Limits...
The cell signaling adaptor protein EPS-8 is essential for C. elegans epidermal elongation and interacts with the ankyrin repeat protein VAB-19.

Ding M, King RS, Berry EC, Wang Y, Hardin J, Chisholm AD - PLoS ONE (2008)

Bottom Line: The epidermal cells of the C. elegans embryo undergo coordinated cell shape changes that result in the morphogenetic process of elongation.The function of EPS-8 in epidermal development involves its N-terminal PTB and central domains, and is independent of its C-terminal SH3 and actin-binding domains.The existence of EPS-8B-like isoforms in Drosophila suggests this function of EPS-8 proteins could be conserved among other organisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology, University of California Santa Cruz, Santa Cruz, California, USA.

ABSTRACT

Background: The epidermal cells of the C. elegans embryo undergo coordinated cell shape changes that result in the morphogenetic process of elongation. The cytoskeletal ankyrin repeat protein VAB-19 is required for cell shape changes and localizes to cell-matrix attachment structures. The molecular functions of VAB-19 in this process are obscure, as no previous interactors for VAB-19 have been described.

Methodology/principal findings: In screens for VAB-19 binding proteins we identified the signaling adaptor EPS-8. Within C. elegans epidermal cells, EPS-8 and VAB-19 colocalize at cell-matrix attachment structures. The central domain of EPS-8 is necessary and sufficient for its interaction with VAB-19. eps-8 mutants, like vab-19 mutants, are defective in epidermal elongation and in epidermal-muscle attachment. The eps-8 locus encodes two isoforms, EPS-8A and EPS-8B, that appear to act redundantly in epidermal elongation. The function of EPS-8 in epidermal development involves its N-terminal PTB and central domains, and is independent of its C-terminal SH3 and actin-binding domains. VAB-19 appears to act earlier in the biogenesis of attachment structures and may recruit EPS-8 to these structures.

Conclusions/significance: EPS-8 and VAB-19 define a novel pathway acting at cell-matrix attachments to regulate epithelial cell shape. This is the first report of a role for EPS-8 proteins in cell-matrix attachments. The existence of EPS-8B-like isoforms in Drosophila suggests this function of EPS-8 proteins could be conserved among other organisms.

Show MeSH
EPS-8 interacts with VAB-19 and colocalizes with VAB-19 at epithelial attachment structures.(A) The VAB-19 ANK repeat domain interacts with the central domain of EPS-8. The initial interacting clone corresponds to almost full length EPS-8A. Deletion derivatives of EPS-8A were screened for interaction with VAB-19ANK in yeast. A fragment containing the central domain (EPS-8A residues 245–502) is sufficient to interact with VAB-19 ANK domains, although constructs containing part of the PTB domain interact more strongly. The PTB domain alone (residues 25–264) does not interact with VAB-19ANK. None of the constructs cause self-activation (pBTM116 controls). (B, C) GFP expressed under the control of a 3.3 kb eps-8 promoter (Peps-8-GFP, juEx526) is first seen in ventral and dorsal epidermal cells at comma stage (350 min) and persists in these tissues throughout larval and adult stages. Peps-8-GFP was also expressed in sublateral neurons (arrow, C). The 3.3 kb eps-8 promoter likely drives a subset of the eps-8 expression pattern, as eps-8 has been shown to be expressed in intestinal and pharyngeal cells [17]. (D) EPS-8B::GFP (juEx700) was localized to circumferential bands in muscle-adjacent epidermis in embryos (not shown) and in larvae. We detected EPS-8B::GFP at other known sites of trans-epidermal attachments: adjacent to mechanosensory neuron processes (arrow, E), and at the uterine seam cell attachment (arrow, F). EPS-8::GFP was also expressed in the uterus and in the excretory cell (not shown). (G) In pharyngeal marginal cells, Pvab-19-EPS-8B::GFP was localized to the apical (lumenal) ends of attachment structures (arrow) and not to the basal surface (arrowhead), in contrast to VAB-19::GFP (juEx433), which is found at both basal and apical ends of pharyngeal marginal cells (arrowhead and arrow, respectively, H). The subcellular localization of EPS-8A::GFP in these tissues was indistinguishable from EPS-8B::GFP (not shown). (I, J) Localization of mCherry:: EPS-8(central) (juEx1784) to embryonic attachment structures (arrow). Truncated mCherry::EPS-8 fusion proteins also displayed aggregation (bright blobs); as these aggregates are not observed with GFP-tagged full length EPS-8 they may result from overexpression of truncated proteins. (K) Co-localization of Pdpy-7-mCherry::EPS-8(central) (juEx1784) and VAB-19::GFP (juEx433) to attachment structures in larval epidermis. Scales, 10 µm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2553197&req=5

