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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 display late-onset defects in epidermal attachment structure development and in the epidermal actin cytoskeleton.(A–D) Localization of epidermal intermediate filaments (MH4 immunostaining) in the wild type and in eps-8(jc36) mutants. At the 1.5-fold stage (A), IFs were restricted in muscle-adjacent region of epidermis. (B) After the twofold stage, IFs localize to regularly spaced circumferential stripes. (C–D) In eps-8 mutants, IFs appear normal until after the twofold stage, when they expand into regions of epidermal cells that are not adjacent to muscle (arrowhead), compared to the wild type. (E–H) Myotactin expression (MH46 immunostaining) in the wild type and in eps-8 mutants. (E, G) During early elongation (1.5 to two-fold), Myotactin appears normal in eps-8 mutants. By the threefold stage, Myotactin localization refines to circumferential stripes in muscle-adjacent regions of epidermis (F). (H) In eps-8 mutants, Myotactin is still localized to muscle-adjacent regions but remains in longitudinal bands rather than circumferential stripes. (I, J) In the wild type, circumferential bundles of F-actin (phalloidin staining) are continuous around the embryo. Insets I' and J' show actin bundles at higher magnification. In eps-8 mutants, actin filaments were more randomly oriented or missing from the apical surface of the epidermis (K, K'). Actin filaments were also disorganized and fragmented in lateral epidermal cells (L, L', asterisk). Scale, 10 µm.
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pone-0003346-g003: eps-8 mutants display late-onset defects in epidermal attachment structure development and in the epidermal actin cytoskeleton.(A–D) Localization of epidermal intermediate filaments (MH4 immunostaining) in the wild type and in eps-8(jc36) mutants. At the 1.5-fold stage (A), IFs were restricted in muscle-adjacent region of epidermis. (B) After the twofold stage, IFs localize to regularly spaced circumferential stripes. (C–D) In eps-8 mutants, IFs appear normal until after the twofold stage, when they expand into regions of epidermal cells that are not adjacent to muscle (arrowhead), compared to the wild type. (E–H) Myotactin expression (MH46 immunostaining) in the wild type and in eps-8 mutants. (E, G) During early elongation (1.5 to two-fold), Myotactin appears normal in eps-8 mutants. By the threefold stage, Myotactin localization refines to circumferential stripes in muscle-adjacent regions of epidermis (F). (H) In eps-8 mutants, Myotactin is still localized to muscle-adjacent regions but remains in longitudinal bands rather than circumferential stripes. (I, J) In the wild type, circumferential bundles of F-actin (phalloidin staining) are continuous around the embryo. Insets I' and J' show actin bundles at higher magnification. In eps-8 mutants, actin filaments were more randomly oriented or missing from the apical surface of the epidermis (K, K'). Actin filaments were also disorganized and fragmented in lateral epidermal cells (L, L', asterisk). Scale, 10 µm.

Mentions: As EPS-8 appears to be a new component of epidermal attachment structures, we examined the effect of eps-8 mutations on other attachment structure components. During early and intermediate (two-fold) stages of epidermal elongation, the localization of intermediate filaments (IFs) was normal in eps-8 mutants (Figure 3A, C). However, during later elongation, IFs became delocalized, expanding into regions not adjacent to body wall muscle with slightly altered patterns of circumferential bands (compare Figure 3B and D). We conclude that EPS-8 is not essential for the initial assembly of attachment structures. We also examined the localization of the receptor-like protein Myotactin, which normally becomes refined at the two-fold stage from broad longitudinal bands adjacent to muscle into circumferential stripes corresponding to attachment structures [24], [25]. The early localization pattern of Myotactin into longitudinal bands was normal in eps-8 embryos (Figure 3E, G). However, Myotactin never reorganized into circumferential stripes (compare Figure 3F and H). These effects on IFs and Myotactin in eps-8 mutant embryos closely resemble those of vab-19 mutants [11]. Expression of another cell junction marker, the apical junctional marker AJM-1 [23] in eps-8(jc36) mutants was similar to wild type (not shown), indicating that epidermal cells are specified correctly and have normal lateral cell–cell contacts.


