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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.


FRAP analysis of NvSNAILA-sYFP2 constructs in different regions ofHeLA cells.A) A cell transfected with NvSNAILA-sYFP2 exhibits enrichednucleoli (prebleach) was bleached in a nucleolus (postbleach). After about40 s the distribution of NvSNAILA-sYFP2 in the cell isalmost completely recovered (endframes, endframes-prebleach). B) Timecourse of the region containing the nucleus (blue) and the bleached circularregion (purple), see main text for details. C,D) FRAP dynamics andradial profiles C) Removal of the FLIP effect from the recoverytraces (n = 6 measurements) shows thatfluorescence in the nucleolus and nucleoplasm is completely recovered within40 s. The recovery curves were fitted with a double exponential,1−a1exp[−t/τ1]−a2exp[−t/τ2], yielding,a1 = 0.46,τ1 = 1.98 s,a2 = 0.18 andτ2 = 11.12 sfor the nucleolus anda1 = 0.31,τ1 = 1.31 s,a2 = 0.23 andτ2 = 7.37 sfor the nucleoplasm. The nucleoplasm recovery is slightly faster thanrecovery in the nucleolus. D) Initial radial profiles just after thebleach shows that the relative mobility of all constructs fused to sYFP2 indifferent regions of the cell is consistent with the FCS measurements.Hence, more shallow gradients indicate higher mobility. The radial profilesshow that NvSNAILA in the nucleolus (nu) has a lower mobility thanthe mobility of NvSNAILA, NvSNAILA-Δ5SNAG andNvSNAILA-Δ20pNLS in the nucleoplasm (np). The latterNvSNAILA-ΔZnf shows similar initial profiles butappear to have a higher mobility compared to the other constructs in thenucleoplasm and in turn it has a lower mobility thanNvSNAILA-ΔZnf in the cytoplasm (cp).
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f9: FRAP analysis of NvSNAILA-sYFP2 constructs in different regions ofHeLA cells.A) A cell transfected with NvSNAILA-sYFP2 exhibits enrichednucleoli (prebleach) was bleached in a nucleolus (postbleach). After about40 s the distribution of NvSNAILA-sYFP2 in the cell isalmost completely recovered (endframes, endframes-prebleach). B) Timecourse of the region containing the nucleus (blue) and the bleached circularregion (purple), see main text for details. C,D) FRAP dynamics andradial profiles C) Removal of the FLIP effect from the recoverytraces (n = 6 measurements) shows thatfluorescence in the nucleolus and nucleoplasm is completely recovered within40 s. The recovery curves were fitted with a double exponential,1−a1exp[−t/τ1]−a2exp[−t/τ2], yielding,a1 = 0.46,τ1 = 1.98 s,a2 = 0.18 andτ2 = 11.12 sfor the nucleolus anda1 = 0.31,τ1 = 1.31 s,a2 = 0.23 andτ2 = 7.37 sfor the nucleoplasm. The nucleoplasm recovery is slightly faster thanrecovery in the nucleolus. D) Initial radial profiles just after thebleach shows that the relative mobility of all constructs fused to sYFP2 indifferent regions of the cell is consistent with the FCS measurements.Hence, more shallow gradients indicate higher mobility. The radial profilesshow that NvSNAILA in the nucleolus (nu) has a lower mobility thanthe mobility of NvSNAILA, NvSNAILA-Δ5SNAG andNvSNAILA-Δ20pNLS in the nucleoplasm (np). The latterNvSNAILA-ΔZnf shows similar initial profiles butappear to have a higher mobility compared to the other constructs in thenucleoplasm and in turn it has a lower mobility thanNvSNAILA-ΔZnf in the cytoplasm (cp).