pone-0003346-g001: EPS-8 interacts with VAB-19 and colocalizes with VAB-19 at epithelial attachment structures.(A) The VAB-19 ANK repeat domain interacts with the central domain of EPS-8. The initial interacting clone corresponds to almost full length EPS-8A. Deletion derivatives of EPS-8A were screened for interaction with VAB-19ANK in yeast. A fragment containing the central domain (EPS-8A residues 245–502) is sufficient to interact with VAB-19 ANK domains, although constructs containing part of the PTB domain interact more strongly. The PTB domain alone (residues 25–264) does not interact with VAB-19ANK. None of the constructs cause self-activation (pBTM116 controls). (B, C) GFP expressed under the control of a 3.3 kb eps-8 promoter (Peps-8-GFP, juEx526) is first seen in ventral and dorsal epidermal cells at comma stage (350 min) and persists in these tissues throughout larval and adult stages. Peps-8-GFP was also expressed in sublateral neurons (arrow, C). The 3.3 kb eps-8 promoter likely drives a subset of the eps-8 expression pattern, as eps-8 has been shown to be expressed in intestinal and pharyngeal cells [17]. (D) EPS-8B::GFP (juEx700) was localized to circumferential bands in muscle-adjacent epidermis in embryos (not shown) and in larvae. We detected EPS-8B::GFP at other known sites of trans-epidermal attachments: adjacent to mechanosensory neuron processes (arrow, E), and at the uterine seam cell attachment (arrow, F). EPS-8::GFP was also expressed in the uterus and in the excretory cell (not shown). (G) In pharyngeal marginal cells, Pvab-19-EPS-8B::GFP was localized to the apical (lumenal) ends of attachment structures (arrow) and not to the basal surface (arrowhead), in contrast to VAB-19::GFP (juEx433), which is found at both basal and apical ends of pharyngeal marginal cells (arrowhead and arrow, respectively, H). The subcellular localization of EPS-8A::GFP in these tissues was indistinguishable from EPS-8B::GFP (not shown). (I, J) Localization of mCherry:: EPS-8(central) (juEx1784) to embryonic attachment structures (arrow). Truncated mCherry::EPS-8 fusion proteins also displayed aggregation (bright blobs); as these aggregates are not observed with GFP-tagged full length EPS-8 they may result from overexpression of truncated proteins. (K) Co-localization of Pdpy-7-mCherry::EPS-8(central) (juEx1784) and VAB-19::GFP (juEx433) to attachment structures in larval epidermis. Scales, 10 µm.

Mentions: To understand how VAB-19 regulates epidermal morphogenesis, we used the C-terminal ankyrin repeat domain of VAB-19 as bait in a yeast two-hybrid screen, and identified C. elegans EPS-8 as a VAB-19 interactor (see Materials and Methods). Eps8 proteins typically contain an N-terminal phosphotyrosine-binding (PTB) domain, a central domain conserved among the Eps8 family and implicated in EGFR binding, an SH3 domain, and a C-terminal ‘effector’ domain that binds actin [19]–[22]. We mapped the interacting domains between VAB-19 and EPS-8 in yeast (Figure 1A) and found that the central domain of EPS-8 (residues 272–502) was necessary for binding VAB-19. Constructs containing only the central domain (residues 245–502) displayed a weaker interaction with VAB-19ANK, suggesting that although the central domain is sufficient for an interaction, the combination of the PTB and central domains may be required for a strong interaction.