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 display late-onset defects in epidermal attachment structure development and in the epidermal actin cytoskeleton.(A–D) Localization of epidermal intermediate filaments (MH4 immunostaining) in the wild type and in eps-8(jc36) mutants. At the 1.5-fold stage (A), IFs were restricted in muscle-adjacent region of epidermis. (B) After the twofold stage, IFs localize to regularly spaced circumferential stripes. (C–D) In eps-8 mutants, IFs appear normal until after the twofold stage, when they expand into regions of epidermal cells that are not adjacent to muscle (arrowhead), compared to the wild type. (E–H) Myotactin expression (MH46 immunostaining) in the wild type and in eps-8 mutants. (E, G) During early elongation (1.5 to two-fold), Myotactin appears normal in eps-8 mutants. By the threefold stage, Myotactin localization refines to circumferential stripes in muscle-adjacent regions of epidermis (F). (H) In eps-8 mutants, Myotactin is still localized to muscle-adjacent regions but remains in longitudinal bands rather than circumferential stripes. (I, J) In the wild type, circumferential bundles of F-actin (phalloidin staining) are continuous around the embryo. Insets I' and J' show actin bundles at higher magnification. In eps-8 mutants, actin filaments were more randomly oriented or missing from the apical surface of the epidermis (K, K'). Actin filaments were also disorganized and fragmented in lateral epidermal cells (L, L', asterisk). Scale, 10 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2553197&req=5

pone-0003346-g003: eps-8 mutants display late-onset defects in epidermal attachment structure development and in the epidermal actin cytoskeleton.(A–D) Localization of epidermal intermediate filaments (MH4 immunostaining) in the wild type and in eps-8(jc36) mutants. At the 1.5-fold stage (A), IFs were restricted in muscle-adjacent region of epidermis. (B) After the twofold stage, IFs localize to regularly spaced circumferential stripes. (C–D) In eps-8 mutants, IFs appear normal until after the twofold stage, when they expand into regions of epidermal cells that are not adjacent to muscle (arrowhead), compared to the wild type. (E–H) Myotactin expression (MH46 immunostaining) in the wild type and in eps-8 mutants. (E, G) During early elongation (1.5 to two-fold), Myotactin appears normal in eps-8 mutants. By the threefold stage, Myotactin localization refines to circumferential stripes in muscle-adjacent regions of epidermis (F). (H) In eps-8 mutants, Myotactin is still localized to muscle-adjacent regions but remains in longitudinal bands rather than circumferential stripes. (I, J) In the wild type, circumferential bundles of F-actin (phalloidin staining) are continuous around the embryo. Insets I' and J' show actin bundles at higher magnification. In eps-8 mutants, actin filaments were more randomly oriented or missing from the apical surface of the epidermis (K, K'). Actin filaments were also disorganized and fragmented in lateral epidermal cells (L, L', asterisk). Scale, 10 µm.
Mentions: As EPS-8 appears to be a new component of epidermal attachment structures, we examined the effect of eps-8 mutations on other attachment structure components. During early and intermediate (two-fold) stages of epidermal elongation, the localization of intermediate filaments (IFs) was normal in eps-8 mutants (Figure 3A, C). However, during later elongation, IFs became delocalized, expanding into regions not adjacent to body wall muscle with slightly altered patterns of circumferential bands (compare Figure 3B and D). We conclude that EPS-8 is not essential for the initial assembly of attachment structures. We also examined the localization of the receptor-like protein Myotactin, which normally becomes refined at the two-fold stage from broad longitudinal bands adjacent to muscle into circumferential stripes corresponding to attachment structures [24], [25]. The early localization pattern of Myotactin into longitudinal bands was normal in eps-8 embryos (Figure 3E, G). However, Myotactin never reorganized into circumferential stripes (compare Figure 3F and H). These effects on IFs and Myotactin in eps-8 mutant embryos closely resemble those of vab-19 mutants [11]. Expression of another cell junction marker, the apical junctional marker AJM-1 [23] in eps-8(jc36) mutants was similar to wild type (not shown), indicating that epidermal cells are specified correctly and have normal lateral cell–cell contacts.

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