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The Caenorhabditis elegans vab-10 spectraplakin isoforms protect the epidermis against internal and external forces.

Bosher JM, Hahn BS, Legouis R, Sookhareea S, Weimer RM, Gansmuller A, Chisholm AD, Rose AM, Bessereau JL, Labouesse M - J. Cell Biol. (2003)

Bottom Line: We suggest that this isoform protects against forces external to the epidermis.In contrast, lack of VAB-10B leads to increased epidermal thickness during embryonic morphogenesis when epidermal cells change shape.We suggest that this isoform protects cells against tension that builds up within the epidermis.

View Article: PubMed Central - PubMed

Affiliation: Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, BP10142, CU de Strasbourg, Illkirch Cedex F-67404, France.

ABSTRACT
Morphogenesis of the Caenorhabditis elegans embryo is driven by actin microfilaments in the epidermis and by sarcomeres in body wall muscles. Both tissues are mechanically coupled, most likely through specialized attachment structures called fibrous organelles (FOs) that connect muscles to the cuticle across the epidermis. Here, we report the identification of new mutations in a gene known as vab-10, which lead to severe morphogenesis defects, and show that vab-10 corresponds to the C. elegans spectraplakin locus. Our analysis of vab-10 reveals novel insights into the role of this plakin subfamily. vab-10 generates isoforms related either to plectin (termed VAB-10A) or to microtubule actin cross-linking factor plakins (termed VAB-10B). Using specific antibodies and mutations, we show that VAB-10A and VAB-10B have distinct distributions and functions in the epidermis. Loss of VAB-10A impairs the integrity of FOs, leading to epidermal detachment from the cuticle and muscles, hence demonstrating that FOs are functionally and molecularly related to hemidesmosomes. We suggest that this isoform protects against forces external to the epidermis. In contrast, lack of VAB-10B leads to increased epidermal thickness during embryonic morphogenesis when epidermal cells change shape. We suggest that this isoform protects cells against tension that builds up within the epidermis.

