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Control of brain patterning by Engrailed paracrine transfer: a new function of the Pbx interaction domain.

Rampon C, Gauron C, Lin T, Meda F, Dupont E, Cosson A, Ipendey E, Frerot A, Aujard I, Le Saux T, Bensimon D, Jullien L, Volovitch M, Vriz S, Joliot A - Development (2015)

Bottom Line: In light of recent reports on the paracrine activity of homeoproteins, including Engrailed, we asked whether Engrailed intercellular transfer was also involved in brain patterning and boundary formation.Both zebrafish Eng2a and Eng2b are competent for intercellular transfer in vivo, but only extracellular endogenous Eng2b, and not Eng2a, participates in DMB positioning.In addition, disruption of the Pbx-interacting motif in Engrailed, known to strongly reduce the gain-of-function phenotype, also downregulates Engrailed transfer, thus revealing an unsuspected participation of the Pbx interaction domain in this pathway.

View Article: PubMed Central - PubMed

Affiliation: Université Paris Diderot, Sorbonne Paris Cité, Paris 75205, Cedex 13, France Center for Interdisciplinary Research in Biology (CIRB) - CNRS UMR 7241, INSERM U1050, Labex MemoLife, PSL Research University, Collège de France, Paris F-75005, France.

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Pbx interaction domain controls En paracrine activity. (A) Phenotypic analysis of hexapeptide-mutated forms of En2 protein. Compared to their wild-type counterparts, the eye phenotype induced by the activation of hexapeptide mutants was drastically reduced. The error bars represent statistical errors. (B-D) Intercellular transfer of En2 hexapeptide mutant ex vivo. HEK293 cells expressing the indicated proteins under the control of doxycycline were cultured for 24 h and cell surface accumulation of the secreted protein was monitored by flow cytometry (B). Internalisation of fluorescein-labelled En2, or En2 WW>KK (green) was visualised (C) and quantified (D) after 1 h incubation at 37°C in the presence of 0.4% Trypan Blue (red) to quench extracellular fluorescence. (E,F) Internalisation of fluorescein-labelled En2, En2HD, Hexa-En2HD was visualised (E) and quantified (F) as described above. The error bars represent statistical errors (A) or the s.e.m. (B,D,F). **P<0.01, ***P<0.001.
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DEV114181F6: Pbx interaction domain controls En paracrine activity. (A) Phenotypic analysis of hexapeptide-mutated forms of En2 protein. Compared to their wild-type counterparts, the eye phenotype induced by the activation of hexapeptide mutants was drastically reduced. The error bars represent statistical errors. (B-D) Intercellular transfer of En2 hexapeptide mutant ex vivo. HEK293 cells expressing the indicated proteins under the control of doxycycline were cultured for 24 h and cell surface accumulation of the secreted protein was monitored by flow cytometry (B). Internalisation of fluorescein-labelled En2, or En2 WW>KK (green) was visualised (C) and quantified (D) after 1 h incubation at 37°C in the presence of 0.4% Trypan Blue (red) to quench extracellular fluorescence. (E,F) Internalisation of fluorescein-labelled En2, En2HD, Hexa-En2HD was visualised (E) and quantified (F) as described above. The error bars represent statistical errors (A) or the s.e.m. (B,D,F). **P<0.01, ***P<0.001.

Mentions: We first assessed the selectivity of the hexapeptide mutation towards Eng2a function by engineering equivalent WW>KK substitutions in the En2, Eng2a and Eng2b constructs. The mRNAs coding for the mutant forms of Engrailed-ERT2 were injected into zebrafish embryos at the one-cell stage, embryos were incubated in cyclofen at 50% epiboly, and eye defects were scored at 30 hpf. Introducing the WW>KK double mutation similarly decreased the occurrence of the eye phenotype for the three Engrailed 2 proteins (Fig. 6A). Although the mutation might appear to selectively affect the intracrine component of Engrailed action, we wondered whether the removal of two tryptophans near the homeodomain might also affect intercellular transfer, given the crucial role of such residues in the internalisation process (Le Roux et al., 1993; Derossi et al., 1994). We tested the ability of the mutant forms of En2 to exit and enter cells using the cell culture quantitative assays described above. When expressed in HEK cells, the mutant and wild-type proteins were secreted to similar extents (Fig. 6B), meaning that the integrity of the hexapeptide is not required for En2 secretion. By contrast, the internalisation of the mutated protein by HEK cells was significantly reduced compared with that of its wild-type counterpart (Fig. 6C,D). We reasoned that, conversely, the presence of the hexapeptide on its own might increase the uptake process. This would corroborate unpublished observations that in this experimental model, full-length En2 homeoprotein uptake was higher than that of an En2 homeodomain fragment (Fig. 6C,E). A 12 amino-acid long peptide bearing the hexapeptide was fused to the N-terminus of the En2 homeodomain and its uptake was evaluated. Addition of the hexapeptide restored the uptake of the En2 homeodomain to levels comparable to those of the full-length homeoprotein (Fig. 6C,E,F). Overexpression of Pbx neither significantly alters En2 location nor interferes with its intercellular transfer (secretion and internalisation) properties (supplementary material Fig. S3), although internalisation was occasionally slightly reduced. We then conclude that the hexapeptide per se potentiates homeodomain uptake and, therefore, that the WW>KK mutation, known to disrupt Pbx-Engrailed transcriptional complexes, also impairs the paracrine component of Engrailed action, by lowering the efficacy of protein trafficking.Fig. 6.