The cell signaling adaptor protein EPS-8 is essential for C. elegans epidermal elongation and interacts with the ankyrin repeat protein VAB-19.

Ding M, King RS, Berry EC, Wang Y, Hardin J, Chisholm AD - PLoS ONE (2008)

EPS-8 interacts with VAB-19 and colocalizes with VAB-19 at epithelial attachment structures.(A) The VAB-19 ANK repeat domain interacts with the central domain of EPS-8. The initial interacting clone corresponds to almost full length EPS-8A. Deletion derivatives of EPS-8A were screened for interaction with VAB-19ANK in yeast. A fragment containing the central domain (EPS-8A residues 245–502) is sufficient to interact with VAB-19 ANK domains, although constructs containing part of the PTB domain interact more strongly. The PTB domain alone (residues 25–264) does not interact with VAB-19ANK. None of the constructs cause self-activation (pBTM116 controls). (B, C) GFP expressed under the control of a 3.3 kb eps-8 promoter (Peps-8-GFP, juEx526) is first seen in ventral and dorsal epidermal cells at comma stage (350 min) and persists in these tissues throughout larval and adult stages. Peps-8-GFP was also expressed in sublateral neurons (arrow, C). The 3.3 kb eps-8 promoter likely drives a subset of the eps-8 expression pattern, as eps-8 has been shown to be expressed in intestinal and pharyngeal cells [17]. (D) EPS-8B::GFP (juEx700) was localized to circumferential bands in muscle-adjacent epidermis in embryos (not shown) and in larvae. We detected EPS-8B::GFP at other known sites of trans-epidermal attachments: adjacent to mechanosensory neuron processes (arrow, E), and at the uterine seam cell attachment (arrow, F). EPS-8::GFP was also expressed in the uterus and in the excretory cell (not shown). (G) In pharyngeal marginal cells, Pvab-19-EPS-8B::GFP was localized to the apical (lumenal) ends of attachment structures (arrow) and not to the basal surface (arrowhead), in contrast to VAB-19::GFP (juEx433), which is found at both basal and apical ends of pharyngeal marginal cells (arrowhead and arrow, respectively, H). The subcellular localization of EPS-8A::GFP in these tissues was indistinguishable from EPS-8B::GFP (not shown). (I, J) Localization of mCherry:: EPS-8(central) (juEx1784) to embryonic attachment structures (arrow). Truncated mCherry::EPS-8 fusion proteins also displayed aggregation (bright blobs); as these aggregates are not observed with GFP-tagged full length EPS-8 they may result from overexpression of truncated proteins. (K) Co-localization of Pdpy-7-mCherry::EPS-8(central) (juEx1784) and VAB-19::GFP (juEx433) to attachment structures in larval epidermis. Scales, 10 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2553197&req=5