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Related in: MedlinePlus

VAB-10A and VAB-10B form alternating circumferential bands in the larval epidermis. (Top) Confocal projections (A, B, C, D, and E–E′′), and optical section through the apico–basal axis along the area marked with a double arrow (A′, B′, C′, and D′) after staining wild-type adult animals (A/A′, D/D′, and E′/E′′) or L1 larvae (B/B′ and C/C′); mAb staining is in red. (A/A′) VAB-10A pAbs and myosin heavy chain–specific mAb 5.6.1.1; note the respective orientations (A) and thickness (A′) of sarcomeres and FOs. (B/B′) VAB-10A pAbs (green) and mAb MH4 (IFs; red); both proteins colocalize. (C/C′) VAB-10A pAbs and mAb MH46 (myotactin); these proteins do not generally colocalize. (D/D′) mAb MH5 (VAB-10A; green) and VAB-10B K22 pAbs (violet); these proteins do not colocalize and VAB-10B extends slightly further than VAB-10A. (E–E′′) Differential interference contrast picture (E) of the animal immunostained with VAB-10B K22 pAbs (E′); the merged image (E′′) shows that VAB-10B is found at the furrows separating annuli (arrowheads; due to the permeabilization treatments, their morphology is rather poor). The K32 antiserum revealed the same pattern, but it was much fainter (not depicted). Bar, 10 μm (A and D), 2.5 μm (B and C), or 5 μm (E). (Bottom) Immunogold- labeled micrographs showing the positions of VAB-10A (F and G), and VAB-10B (H) obtained with 4F2 and K22 antibodies, respectively. In the epidermis, VAB-10A (but not VAB-10B) is enriched in FOs (arrowheads, gold particles; white arrows, dense bodies; F and G are from different sections). The specificity of staining is indicated by the signal-to-noise ratio measured by counting gold beads in sections stained with 4F2 or K22 primary antibodies, versus in control sections without primary antibody. 4F2: epidermis, 36.7 ± 4.6, and muscle, 0.9 ± 0.2; K22: epidermis, 4.3 ± 0.5, and muscle, 3.7 ± 0.6 (n = 2). Bars: 500 nm (F and H) and 100 nm (G).
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fig5: VAB-10A and VAB-10B form alternating circumferential bands in the larval epidermis. (Top) Confocal projections (A, B, C, D, and E–E′′), and optical section through the apico–basal axis along the area marked with a double arrow (A′, B′, C′, and D′) after staining wild-type adult animals (A/A′, D/D′, and E′/E′′) or L1 larvae (B/B′ and C/C′); mAb staining is in red. (A/A′) VAB-10A pAbs and myosin heavy chain–specific mAb 5.6.1.1; note the respective orientations (A) and thickness (A′) of sarcomeres and FOs. (B/B′) VAB-10A pAbs (green) and mAb MH4 (IFs; red); both proteins colocalize. (C/C′) VAB-10A pAbs and mAb MH46 (myotactin); these proteins do not generally colocalize. (D/D′) mAb MH5 (VAB-10A; green) and VAB-10B K22 pAbs (violet); these proteins do not colocalize and VAB-10B extends slightly further than VAB-10A. (E–E′′) Differential interference contrast picture (E) of the animal immunostained with VAB-10B K22 pAbs (E′); the merged image (E′′) shows that VAB-10B is found at the furrows separating annuli (arrowheads; due to the permeabilization treatments, their morphology is rather poor). The K32 antiserum revealed the same pattern, but it was much fainter (not depicted). Bar, 10 μm (A and D), 2.5 μm (B and C), or 5 μm (E). (Bottom) Immunogold- labeled micrographs showing the positions of VAB-10A (F and G), and VAB-10B (H) obtained with 4F2 and K22 antibodies, respectively. In the epidermis, VAB-10A (but not VAB-10B) is enriched in FOs (arrowheads, gold particles; white arrows, dense bodies; F and G are from different sections). The specificity of staining is indicated by the signal-to-noise ratio measured by counting gold beads in sections stained with 4F2 or K22 primary antibodies, versus in control sections without primary antibody. 4F2: epidermis, 36.7 ± 4.6, and muscle, 0.9 ± 0.2; K22: epidermis, 4.3 ± 0.5, and muscle, 3.7 ± 0.6 (n = 2). Bars: 500 nm (F and H) and 100 nm (G).

Mentions: In embryos, VAB-10A and VAB-10B antibodies first detected a signal at the basal and apical plasma membranes of dorsal and ventral epidermal cells, soon after the onset of differentiation (Fig. 4, A and F). As these cells became thinner during morphogenesis, staining appeared as four longitudinal rows (Fig. 4, B–E and G–L) and progressively evolved into circumferentially oriented bands located above muscle sarcomeres (Fig. 5 A). We also detected VAB-10A basally and apically in the pharynx and along mechanosensory axons as described previously (Francis and Waterston, 1991). VAB-10B was also present in the pharynx lumen, intestine lumen, nerve ring, and in body wall muscles and somatic gonad of larvae (Fig. 4, F–H, and unpublished data; a detailed account of VAB-10 distribution and role in nonepidermal tissues is hoped to be presented elsewhere). These patterns are specific because MH5 and polyclonal VAB-10A antibodies failed to detect a signal in vab-10(h1356) and vab-10A–deficient embryos (Fig. 4, D–E, and Fig. 6 H). Likewise, VAB-10B antibodies could only detect a weak signal in the intestine and pharynx of mid-staged vab-10B(mc44) and vab-10(h1356) embryos (Fig. 4, J and L). Although we cannot exclude that VAB-10B antisera recognize a cross-reacting protein, we believe that VAB-10B is expressed in the pharynx and the intestine because surviving vab-10B(mc44) larvae had an abnormal intestine (unpublished data).


