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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.

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eps-8  mutants are defective in embryonic morphogenesis and muscle attachment.(A) Structure of EPS-8 isoforms and predicted effects of by160 and jc36 deletions. The location of the ok539 intron deletion is shown in Croce et al., 2004 [17]. (B) Three-fold arrest with detached body wall muscles (arrow) in eps-8(RNAi) animal. (C) Arrested eps-8(by160) larva with normal epidermal morphology. (D–F) Frames from 4D time lapse movies of wild type (D), eps-8(jc36) mutants (E), and vab-19(ju406)/Df embryos (F) at time points equivalent to wild-type comma stage (395 minutes after first cleavage), two-fold (∼450 minutes), and three-fold (∼500 minutes). Like vab-19 embryos, eps-8(jc36) mutants elongate at normal rates to the two-fold stage (n = 12 embryos recorded). vab-19 embryos stop elongating within 5–10 minutes of the twofold stage, whereas all eps-8(jc36) embryos elongate to a 2.5- to 3-fold stage, stop elongating about an hour after the twofold stage, and then partly retract. eps-8(jc36) embryos show normal muscle twitching and, unlike vab-19, show vigorous movements within the eggshell; these movements stop after elongation arrest. Unlike vab-19 mutants, which typically hatch as lumpy two-fold embryos, most jc36 embryos do not hatch. Scales, 10 µm. (G, H) Rescue of eps-8(jc36) elongation defects by Pvab-19-EPS-8B::GFP (juEx703); GFP (G) and DIC (H). (I) Enhancement of vab-19(e1036cs) embryonic lethality by the eps-8b(ok539) allele; bars show mean±SEM for 5 complete broods for each genotype; P<0.001 by two-tailed t test. (J) Typical vab-19(e1036) eps-8(ok539) embryo from parents raised at 15°C, showing two-fold arrest and deformed head epidermis (arrow).
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pone-0003346-g002: eps-8 mutants are defective in embryonic morphogenesis and muscle attachment.(A) Structure of EPS-8 isoforms and predicted effects of by160 and jc36 deletions. The location of the ok539 intron deletion is shown in Croce et al., 2004 [17]. (B) Three-fold arrest with detached body wall muscles (arrow) in eps-8(RNAi) animal. (C) Arrested eps-8(by160) larva with normal epidermal morphology. (D–F) Frames from 4D time lapse movies of wild type (D), eps-8(jc36) mutants (E), and vab-19(ju406)/Df embryos (F) at time points equivalent to wild-type comma stage (395 minutes after first cleavage), two-fold (∼450 minutes), and three-fold (∼500 minutes). Like vab-19 embryos, eps-8(jc36) mutants elongate at normal rates to the two-fold stage (n = 12 embryos recorded). vab-19 embryos stop elongating within 5–10 minutes of the twofold stage, whereas all eps-8(jc36) embryos elongate to a 2.5- to 3-fold stage, stop elongating about an hour after the twofold stage, and then partly retract. eps-8(jc36) embryos show normal muscle twitching and, unlike vab-19, show vigorous movements within the eggshell; these movements stop after elongation arrest. Unlike vab-19 mutants, which typically hatch as lumpy two-fold embryos, most jc36 embryos do not hatch. Scales, 10 µm. (G, H) Rescue of eps-8(jc36) elongation defects by Pvab-19-EPS-8B::GFP (juEx703); GFP (G) and DIC (H). (I) Enhancement of vab-19(e1036cs) embryonic lethality by the eps-8b(ok539) allele; bars show mean±SEM for 5 complete broods for each genotype; P<0.001 by two-tailed t test. (J) Typical vab-19(e1036) eps-8(ok539) embryo from parents raised at 15°C, showing two-fold arrest and deformed head epidermis (arrow).

Mentions: vab-19 is expressed in dorsal and ventral epidermal cells from early elongation stages onwards [11]. We found that eps-8 transcriptional reporters were likewise expressed in dorsal and ventral embryonic epidermal cells beginning soon after epidermal enclosure (Figure 1B) and persisting in larval and adult stages (Figure 1C). To examine subcellular localization of EPS-8 within epidermal cells we inserted GFP into the N-terminus of EPS-8 and expressed it under the control of the epidermal-specific vab-19 promoter. The eps-8 locus generates two isoforms that differ in their C-termini, as a result of alternative splicing: EPS-8A contains the C-terminal actin binding ‘effector’ domain, whereas EPS-8B does not [17] (Figure 2A). Both EPS-8::GFP fusion proteins displayed indistinguishable patterns in the epidermis, and are referred to generically as EPS-8::GFP. In post-embryonic stages EPS-8::GFP was localized to circumferential bands in epidermis adjacent to muscles (Figure 1D), a pattern corresponding to trans-epidermal attachments. We also found EPS-8::GFP localized to other sites of trans-epidermal attachments (Figure 1E, F). These results indicate that in epidermal cells EPS-8, like VAB-19, is localized to sites of cell-matrix attachments.


