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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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vab-10 encodes two distinct plakins. (A) The first two lines represent the chromosomal region where vab-10 maps and shows the approximate (dotted areas) endpoints of the deficiencies hDf17 and hDf16 used to refine vab-10 position. vab-10 spreads over two overlapping cosmids (F53B8 and ZK1151) and a yeast artificial chromosome (Y47H9B). The first 15 exons (dark gray boxes, corresponding to a predicted gene known as ZK1151.1) are common to vab-10A and vab-10B isoforms; exons 16–17 (medium gray boxes, corresponding to a predicted gene known as ZK1151.3) are unique to vab-10A isoforms, exons 18–32 (light gray boxes, corresponding to a predicted gene known as ZK1151.2) are unique to vab-10B isoforms. Numbered exons (arrowheads) can be alternatively spliced (see Fig. S1). Arrows mark the positions of vab-10 mutations as follows: h1356 is a G to A transition in the GT consensus donor splice site of intron 10, leading to a premature stop codon downstream; ju281 is a G to A transition (nucleotide 20242 of ZK1151) changing the Gly1560 of the longest VAB-10A isoform into a Glu; e698 is a G to A transition (nucleotide 19925 of ZK1151) changing the Pro1666 of the longest VAB-10A isoform into a Ser; mc44 is a 1033-nucleotide deletion (spanning nucleotides 2636–1605 of ZK1151) truncating VAB-10B isoforms after residue 3515. Shown below are the functional domains predicted by the SMART program (http://smart.embl-heidelberg.de) in VAB-10A, VAB-10B, and the two most closely related vertebrate plakins. ABD, actin-binding domain (exons encoding the ABD are surrounded by a dotted box); CH, calponin homology; SH3, Src-homology domain 3; SR, spectrin repeat; PR, plectin repeat; GAR, growth-arrest protein 2-related homology; EF-hand, calcium-binding motif. (B) Complementation tests among vab-10 alleles bearing on at least 100 individuals of each genotype. Only the most common phenotypes are mentioned (<C, 1.7F, 2F, 2.5F: arrest at the comma, 1.7-fold, twofold, and 2.5-fold stages, respectively; L1: L1 arrest; Vab, variably abnormal; WT, wild-type; ND, not determined).
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fig2: vab-10 encodes two distinct plakins. (A) The first two lines represent the chromosomal region where vab-10 maps and shows the approximate (dotted areas) endpoints of the deficiencies hDf17 and hDf16 used to refine vab-10 position. vab-10 spreads over two overlapping cosmids (F53B8 and ZK1151) and a yeast artificial chromosome (Y47H9B). The first 15 exons (dark gray boxes, corresponding to a predicted gene known as ZK1151.1) are common to vab-10A and vab-10B isoforms; exons 16–17 (medium gray boxes, corresponding to a predicted gene known as ZK1151.3) are unique to vab-10A isoforms, exons 18–32 (light gray boxes, corresponding to a predicted gene known as ZK1151.2) are unique to vab-10B isoforms. Numbered exons (arrowheads) can be alternatively spliced (see Fig. S1). Arrows mark the positions of vab-10 mutations as follows: h1356 is a G to A transition in the GT consensus donor splice site of intron 10, leading to a premature stop codon downstream; ju281 is a G to A transition (nucleotide 20242 of ZK1151) changing the Gly1560 of the longest VAB-10A isoform into a Glu; e698 is a G to A transition (nucleotide 19925 of ZK1151) changing the Pro1666 of the longest VAB-10A isoform into a Ser; mc44 is a 1033-nucleotide deletion (spanning nucleotides 2636–1605 of ZK1151) truncating VAB-10B isoforms after residue 3515. Shown below are the functional domains predicted by the SMART program (http://smart.embl-heidelberg.de) in VAB-10A, VAB-10B, and the two most closely related vertebrate plakins. ABD, actin-binding domain (exons encoding the ABD are surrounded by a dotted box); CH, calponin homology; SH3, Src-homology domain 3; SR, spectrin repeat; PR, plectin repeat; GAR, growth-arrest protein 2-related homology; EF-hand, calcium-binding motif. (B) Complementation tests among vab-10 alleles bearing on at least 100 individuals of each genotype. Only the most common phenotypes are mentioned (<C, 1.7F, 2F, 2.5F: arrest at the comma, 1.7-fold, twofold, and 2.5-fold stages, respectively; L1: L1 arrest; Vab, variably abnormal; WT, wild-type; ND, not determined).

Mentions: We molecularly identified vab-10 by a positional cloning strategy (see Materials and methods). vab-10 is a complex locus that generates two distinct sets of isoforms by alternative splicing of a common 5′ region to two distinct 3′ regions. The locus spans a region previously predicted to contain three genes (ZK1151.1, ZK1151.2, and ZK1151.3; Fig. 2 A). Using RT-PCR, we identified two distinct sets of isoforms that all contained ZK1151.1 exons (dark gray) spliced either to ZK1151.3 (medium gray) or to ZK1151.2 (light gray) exons (Fig. 2 A and Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200302151/DC1); these isoforms will subsequently be referred to as vab-10A and vab-10B, respectively. We identified additional potential splice variants affecting exons 5 and 9 (common region), exon 16 (vab-10A–specific exon), and exons 21–23 and 27 (vab-10B–specific exons), but could not identify isoforms containing both ZK1151.3 and ZK1151.2 exons (Fig. 2 A and Fig. S1). We did not try to determine if all potential combinations of splice variants exist in vivo.