The Caenorhabditis elegans vab-10 spectraplakin isoforms protect the epidermis against internal and external forces.

Bosher JM, Hahn BS, Legouis R, Sookhareea S, Weimer RM, Gansmuller A, Chisholm AD, Rose AM, Bessereau JL, Labouesse M - J. Cell Biol. (2003)

VAB-10A and VAB-10B form alternating circumferential bands in the larval epidermis. (Top) Confocal projections (A, B, C, D, and E–E′′), and optical section through the apico–basal axis along the area marked with a double arrow (A′, B′, C′, and D′) after staining wild-type adult animals (A/A′, D/D′, and E′/E′′) or L1 larvae (B/B′ and C/C′); mAb staining is in red. (A/A′) VAB-10A pAbs and myosin heavy chain–specific mAb 5.6.1.1; note the respective orientations (A) and thickness (A′) of sarcomeres and FOs. (B/B′) VAB-10A pAbs (green) and mAb MH4 (IFs; red); both proteins colocalize. (C/C′) VAB-10A pAbs and mAb MH46 (myotactin); these proteins do not generally colocalize. (D/D′) mAb MH5 (VAB-10A; green) and VAB-10B K22 pAbs (violet); these proteins do not colocalize and VAB-10B extends slightly further than VAB-10A. (E–E′′) Differential interference contrast picture (E) of the animal immunostained with VAB-10B K22 pAbs (E′); the merged image (E′′) shows that VAB-10B is found at the furrows separating annuli (arrowheads; due to the permeabilization treatments, their morphology is rather poor). The K32 antiserum revealed the same pattern, but it was much fainter (not depicted). Bar, 10 μm (A and D), 2.5 μm (B and C), or 5 μm (E). (Bottom) Immunogold- labeled micrographs showing the positions of VAB-10A (F and G), and VAB-10B (H) obtained with 4F2 and K22 antibodies, respectively. In the epidermis, VAB-10A (but not VAB-10B) is enriched in FOs (arrowheads, gold particles; white arrows, dense bodies; F and G are from different sections). The specificity of staining is indicated by the signal-to-noise ratio measured by counting gold beads in sections stained with 4F2 or K22 primary antibodies, versus in control sections without primary antibody. 4F2: epidermis, 36.7 ± 4.6, and muscle, 0.9 ± 0.2; K22: epidermis, 4.3 ± 0.5, and muscle, 3.7 ± 0.6 (n = 2). Bars: 500 nm (F and H) and 100 nm (G).
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fig5: VAB-10A and VAB-10B form alternating circumferential bands in the larval epidermis. (Top) Confocal projections (A, B, C, D, and E–E′′), and optical section through the apico–basal axis along the area marked with a double arrow (A′, B′, C′, and D′) after staining wild-type adult animals (A/A′, D/D′, and E′/E′′) or L1 larvae (B/B′ and C/C′); mAb staining is in red. (A/A′) VAB-10A pAbs and myosin heavy chain–specific mAb 5.6.1.1; note the respective orientations (A) and thickness (A′) of sarcomeres and FOs. (B/B′) VAB-10A pAbs (green) and mAb MH4 (IFs; red); both proteins colocalize. (C/C′) VAB-10A pAbs and mAb MH46 (myotactin); these proteins do not generally colocalize. (D/D′) mAb MH5 (VAB-10A; green) and VAB-10B K22 pAbs (violet); these proteins do not colocalize and VAB-10B extends slightly further than VAB-10A. (E–E′′) Differential interference contrast picture (E) of the animal immunostained with VAB-10B K22 pAbs (E′); the merged image (E′′) shows that VAB-10B is found at the furrows separating annuli (arrowheads; due to the permeabilization treatments, their morphology is rather poor). The K32 antiserum revealed the same pattern, but it was much fainter (not depicted). Bar, 10 μm (A and D), 2.5 μm (B and C), or 5 μm (E). (Bottom) Immunogold- labeled micrographs showing the positions of VAB-10A (F and G), and VAB-10B (H) obtained with 4F2 and K22 antibodies, respectively. In the epidermis, VAB-10A (but not VAB-10B) is enriched in FOs (arrowheads, gold particles; white arrows, dense bodies; F and G are from different sections). The specificity of staining is indicated by the signal-to-noise ratio measured by counting gold beads in sections stained with 4F2 or K22 primary antibodies, versus in control sections without primary antibody. 4F2: epidermis, 36.7 ± 4.6, and muscle, 0.9 ± 0.2; K22: epidermis, 4.3 ± 0.5, and muscle, 3.7 ± 0.6 (n = 2). Bars: 500 nm (F and H) and 100 nm (G).
Mentions: In embryos, VAB-10A and VAB-10B antibodies first detected a signal at the basal and apical plasma membranes of dorsal and ventral epidermal cells, soon after the onset of differentiation (Fig. 4, A and F). As these cells became thinner during morphogenesis, staining appeared as four longitudinal rows (Fig. 4, B–E and G–L) and progressively evolved into circumferentially oriented bands located above muscle sarcomeres (Fig. 5 A). We also detected VAB-10A basally and apically in the pharynx and along mechanosensory axons as described previously (Francis and Waterston, 1991). VAB-10B was also present in the pharynx lumen, intestine lumen, nerve ring, and in body wall muscles and somatic gonad of larvae (Fig. 4, F–H, and unpublished data; a detailed account of VAB-10 distribution and role in nonepidermal tissues is hoped to be presented elsewhere). These patterns are specific because MH5 and polyclonal VAB-10A antibodies failed to detect a signal in vab-10(h1356) and vab-10A–deficient embryos (Fig. 4, D–E, and Fig. 6 H). Likewise, VAB-10B antibodies could only detect a weak signal in the intestine and pharynx of mid-staged vab-10B(mc44) and vab-10(h1356) embryos (Fig. 4, J and L). Although we cannot exclude that VAB-10B antisera recognize a cross-reacting protein, we believe that VAB-10B is expressed in the pharynx and the intestine because surviving vab-10B(mc44) larvae had an abnormal intestine (unpublished data).

