required for nucleolar enrichment of NvSNAILA. Differently from SNAIL transcriptional factors described before, NvSNAILA is specifically enriched in the nucleoli co-localizing with nucleolar markers even after nucleolar disruption. Our findings implicate additional roles for SNAG and zinc-finger domains, suggesting a role for NvSNAILA in the nucleolus.

No MeSH data available.


Cellular localisation of NvSNAILA and B constructs and their truncatedvariants in HeLa cells.A–D) Cellular localisation by confocal imaging ofthe full length (FL) constructs of NvSNAILA, B and HsSNAIL1, 2fused to mTurquoise2 at the C-terminus in HeLa cells together with thenucleolar marker sYFP2-HsFIB, white arrows indicate position ofnucleoli. E) Constructs analysed for both NvSNAILA andNvSNAILB by truncation of respectively the first 5 amino acids ofthe SNAG domain alone (NvSNAILA/B-Δ5SNAG), SNAG domaintogether with the predicted NLS (NvSNAILA/B-Δ20p.NLS) andof the entire zinc-finger domain (NvSNAILA/B-ΔZnf).F–K) Confocal imaging of the truncatedproteins transfected in HeLa cells fused to mTurquoise2 at the C-terminustogether with the nucleolar marker sYFP2-HsFIB, white arrows indicateposition of nucleoli and yellow arrows the cytoplasm. Scale bar10 μm. L) Quantification of nucleolarlocalisation expressed as the ratio of the fluorescence intensity in thenucleolus over nucleoplasm. Ratios are corrected for non-specificlocalisation using mCherry channel, zero values indicate non-specificlocalisation. Error bars are confidence intervals at 95% confidence level.M) Cytoplasm/nucleoplasm fluorescence ratios for different SNAILconstructs. All ratios are normalised with the ratio of mCherry, in this wayany variation coming from geometrical differences or organelle distributionare removed from the ratio. If the ratio is equal to one, then thecytoplasm/nucleoplasm ratio is indistinguishable from mCherry. Error barsrepresent 95% confidence intervals.
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f3: Cellular localisation of NvSNAILA and B constructs and their truncatedvariants in HeLa cells.A–D) Cellular localisation by confocal imaging ofthe full length (FL) constructs of NvSNAILA, B and HsSNAIL1, 2fused to mTurquoise2 at the C-terminus in HeLa cells together with thenucleolar marker sYFP2-HsFIB, white arrows indicate position ofnucleoli. E) Constructs analysed for both NvSNAILA andNvSNAILB by truncation of respectively the first 5 amino acids ofthe SNAG domain alone (NvSNAILA/B-Δ5SNAG), SNAG domaintogether with the predicted NLS (NvSNAILA/B-Δ20p.NLS) andof the entire zinc-finger domain (NvSNAILA/B-ΔZnf).F–K) Confocal imaging of the truncatedproteins transfected in HeLa cells fused to mTurquoise2 at the C-terminustogether with the nucleolar marker sYFP2-HsFIB, white arrows indicateposition of nucleoli and yellow arrows the cytoplasm. Scale bar10 μm. L) Quantification of nucleolarlocalisation expressed as the ratio of the fluorescence intensity in thenucleolus over nucleoplasm. Ratios are corrected for non-specificlocalisation using mCherry channel, zero values indicate non-specificlocalisation. Error bars are confidence intervals at 95% confidence level.M) Cytoplasm/nucleoplasm fluorescence ratios for different SNAILconstructs. All ratios are normalised with the ratio of mCherry, in this wayany variation coming from geometrical differences or organelle distributionare removed from the ratio. If the ratio is equal to one, then thecytoplasm/nucleoplasm ratio is indistinguishable from mCherry. Error barsrepresent 95% confidence intervals.