Control of brain patterning by Engrailed paracrine transfer: a new function of the Pbx interaction domain.

Rampon C, Gauron C, Lin T, Meda F, Dupont E, Cosson A, Ipendey E, Frerot A, Aujard I, Le Saux T, Bensimon D, Jullien L, Volovitch M, Vriz S, Joliot A - Development (2015)

Pbx interaction domain controls En paracrine activity. (A) Phenotypic analysis of hexapeptide-mutated forms of En2 protein. Compared to their wild-type counterparts, the eye phenotype induced by the activation of hexapeptide mutants was drastically reduced. The error bars represent statistical errors. (B-D) Intercellular transfer of En2 hexapeptide mutant ex vivo. HEK293 cells expressing the indicated proteins under the control of doxycycline were cultured for 24 h and cell surface accumulation of the secreted protein was monitored by flow cytometry (B). Internalisation of fluorescein-labelled En2, or En2 WW>KK (green) was visualised (C) and quantified (D) after 1 h incubation at 37°C in the presence of 0.4% Trypan Blue (red) to quench extracellular fluorescence. (E,F) Internalisation of fluorescein-labelled En2, En2HD, Hexa-En2HD was visualised (E) and quantified (F) as described above. The error bars represent statistical errors (A) or the s.e.m. (B,D,F). **P<0.01, ***P<0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4440920&req=5

DEV114181F6: Pbx interaction domain controls En paracrine activity. (A) Phenotypic analysis of hexapeptide-mutated forms of En2 protein. Compared to their wild-type counterparts, the eye phenotype induced by the activation of hexapeptide mutants was drastically reduced. The error bars represent statistical errors. (B-D) Intercellular transfer of En2 hexapeptide mutant ex vivo. HEK293 cells expressing the indicated proteins under the control of doxycycline were cultured for 24 h and cell surface accumulation of the secreted protein was monitored by flow cytometry (B). Internalisation of fluorescein-labelled En2, or En2 WW>KK (green) was visualised (C) and quantified (D) after 1 h incubation at 37°C in the presence of 0.4% Trypan Blue (red) to quench extracellular fluorescence. (E,F) Internalisation of fluorescein-labelled En2, En2HD, Hexa-En2HD was visualised (E) and quantified (F) as described above. The error bars represent statistical errors (A) or the s.e.m. (B,D,F). **P<0.01, ***P<0.001.
Mentions: We first assessed the selectivity of the hexapeptide mutation towards Eng2a function by engineering equivalent WW>KK substitutions in the En2, Eng2a and Eng2b constructs. The mRNAs coding for the mutant forms of Engrailed-ERT2 were injected into zebrafish embryos at the one-cell stage, embryos were incubated in cyclofen at 50% epiboly, and eye defects were scored at 30 hpf. Introducing the WW>KK double mutation similarly decreased the occurrence of the eye phenotype for the three Engrailed 2 proteins (Fig. 6A). Although the mutation might appear to selectively affect the intracrine component of Engrailed action, we wondered whether the removal of two tryptophans near the homeodomain might also affect intercellular transfer, given the crucial role of such residues in the internalisation process (Le Roux et al., 1993; Derossi et al., 1994). We tested the ability of the mutant forms of En2 to exit and enter cells using the cell culture quantitative assays described above. When expressed in HEK cells, the mutant and wild-type proteins were secreted to similar extents (Fig. 6B), meaning that the integrity of the hexapeptide is not required for En2 secretion. By contrast, the internalisation of the mutated protein by HEK cells was significantly reduced compared with that of its wild-type counterpart (Fig. 6C,D). We reasoned that, conversely, the presence of the hexapeptide on its own might increase the uptake process. This would corroborate unpublished observations that in this experimental model, full-length En2 homeoprotein uptake was higher than that of an En2 homeodomain fragment (Fig. 6C,E). A 12 amino-acid long peptide bearing the hexapeptide was fused to the N-terminus of the En2 homeodomain and its uptake was evaluated. Addition of the hexapeptide restored the uptake of the En2 homeodomain to levels comparable to those of the full-length homeoprotein (Fig. 6C,E,F). Overexpression of Pbx neither significantly alters En2 location nor interferes with its intercellular transfer (secretion and internalisation) properties (supplementary material Fig. S3), although internalisation was occasionally slightly reduced. We then conclude that the hexapeptide per se potentiates homeodomain uptake and, therefore, that the WW>KK mutation, known to disrupt Pbx-Engrailed transcriptional complexes, also impairs the paracrine component of Engrailed action, by lowering the efficacy of protein trafficking.Fig. 6.

Bottom Line: In light of recent reports on the paracrine activity of homeoproteins, including Engrailed, we asked whether Engrailed intercellular transfer was also involved in brain patterning and boundary formation.Both zebrafish Eng2a and Eng2b are competent for intercellular transfer in vivo, but only extracellular endogenous Eng2b, and not Eng2a, participates in DMB positioning.In addition, disruption of the Pbx-interacting motif in Engrailed, known to strongly reduce the gain-of-function phenotype, also downregulates Engrailed transfer, thus revealing an unsuspected participation of the Pbx interaction domain in this pathway.

View Article: PubMed Central - PubMed

Affiliation: Université Paris Diderot, Sorbonne Paris Cité, Paris 75205, Cedex 13, France Center for Interdisciplinary Research in Biology (CIRB) - CNRS UMR 7241, INSERM U1050, Labex MemoLife, PSL Research University, Collège de France, Paris F-75005, France.

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