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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

Characterization of four VAB-10–specific antibodies. (Top) Positions and names (Prot) of the peptides (horizontal double arrows; domain symbols are as in Fig. 2 A) used to raise antibodies (Ab), and of the epitope recognized by the mAb MH5 (vertical arrow). (Bottom) Western blots of total worm extracts probed with immunopurified antibodies (Ab) in the absence (−) or in the presence of a competing protein (C.P.). Competition with 4fr, kh2, and kh3 recombinant peptides were effective only with the cognate antibodies (e.g., 4fr did not compete interaction with mAb MH5).
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fig3: Characterization of four VAB-10–specific antibodies. (Top) Positions and names (Prot) of the peptides (horizontal double arrows; domain symbols are as in Fig. 2 A) used to raise antibodies (Ab), and of the epitope recognized by the mAb MH5 (vertical arrow). (Bottom) Western blots of total worm extracts probed with immunopurified antibodies (Ab) in the absence (−) or in the presence of a competing protein (C.P.). Competition with 4fr, kh2, and kh3 recombinant peptides were effective only with the cognate antibodies (e.g., 4fr did not compete interaction with mAb MH5).

Mentions: The preceding molecular and genetic data predict that VAB-10A and VAB-10B isoforms should fulfil distinct functions. To address this hypothesis, we set out to examine whether their cellular and subcellular distributions are similar or different. We raised pAbs against one VAB-10A–specific and two VAB-10B–specific domains (Fig. 3). In addition, we used the mAb MH5, known to recognize an FO component (Francis and Waterston, 1991). While characterizing vab-10, we noticed that MH5 failed to stain vab-10(h1356) embryos (Fig. 4 D), suggesting that it might recognize a VAB-10 isoform. Several lines of evidence establish the specificity of these antibodies and demonstrate that MH5 specifically recognizes VAB-10A. First, rabbit polyclonal VAB-10A antibodies detected a band on Western blots that co-migrated with the band detected by MH5 in the 300–400 kD range, in agreement with previous estimates (Francis and Waterston, 1991). Second, VAB-10B antibodies detected a band that migrated above VAB-10A (Fig. 3), consistent with the predicted sizes of both plakins. Third, addition of the polypeptide used to raise the antibodies eliminated the signal recognized by VAB-10A and VAB-10B pAbs (Fig. 3), attesting to their specificity. Last, we could further map the epitope recognized by MH5 within the VAB-10A–specific alternative exon 16 (Fig. S3).


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)

Characterization of four VAB-10–specific antibodies. (Top) Positions and names (Prot) of the peptides (horizontal double arrows; domain symbols are as in Fig. 2 A) used to raise antibodies (Ab), and of the epitope recognized by the mAb MH5 (vertical arrow). (Bottom) Western blots of total worm extracts probed with immunopurified antibodies (Ab) in the absence (−) or in the presence of a competing protein (C.P.). Competition with 4fr, kh2, and kh3 recombinant peptides were effective only with the cognate antibodies (e.g., 4fr did not compete interaction with mAb MH5).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199363&req=5

fig3: Characterization of four VAB-10–specific antibodies. (Top) Positions and names (Prot) of the peptides (horizontal double arrows; domain symbols are as in Fig. 2 A) used to raise antibodies (Ab), and of the epitope recognized by the mAb MH5 (vertical arrow). (Bottom) Western blots of total worm extracts probed with immunopurified antibodies (Ab) in the absence (−) or in the presence of a competing protein (C.P.). Competition with 4fr, kh2, and kh3 recombinant peptides were effective only with the cognate antibodies (e.g., 4fr did not compete interaction with mAb MH5).
Mentions: The preceding molecular and genetic data predict that VAB-10A and VAB-10B isoforms should fulfil distinct functions. To address this hypothesis, we set out to examine whether their cellular and subcellular distributions are similar or different. We raised pAbs against one VAB-10A–specific and two VAB-10B–specific domains (Fig. 3). In addition, we used the mAb MH5, known to recognize an FO component (Francis and Waterston, 1991). While characterizing vab-10, we noticed that MH5 failed to stain vab-10(h1356) embryos (Fig. 4 D), suggesting that it might recognize a VAB-10 isoform. Several lines of evidence establish the specificity of these antibodies and demonstrate that MH5 specifically recognizes VAB-10A. First, rabbit polyclonal VAB-10A antibodies detected a band on Western blots that co-migrated with the band detected by MH5 in the 300–400 kD range, in agreement with previous estimates (Francis and Waterston, 1991). Second, VAB-10B antibodies detected a band that migrated above VAB-10A (Fig. 3), consistent with the predicted sizes of both plakins. Third, addition of the polypeptide used to raise the antibodies eliminated the signal recognized by VAB-10A and VAB-10B pAbs (Fig. 3), attesting to their specificity. Last, we could further map the epitope recognized by MH5 within the VAB-10A–specific alternative exon 16 (Fig. S3).

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