Mentions: The localisation analysis showed that NvSNAILA is significantly enrichedin the nucleolus and the FCS analysis indicated that it has a low apparentdiffusion coefficient in the nucleolus compared to the nucleoplasm. The FCSanalysis gives information about movement in and out of the detection volume;however it does not necessary give information about transport into and out ofthe nucleolus37. In order to study this process, a FRAP analysiswas performed. For this experiment, the full length NvSNAILA was fused tothe yellow fluorescent protein sYFP2 (NvSNAILA-sYFP2), which can bebleached more efficiently than mTurquoise228. A circular regionwith a diameter of 2.484 μm was bleached for one second,after which recovery was monitored over a period of about forty seconds (seeMethods). In Fig. 9A nucleolar enrichment ofNvSNAILA-sYFP2 is clearly visible in the averaged frame before bleaching(pre-bleach). Directly after bleaching the nucleolus is almost completelybleached (post-bleach) and after a period of about forty seconds the cell showsvirtually the same distribution (end-frames and difference image end-framesminus pre-bleach, supplementary video S1). Because the nucleus is a confinedcompartment, the total fluorescence in the nucleus is reduced, which is apparentas a step change in the time trace of the nucleus region (Fig.9B, blue trace); the part that is not recovered in the FRAP region(Fig. 9B, purple trace) can be completely explained bythis loss of total nuclear fluorescence. If the total loss of nuclearfluorescence is removed from the FRAP region traces, residual traces can beobtained (Fig. 9C). These residual traces(n = 6 measurements) show that the nucleoluscompletely recovers and does not appear to have any significant immobilefraction and that the long term recovery in the nucleolus has a similar timecourse as recovery in the nucleoplasm. This suggests that exchange ofNvSNAILA-sYFP2 between the nucleolus and the nucleoplasm is relativelyfast and that its long-term recovery is limited by diffusion from thenucleoplasm to the nucleolus and largely determined by the low diffusioncoefficient in the nucleoplasm and the geometry of the nucleus. Further FRAPanalysis was performed on NvSNAILA-Δ5SNAG,NvSNAILA-Δ20pNLS and NvSNAILA-ΔZnf both inthe nucleoplasm as well as the cytoplasm. The steepness of the radial profilesaround the centre of the bleached area just after the bleach was determined(Fig. 9D), which represent an indication of themobility of the different constructs and show that the FRAP results are in closeagreement with the apparent diffusion coefficients that were obtained from theFCS analysis.


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)

FRAP analysis of NvSNAILA-sYFP2 constructs in different regions ofHeLA cells.A) A cell transfected with NvSNAILA-sYFP2 exhibits enrichednucleoli (prebleach) was bleached in a nucleolus (postbleach). After about40 s the distribution of NvSNAILA-sYFP2 in the cell isalmost completely recovered (endframes, endframes-prebleach). B) Timecourse of the region containing the nucleus (blue) and the bleached circularregion (purple), see main text for details. C,D) FRAP dynamics andradial profiles C) Removal of the FLIP effect from the recoverytraces (n = 6 measurements) shows thatfluorescence in the nucleolus and nucleoplasm is completely recovered within40 s. The recovery curves were fitted with a double exponential,1−a1exp[−t/τ1]−a2exp[−t/τ2], yielding,a1 = 0.46,τ1 = 1.98 s,a2 = 0.18 andτ2 = 11.12 sfor the nucleolus anda1 = 0.31,τ1 = 1.31 s,a2 = 0.23 andτ2 = 7.37 sfor the nucleoplasm. The nucleoplasm recovery is slightly faster thanrecovery in the nucleolus. D) Initial radial profiles just after thebleach shows that the relative mobility of all constructs fused to sYFP2 indifferent regions of the cell is consistent with the FCS measurements.Hence, more shallow gradients indicate higher mobility. The radial profilesshow that NvSNAILA in the nucleolus (nu) has a lower mobility thanthe mobility of NvSNAILA, NvSNAILA-Δ5SNAG andNvSNAILA-Δ20pNLS in the nucleoplasm (np). The latterNvSNAILA-ΔZnf shows similar initial profiles butappear to have a higher mobility compared to the other constructs in thenucleoplasm and in turn it has a lower mobility thanNvSNAILA-ΔZnf in the cytoplasm (cp).
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Related In: Results  -  Collection