Mentions: In order to study the cellular localization of Nematostella vectenis snailproteins in comparison with human homologues, we transfected plasmids encodingNvSNAILA, NvSNAILB, HsSNAIL1 and HsSNAIL2 fusedto mTurquoise228 at the C-terminus in HeLa cells. This cell typewas used as a heterologous model system for our comparison studies because manyexperimental cycles can be performed easily (transfection and advancedmicroscopy), constituting a convenient and powerful tool for protein analysis tocombine with Nematostella vectenis embryological experiments. Previousstudies have shown that the concentration levels of transcription factors inanimal cells are found in a range of 10–300 nM29. These values are comparable with the values found withFluorescence Correlation Spectroscopy (FCS) experiments (see Methods); valuesare about 5 fold higher for experiments performed with confocal microscopy. Allover-expressed full-length SNAIL constructs exhibit strong nuclear localizationcompared to non-specific localisation of mCherry. The cytoplasm/nucleoplasmratio of mCherry isRcp−np = 0.89 ± 0.01(n = 275) and relative to this ratio the fulllength constructs are about five-fold higher in the nucleoplasm (Fig. 3M and supplemental Table S2, see Fig.4B,F for typical mCherry localisation). Furthermore, HsSNAIL1and 2 (Fig. 3A,B) show the same nuclear localization aspreviously described2125. Because the protein levels in thenucleolus varied for the different SNAIL constructs (Fig.3A–D) we quantified the ratios of nucleolus (nu)and nucleoplasm (np) (supplemental Table S2, Fig.3L). With the aid of the nucleolar marker HsFIB fused to thesYFP230 and mCherry alone we extracted thenucleolus/nucleoplasm ratios of the constructs of interest and corrected fornon-specific localisation to increase the sensitivity of the quantitativeanalysis (Fig. 4, see Methods). The quantitative analysisreveals that NvSNAILA (Fig. 3A) is significantlyenriched in the nucleoli (Fig. 3L),Rnu−np = 2.46 ± 0.25(n = 27). This was also observed in otherhuman cell lines such as HEK293 and U2OS (data not shown). We found that theN-terminal fusion construct, mTurquoise2-NvSNAILA exhibited a similarenrichment level in nucleoli (data not shown) suggesting that the fusedfluorescent protein is not interfering with the sub-cellular localisationmechanism. HsSNAIL1 (Fig. 3A) exhibitssignificantly lower levelsRnu−np = 0.27 ± 0.09(n = 31). NvSNAILB (Fig. 3D) and HsSNAIL2 (Fig. 3B) exhibitintermediate levels with ratiosRnu−np = 0.90 ± 0.15(n = 14) andRnu−np = 0.52 ± 0.09(n = 32) respectively. Summarizing,NvSNAILA displays strong nucleolar localization compared to the otherconstructs, while NvSNAILB as well as HsSNAIL1 and 2 are detectedat much lower levels in this sub-nuclear structure and in fact are relativelyexcluded.


Domain analysis of the Nematostella vectensis SNAIL ortholog reveals unique nucleolar localization that depends on the zinc-finger domains.

Dattoli AA, Hink MA, DuBuc TQ, Teunisse BJ, Goedhart J, Röttinger E, Postma M - Sci Rep (2015)

Cellular localisation of NvSNAILA and B constructs and their truncatedvariants in HeLa cells.A–D) Cellular localisation by confocal imaging ofthe full length (FL) constructs of NvSNAILA, B and HsSNAIL1, 2fused to mTurquoise2 at the C-terminus in HeLa cells together with thenucleolar marker sYFP2-HsFIB, white arrows indicate position ofnucleoli. E) Constructs analysed for both NvSNAILA andNvSNAILB by truncation of respectively the first 5 amino acids ofthe SNAG domain alone (NvSNAILA/B-Δ5SNAG), SNAG domaintogether with the predicted NLS (NvSNAILA/B-Δ20p.NLS) andof the entire zinc-finger domain (NvSNAILA/B-ΔZnf).F–K) Confocal imaging of the truncatedproteins transfected in HeLa cells fused to mTurquoise2 at the C-terminustogether with the nucleolar marker sYFP2-HsFIB, white arrows indicateposition of nucleoli and yellow arrows the cytoplasm. Scale bar10 μm. L) Quantification of nucleolarlocalisation expressed as the ratio of the fluorescence intensity in thenucleolus over nucleoplasm. Ratios are corrected for non-specificlocalisation using mCherry channel, zero values indicate non-specificlocalisation. Error bars are confidence intervals at 95% confidence level.M) Cytoplasm/nucleoplasm fluorescence ratios for different SNAILconstructs. All ratios are normalised with the ratio of mCherry, in this wayany variation coming from geometrical differences or organelle distributionare removed from the ratio. If the ratio is equal to one, then thecytoplasm/nucleoplasm ratio is indistinguishable from mCherry. Error barsrepresent 95% confidence intervals.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4507178&req=5