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-10 encodes two distinct plakins. (A) The first two lines represent the chromosomal region where vab-10 maps and shows the approximate (dotted areas) endpoints of the deficiencies hDf17 and hDf16 used to refine vab-10 position. vab-10 spreads over two overlapping cosmids (F53B8 and ZK1151) and a yeast artificial chromosome (Y47H9B). The first 15 exons (dark gray boxes, corresponding to a predicted gene known as ZK1151.1) are common to vab-10A and vab-10B isoforms; exons 16–17 (medium gray boxes, corresponding to a predicted gene known as ZK1151.3) are unique to vab-10A isoforms, exons 18–32 (light gray boxes, corresponding to a predicted gene known as ZK1151.2) are unique to vab-10B isoforms. Numbered exons (arrowheads) can be alternatively spliced (see Fig. S1). Arrows mark the positions of vab-10 mutations as follows: h1356 is a G to A transition in the GT consensus donor splice site of intron 10, leading to a premature stop codon downstream; ju281 is a G to A transition (nucleotide 20242 of ZK1151) changing the Gly1560 of the longest VAB-10A isoform into a Glu; e698 is a G to A transition (nucleotide 19925 of ZK1151) changing the Pro1666 of the longest VAB-10A isoform into a Ser; mc44 is a 1033-nucleotide deletion (spanning nucleotides 2636–1605 of ZK1151) truncating VAB-10B isoforms after residue 3515. Shown below are the functional domains predicted by the SMART program (http://smart.embl-heidelberg.de) in VAB-10A, VAB-10B, and the two most closely related vertebrate plakins. ABD, actin-binding domain (exons encoding the ABD are surrounded by a dotted box); CH, calponin homology; SH3, Src-homology domain 3; SR, spectrin repeat; PR, plectin repeat; GAR, growth-arrest protein 2-related homology; EF-hand, calcium-binding motif. (B) Complementation tests among vab-10 alleles bearing on at least 100 individuals of each genotype. Only the most common phenotypes are mentioned (<C, 1.7F, 2F, 2.5F: arrest at the comma, 1.7-fold, twofold, and 2.5-fold stages, respectively; L1: L1 arrest; Vab, variably abnormal; WT, wild-type; ND, not determined).
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fig2: vab-10 encodes two distinct plakins. (A) The first two lines represent the chromosomal region where vab-10 maps and shows the approximate (dotted areas) endpoints of the deficiencies hDf17 and hDf16 used to refine vab-10 position. vab-10 spreads over two overlapping cosmids (F53B8 and ZK1151) and a yeast artificial chromosome (Y47H9B). The first 15 exons (dark gray boxes, corresponding to a predicted gene known as ZK1151.1) are common to vab-10A and vab-10B isoforms; exons 16–17 (medium gray boxes, corresponding to a predicted gene known as ZK1151.3) are unique to vab-10A isoforms, exons 18–32 (light gray boxes, corresponding to a predicted gene known as ZK1151.2) are unique to vab-10B isoforms. Numbered exons (arrowheads) can be alternatively spliced (see Fig. S1). Arrows mark the positions of vab-10 mutations as follows: h1356 is a G to A transition in the GT consensus donor splice site of intron 10, leading to a premature stop codon downstream; ju281 is a G to A transition (nucleotide 20242 of ZK1151) changing the Gly1560 of the longest VAB-10A isoform into a Glu; e698 is a G to A transition (nucleotide 19925 of ZK1151) changing the Pro1666 of the longest VAB-10A isoform into a Ser; mc44 is a 1033-nucleotide deletion (spanning nucleotides 2636–1605 of ZK1151) truncating VAB-10B isoforms after residue 3515. Shown below are the functional domains predicted by the SMART program (http://smart.embl-heidelberg.de) in VAB-10A, VAB-10B, and the two most closely related vertebrate plakins. ABD, actin-binding domain (exons encoding the ABD are surrounded by a dotted box); CH, calponin homology; SH3, Src-homology domain 3; SR, spectrin repeat; PR, plectin repeat; GAR, growth-arrest protein 2-related homology; EF-hand, calcium-binding motif. (B) Complementation tests among vab-10 alleles bearing on at least 100 individuals of each genotype. Only the most common phenotypes are mentioned (<C, 1.7F, 2F, 2.5F: arrest at the comma, 1.7-fold, twofold, and 2.5-fold stages, respectively; L1: L1 arrest; Vab, variably abnormal; WT, wild-type; ND, not determined).
Mentions: We molecularly identified vab-10 by a positional cloning strategy (see Materials and methods). vab-10 is a complex locus that generates two distinct sets of isoforms by alternative splicing of a common 5′ region to two distinct 3′ regions. The locus spans a region previously predicted to contain three genes (ZK1151.1, ZK1151.2, and ZK1151.3; Fig. 2 A). Using RT-PCR, we identified two distinct sets of isoforms that all contained ZK1151.1 exons (dark gray) spliced either to ZK1151.3 (medium gray) or to ZK1151.2 (light gray) exons (Fig. 2 A and Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200302151/DC1); these isoforms will subsequently be referred to as vab-10A and vab-10B, respectively. We identified additional potential splice variants affecting exons 5 and 9 (common region), exon 16 (vab-10A–specific exon), and exons 21–23 and 27 (vab-10B–specific exons), but could not identify isoforms containing both ZK1151.3 and ZK1151.2 exons (Fig. 2 A and Fig. S1). We did not try to determine if all potential combinations of splice variants exist in vivo.

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