Bottom Line: We suggest that this isoform protects against forces external to the epidermis.In contrast, lack of VAB-10B leads to increased epidermal thickness during embryonic morphogenesis when epidermal cells change shape.We suggest that this isoform protects cells against tension that builds up within the epidermis.

View Article: PubMed Central - PubMed

Affiliation: Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, BP10142, CU de Strasbourg, Illkirch Cedex F-67404, France.

ABSTRACT
Morphogenesis of the Caenorhabditis elegans embryo is driven by actin microfilaments in the epidermis and by sarcomeres in body wall muscles. Both tissues are mechanically coupled, most likely through specialized attachment structures called fibrous organelles (FOs) that connect muscles to the cuticle across the epidermis. Here, we report the identification of new mutations in a gene known as vab-10, which lead to severe morphogenesis defects, and show that vab-10 corresponds to the C. elegans spectraplakin locus. Our analysis of vab-10 reveals novel insights into the role of this plakin subfamily. vab-10 generates isoforms related either to plectin (termed VAB-10A) or to microtubule actin cross-linking factor plakins (termed VAB-10B). Using specific antibodies and mutations, we show that VAB-10A and VAB-10B have distinct distributions and functions in the epidermis. Loss of VAB-10A impairs the integrity of FOs, leading to epidermal detachment from the cuticle and muscles, hence demonstrating that FOs are functionally and molecularly related to hemidesmosomes. We suggest that this isoform protects against forces external to the epidermis. In contrast, lack of VAB-10B leads to increased epidermal thickness during embryonic morphogenesis when epidermal cells change shape. We suggest that this isoform protects cells against tension that builds up within the epidermis.

Show MeSH
Related in: MedlinePlus