pone-0003346-g001: EPS-8 interacts with VAB-19 and colocalizes with VAB-19 at epithelial attachment structures.(A) The VAB-19 ANK repeat domain interacts with the central domain of EPS-8. The initial interacting clone corresponds to almost full length EPS-8A. Deletion derivatives of EPS-8A were screened for interaction with VAB-19ANK in yeast. A fragment containing the central domain (EPS-8A residues 245–502) is sufficient to interact with VAB-19 ANK domains, although constructs containing part of the PTB domain interact more strongly. The PTB domain alone (residues 25–264) does not interact with VAB-19ANK. None of the constructs cause self-activation (pBTM116 controls). (B, C) GFP expressed under the control of a 3.3 kb eps-8 promoter (Peps-8-GFP, juEx526) is first seen in ventral and dorsal epidermal cells at comma stage (350 min) and persists in these tissues throughout larval and adult stages. Peps-8-GFP was also expressed in sublateral neurons (arrow, C). The 3.3 kb eps-8 promoter likely drives a subset of the eps-8 expression pattern, as eps-8 has been shown to be expressed in intestinal and pharyngeal cells [17]. (D) EPS-8B::GFP (juEx700) was localized to circumferential bands in muscle-adjacent epidermis in embryos (not shown) and in larvae. We detected EPS-8B::GFP at other known sites of trans-epidermal attachments: adjacent to mechanosensory neuron processes (arrow, E), and at the uterine seam cell attachment (arrow, F). EPS-8::GFP was also expressed in the uterus and in the excretory cell (not shown). (G) In pharyngeal marginal cells, Pvab-19-EPS-8B::GFP was localized to the apical (lumenal) ends of attachment structures (arrow) and not to the basal surface (arrowhead), in contrast to VAB-19::GFP (juEx433), which is found at both basal and apical ends of pharyngeal marginal cells (arrowhead and arrow, respectively, H). The subcellular localization of EPS-8A::GFP in these tissues was indistinguishable from EPS-8B::GFP (not shown). (I, J) Localization of mCherry:: EPS-8(central) (juEx1784) to embryonic attachment structures (arrow). Truncated mCherry::EPS-8 fusion proteins also displayed aggregation (bright blobs); as these aggregates are not observed with GFP-tagged full length EPS-8 they may result from overexpression of truncated proteins. (K) Co-localization of Pdpy-7-mCherry::EPS-8(central) (juEx1784) and VAB-19::GFP (juEx433) to attachment structures in larval epidermis. Scales, 10 µm.
Mentions: To understand how VAB-19 regulates epidermal morphogenesis, we used the C-terminal ankyrin repeat domain of VAB-19 as bait in a yeast two-hybrid screen, and identified C. elegans EPS-8 as a VAB-19 interactor (see Materials and Methods). Eps8 proteins typically contain an N-terminal phosphotyrosine-binding (PTB) domain, a central domain conserved among the Eps8 family and implicated in EGFR binding, an SH3 domain, and a C-terminal ‘effector’ domain that binds actin [19]–[22]. We mapped the interacting domains between VAB-19 and EPS-8 in yeast (Figure 1A) and found that the central domain of EPS-8 (residues 272–502) was necessary for binding VAB-19. Constructs containing only the central domain (residues 245–502) displayed a weaker interaction with VAB-19ANK, suggesting that although the central domain is sufficient for an interaction, the combination of the PTB and central domains may be required for a strong interaction.

Bottom Line: The epidermal cells of the C. elegans embryo undergo coordinated cell shape changes that result in the morphogenetic process of elongation.The function of EPS-8 in epidermal development involves its N-terminal PTB and central domains, and is independent of its C-terminal SH3 and actin-binding domains.The existence of EPS-8B-like isoforms in Drosophila suggests this function of EPS-8 proteins could be conserved among other organisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology, University of California Santa Cruz, Santa Cruz, California, USA.

ABSTRACT

Background: The epidermal cells of the C. elegans embryo undergo coordinated cell shape changes that result in the morphogenetic process of elongation. The cytoskeletal ankyrin repeat protein VAB-19 is required for cell shape changes and localizes to cell-matrix attachment structures. The molecular functions of VAB-19 in this process are obscure, as no previous interactors for VAB-19 have been described.

Methodology/principal findings: In screens for VAB-19 binding proteins we identified the signaling adaptor EPS-8. Within C. elegans epidermal cells, EPS-8 and VAB-19 colocalize at cell-matrix attachment structures. The central domain of EPS-8 is necessary and sufficient for its interaction with VAB-19. eps-8 mutants, like vab-19 mutants, are defective in epidermal elongation and in epidermal-muscle attachment. The eps-8 locus encodes two isoforms, EPS-8A and EPS-8B, that appear to act redundantly in epidermal elongation. The function of EPS-8 in epidermal development involves its N-terminal PTB and central domains, and is independent of its C-terminal SH3 and actin-binding domains. VAB-19 appears to act earlier in the biogenesis of attachment structures and may recruit EPS-8 to these structures.

Conclusions/significance: EPS-8 and VAB-19 define a novel pathway acting at cell-matrix attachments to regulate epithelial cell shape. This is the first report of a role for EPS-8 proteins in cell-matrix attachments. The existence of EPS-8B-like isoforms in Drosophila suggests this function of EPS-8 proteins could be conserved among other organisms.

Show MeSH