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Show All Figures
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f9: FRAP analysis of NvSNAILA-sYFP2 constructs in different regions ofHeLA cells.A) A cell transfected with NvSNAILA-sYFP2 exhibits enrichednucleoli (prebleach) was bleached in a nucleolus (postbleach). After about40 s the distribution of NvSNAILA-sYFP2 in the cell isalmost completely recovered (endframes, endframes-prebleach). B) Timecourse of the region containing the nucleus (blue) and the bleached circularregion (purple), see main text for details. C,D) FRAP dynamics andradial profiles C) Removal of the FLIP effect from the recoverytraces (n = 6 measurements) shows thatfluorescence in the nucleolus and nucleoplasm is completely recovered within40 s. The recovery curves were fitted with a double exponential,1−a1exp[−t/τ1]−a2exp[−t/τ2], yielding,a1 = 0.46,τ1 = 1.98 s,a2 = 0.18 andτ2 = 11.12 sfor the nucleolus anda1 = 0.31,τ1 = 1.31 s,a2 = 0.23 andτ2 = 7.37 sfor the nucleoplasm. The nucleoplasm recovery is slightly faster thanrecovery in the nucleolus. D) Initial radial profiles just after thebleach shows that the relative mobility of all constructs fused to sYFP2 indifferent regions of the cell is consistent with the FCS measurements.Hence, more shallow gradients indicate higher mobility. The radial profilesshow that NvSNAILA in the nucleolus (nu) has a lower mobility thanthe mobility of NvSNAILA, NvSNAILA-Δ5SNAG andNvSNAILA-Δ20pNLS in the nucleoplasm (np). The latterNvSNAILA-ΔZnf shows similar initial profiles butappear to have a higher mobility compared to the other constructs in thenucleoplasm and in turn it has a lower mobility thanNvSNAILA-ΔZnf in the cytoplasm (cp).
Mentions: The localisation analysis showed that NvSNAILA is significantly enrichedin the nucleolus and the FCS analysis indicated that it has a low apparentdiffusion coefficient in the nucleolus compared to the nucleoplasm. The FCSanalysis gives information about movement in and out of the detection volume;however it does not necessary give information about transport into and out ofthe nucleolus37. In order to study this process, a FRAP analysiswas performed. For this experiment, the full length NvSNAILA was fused tothe yellow fluorescent protein sYFP2 (NvSNAILA-sYFP2), which can bebleached more efficiently than mTurquoise228. A circular regionwith a diameter of 2.484 μm was bleached for one second,after which recovery was monitored over a period of about forty seconds (seeMethods). In Fig. 9A nucleolar enrichment ofNvSNAILA-sYFP2 is clearly visible in the averaged frame before bleaching(pre-bleach). Directly after bleaching the nucleolus is almost completelybleached (post-bleach) and after a period of about forty seconds the cell showsvirtually the same distribution (end-frames and difference image end-framesminus pre-bleach, supplementary video S1). Because the nucleus is a confinedcompartment, the total fluorescence in the nucleus is reduced, which is apparentas a step change in the time trace of the nucleus region (Fig.9B, blue trace); the part that is not recovered in the FRAP region(Fig. 9B, purple trace) can be completely explained bythis loss of total nuclear fluorescence. If the total loss of nuclearfluorescence is removed from the FRAP region traces, residual traces can beobtained (Fig. 9C). These residual traces(n = 6 measurements) show that the nucleoluscompletely recovers and does not appear to have any significant immobilefraction and that the long term recovery in the nucleolus has a similar timecourse as recovery in the nucleoplasm. This suggests that exchange ofNvSNAILA-sYFP2 between the nucleolus and the nucleoplasm is relativelyfast and that its long-term recovery is limited by diffusion from thenucleoplasm to the nucleolus and largely determined by the low diffusioncoefficient in the nucleoplasm and the geometry of the nucleus. Further FRAPanalysis was performed on NvSNAILA-Δ5SNAG,NvSNAILA-Δ20pNLS and NvSNAILA-ΔZnf both inthe nucleoplasm as well as the cytoplasm. The steepness of the radial profilesaround the centre of the bleached area just after the bleach was determined(Fig. 9D), which represent an indication of themobility of the different constructs and show that the FRAP results are in closeagreement with the apparent diffusion coefficients that were obtained from theFCS analysis.

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.