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  mutants are defective in embryonic morphogenesis and muscle attachment.(A) Structure of EPS-8 isoforms and predicted effects of by160 and jc36 deletions. The location of the ok539 intron deletion is shown in Croce et al., 2004 [17]. (B) Three-fold arrest with detached body wall muscles (arrow) in eps-8(RNAi) animal. (C) Arrested eps-8(by160) larva with normal epidermal morphology. (D–F) Frames from 4D time lapse movies of wild type (D), eps-8(jc36) mutants (E), and vab-19(ju406)/Df embryos (F) at time points equivalent to wild-type comma stage (395 minutes after first cleavage), two-fold (∼450 minutes), and three-fold (∼500 minutes). Like vab-19 embryos, eps-8(jc36) mutants elongate at normal rates to the two-fold stage (n = 12 embryos recorded). vab-19 embryos stop elongating within 5–10 minutes of the twofold stage, whereas all eps-8(jc36) embryos elongate to a 2.5- to 3-fold stage, stop elongating about an hour after the twofold stage, and then partly retract. eps-8(jc36) embryos show normal muscle twitching and, unlike vab-19, show vigorous movements within the eggshell; these movements stop after elongation arrest. Unlike vab-19 mutants, which typically hatch as lumpy two-fold embryos, most jc36 embryos do not hatch. Scales, 10 µm. (G, H) Rescue of eps-8(jc36) elongation defects by Pvab-19-EPS-8B::GFP (juEx703); GFP (G) and DIC (H). (I) Enhancement of vab-19(e1036cs) embryonic lethality by the eps-8b(ok539) allele; bars show mean±SEM for 5 complete broods for each genotype; P<0.001 by two-tailed t test. (J) Typical vab-19(e1036) eps-8(ok539) embryo from parents raised at 15°C, showing two-fold arrest and deformed head epidermis (arrow).
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pone-0003346-g002: eps-8 mutants are defective in embryonic morphogenesis and muscle attachment.(A) Structure of EPS-8 isoforms and predicted effects of by160 and jc36 deletions. The location of the ok539 intron deletion is shown in Croce et al., 2004 [17]. (B) Three-fold arrest with detached body wall muscles (arrow) in eps-8(RNAi) animal. (C) Arrested eps-8(by160) larva with normal epidermal morphology. (D–F) Frames from 4D time lapse movies of wild type (D), eps-8(jc36) mutants (E), and vab-19(ju406)/Df embryos (F) at time points equivalent to wild-type comma stage (395 minutes after first cleavage), two-fold (∼450 minutes), and three-fold (∼500 minutes). Like vab-19 embryos, eps-8(jc36) mutants elongate at normal rates to the two-fold stage (n = 12 embryos recorded). vab-19 embryos stop elongating within 5–10 minutes of the twofold stage, whereas all eps-8(jc36) embryos elongate to a 2.5- to 3-fold stage, stop elongating about an hour after the twofold stage, and then partly retract. eps-8(jc36) embryos show normal muscle twitching and, unlike vab-19, show vigorous movements within the eggshell; these movements stop after elongation arrest. Unlike vab-19 mutants, which typically hatch as lumpy two-fold embryos, most jc36 embryos do not hatch. Scales, 10 µm. (G, H) Rescue of eps-8(jc36) elongation defects by Pvab-19-EPS-8B::GFP (juEx703); GFP (G) and DIC (H). (I) Enhancement of vab-19(e1036cs) embryonic lethality by the eps-8b(ok539) allele; bars show mean±SEM for 5 complete broods for each genotype; P<0.001 by two-tailed t test. (J) Typical vab-19(e1036) eps-8(ok539) embryo from parents raised at 15°C, showing two-fold arrest and deformed head epidermis (arrow).
Mentions: vab-19 is expressed in dorsal and ventral epidermal cells from early elongation stages onwards [11]. We found that eps-8 transcriptional reporters were likewise expressed in dorsal and ventral embryonic epidermal cells beginning soon after epidermal enclosure (Figure 1B) and persisting in larval and adult stages (Figure 1C). To examine subcellular localization of EPS-8 within epidermal cells we inserted GFP into the N-terminus of EPS-8 and expressed it under the control of the epidermal-specific vab-19 promoter. The eps-8 locus generates two isoforms that differ in their C-termini, as a result of alternative splicing: EPS-8A contains the C-terminal actin binding ‘effector’ domain, whereas EPS-8B does not [17] (Figure 2A). Both EPS-8::GFP fusion proteins displayed indistinguishable patterns in the epidermis, and are referred to generically as EPS-8::GFP. In post-embryonic stages EPS-8::GFP was localized to circumferential bands in epidermis adjacent to muscles (Figure 1D), a pattern corresponding to trans-epidermal attachments. We also found EPS-8::GFP localized to other sites of trans-epidermal attachments (Figure 1E, F). These results indicate that in epidermal cells EPS-8, like VAB-19, is localized to sites of cell-matrix attachments.

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
Related in: MedlinePlus