f3: Cellular localisation of NvSNAILA and B constructs and their truncatedvariants in HeLa cells.A–D) Cellular localisation by confocal imaging ofthe full length (FL) constructs of NvSNAILA, B and HsSNAIL1, 2fused to mTurquoise2 at the C-terminus in HeLa cells together with thenucleolar marker sYFP2-HsFIB, white arrows indicate position ofnucleoli. E) Constructs analysed for both NvSNAILA andNvSNAILB by truncation of respectively the first 5 amino acids ofthe SNAG domain alone (NvSNAILA/B-Δ5SNAG), SNAG domaintogether with the predicted NLS (NvSNAILA/B-Δ20p.NLS) andof the entire zinc-finger domain (NvSNAILA/B-ΔZnf).F–K) Confocal imaging of the truncatedproteins transfected in HeLa cells fused to mTurquoise2 at the C-terminustogether with the nucleolar marker sYFP2-HsFIB, white arrows indicateposition of nucleoli and yellow arrows the cytoplasm. Scale bar10 μm. L) Quantification of nucleolarlocalisation expressed as the ratio of the fluorescence intensity in thenucleolus over nucleoplasm. Ratios are corrected for non-specificlocalisation using mCherry channel, zero values indicate non-specificlocalisation. Error bars are confidence intervals at 95% confidence level.M) Cytoplasm/nucleoplasm fluorescence ratios for different SNAILconstructs. All ratios are normalised with the ratio of mCherry, in this wayany variation coming from geometrical differences or organelle distributionare removed from the ratio. If the ratio is equal to one, then thecytoplasm/nucleoplasm ratio is indistinguishable from mCherry. Error barsrepresent 95% confidence intervals.
Mentions: In order to study the cellular localization of Nematostella vectenis snailproteins in comparison with human homologues, we transfected plasmids encodingNvSNAILA, NvSNAILB, HsSNAIL1 and HsSNAIL2 fusedto mTurquoise228 at the C-terminus in HeLa cells. This cell typewas used as a heterologous model system for our comparison studies because manyexperimental cycles can be performed easily (transfection and advancedmicroscopy), constituting a convenient and powerful tool for protein analysis tocombine with Nematostella vectenis embryological experiments. Previousstudies have shown that the concentration levels of transcription factors inanimal cells are found in a range of 10–300 nM29. These values are comparable with the values found withFluorescence Correlation Spectroscopy (FCS) experiments (see Methods); valuesare about 5 fold higher for experiments performed with confocal microscopy. Allover-expressed full-length SNAIL constructs exhibit strong nuclear localizationcompared to non-specific localisation of mCherry. The cytoplasm/nucleoplasmratio of mCherry isRcp−np = 0.89 ± 0.01(n = 275) and relative to this ratio the fulllength constructs are about five-fold higher in the nucleoplasm (Fig. 3M and supplemental Table S2, see Fig.4B,F for typical mCherry localisation). Furthermore, HsSNAIL1and 2 (Fig. 3A,B) show the same nuclear localization aspreviously described2125. Because the protein levels in thenucleolus varied for the different SNAIL constructs (Fig.3A–D) we quantified the ratios of nucleolus (nu)and nucleoplasm (np) (supplemental Table S2, Fig.3L). With the aid of the nucleolar marker HsFIB fused to thesYFP230 and mCherry alone we extracted thenucleolus/nucleoplasm ratios of the constructs of interest and corrected fornon-specific localisation to increase the sensitivity of the quantitativeanalysis (Fig. 4, see Methods). The quantitative analysisreveals that NvSNAILA (Fig. 3A) is significantlyenriched in the nucleoli (Fig. 3L),Rnu−np = 2.46 ± 0.25(n = 27). This was also observed in otherhuman cell lines such as HEK293 and U2OS (data not shown). We found that theN-terminal fusion construct, mTurquoise2-NvSNAILA exhibited a similarenrichment level in nucleoli (data not shown) suggesting that the fusedfluorescent protein is not interfering with the sub-cellular localisationmechanism. HsSNAIL1 (Fig. 3A) exhibitssignificantly lower levelsRnu−np = 0.27 ± 0.09(n = 31). NvSNAILB (Fig. 3D) and HsSNAIL2 (Fig. 3B) exhibitintermediate levels with ratiosRnu−np = 0.90 ± 0.15(n = 14) andRnu−np = 0.52 ± 0.09(n = 32) respectively. Summarizing,NvSNAILA displays strong nucleolar localization compared to the otherconstructs, while NvSNAILB as well as HsSNAIL1 and 2 are detectedat much lower levels in this sub-nuclear structure and in fact are relativelyexcluded.

Bottom Line: Truncation of the N-terminal SNAG domain, reported to contain Nuclear Localization Signals, markedly reduces nucleolar levels, without effecting nuclear localization or mobility.Truncation of the C-terminal zinc-fingers, involved in DNA binding in higher organisms, significantly affects subcellular localization and mobility.Our findings implicate additional roles for SNAG and zinc-finger domains, suggesting a role for NvSNAILA in the nucleolus.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam Science Park 904, NL-1098 XH Amsterdam The Netherlands.

ABSTRACT
SNAIL transcriptional factors are key regulators during development and disease. They arose early during evolution, and in cnidarians such as Nematostella vectensis, NvSNAILA/B are detected in invaginating tissues during gastrulation. The function of SNAIL proteins is well established in bilaterians but their roles in cnidarians remain unknown. The structure of NvSNAILA and B is similar to the human SNAIL1 and 2, including SNAG and zinc-finger domains. Here, we performed a molecular analysis on localization and mobility of NvSNAILA/B using mammalian cells and Nematostella embryos. NvSNAILA/B display nuclear localization and mobility similar to HsSNAIL1/2. Strikingly, NvSNAILA is highly enriched in the nucleoli and shuttles between the nucleoli and the nucleoplasm. Truncation of the N-terminal SNAG domain, reported to contain Nuclear Localization Signals, markedly reduces nucleolar levels, without effecting nuclear localization or mobility. Truncation of the C-terminal zinc-fingers, involved in DNA binding in higher organisms, significantly affects subcellular localization and mobility. Specifically, the zinc-finger domains are required for nucleolar enrichment of NvSNAILA. Differently from SNAIL transcriptional factors described before, NvSNAILA is specifically enriched in the nucleoli co-localizing with nucleolar markers even after nucleolar disruption. Our findings implicate additional roles for SNAG and zinc-finger domains, suggesting a role for NvSNAILA in the nucleolus.

No MeSH data available.