Limits...
LAD-1, the Caenorhabditis elegans L1CAM homologue, participates in embryonic and gonadal morphogenesis and is a substrate for fibroblast growth factor receptor pathway-dependent phosphotyrosine-based signaling.

Chen L, Ong B, Bennett V - J. Cell Biol. (2001)

Bottom Line: In addition, the ubiquitously expressed LAD-1, which binds to ankyrin-G, colocalizes with the C. elegans ankyrin, UNC-44, in multiple tissues at sites of cell-cell contact.Immunofluorescence studies revealed that FIGQY-tyrosine-phosphorylated LAD-1 does not colocalize with nonphosphorylated LAD-1 or UNC-44 ankyrin but instead is localized to sites that undergo mechanical stress in polarized epithelia and axon-body wall muscle junctions.These findings suggest a novel ankyrin-independent role for LAD-1 related to FGFR signaling.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Cell Biology, and Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA. l.chen@cellbio.duke.edu

ABSTRACT
This study shows that L1-like adhesion (LAD-1), the sole Caenorhabditis elegans homologue of the L1 family of neuronal adhesion molecules, is required for proper development of the germline and the early embryo and embryonic and gonadal morphogenesis. In addition, the ubiquitously expressed LAD-1, which binds to ankyrin-G, colocalizes with the C. elegans ankyrin, UNC-44, in multiple tissues at sites of cell-cell contact. Finally, we show that LAD-1 is phosphorylated in a fibroblast growth factor receptor (FGFR) pathway-dependent manner on a tyrosine residue in the highly conserved ankyrin-binding motif, FIGQY, which was shown previously to abolish the L1 family of cell adhesion molecule (L1CAM) binding to ankyrin in cultured cells. Immunofluorescence studies revealed that FIGQY-tyrosine-phosphorylated LAD-1 does not colocalize with nonphosphorylated LAD-1 or UNC-44 ankyrin but instead is localized to sites that undergo mechanical stress in polarized epithelia and axon-body wall muscle junctions. These findings suggest a novel ankyrin-independent role for LAD-1 related to FGFR signaling. Taken together, these results indicate that L1CAMs constitute a family of ubiquitous adhesion molecules, which participate in tissue morphogenesis and maintaining tissue integrity in metazoans.

Show MeSH

Related in: MedlinePlus

Phosphorylation of LAD-1 at the conserved tyrosine residue of the ankyrin-binding motif, FIGQY, occurs in vivo, and LAD-1P localizes to polarized sites in epithelial tissues. (A) Immunoblots of BSA-coupled LAD-1 peptides containing phosphorylated or nonphosphorylated FIGQY-tyrosine (+P and −P, respectively): i, LAD-1P antibody; ii, LAD-1NP antibody. (B) Immunoblots of total C. elegans lysates blotted with antibodies against the LAD-1 cytoplasmic tail (lane 2), LAD-1P (lane 4), LAD-1NP (lane 5), and 125I-labeled protein A (lane 3). Lane 5 shows that the 185 and 65 kD LAD-1 polypeptides contain the FIGQY epitope; only the 185-kD product is phosphorylated (lane 4, arrow). Lane 1 shows a Coomassie blue–stained C. elegans lysate and relative molecular weight markers. (C) Control nDf41 homozygous embryos, which contains the lad-1 gene, show LAD-1P (i) and LAD-1NP (ii) staining (green) unlike embryos homozygous for stDf7, which lacks the lad-1 gene; positive staining of JAM-1 is in red. (D) Preincubation of the LAD-1P antibody with the phosphorylated FIGQY peptide eliminated LAD-1P signal in stained animals; only JAM-1 immunostaining in red was detected (i). LAD-1P signal (green) is not displaced by preincubation of the LAD-1P antibody with the nonphosphorylated FIGQY peptide (ii). (E) Immunodetection of LAD-1P is localized in the pharynx and intestine (i, long and short arrow, respectively) and in the hypodermis (ii, arrow). LAD-1P (green) colocalized with JAM-1 (red) at the adherens junctions of hypodermal and intestinal cells. This colocalization is seen in the overlay panels as a yellow signal. Bar: (C and D) 10 μm; (E) 20 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196473&req=5

fig4: Phosphorylation of LAD-1 at the conserved tyrosine residue of the ankyrin-binding motif, FIGQY, occurs in vivo, and LAD-1P localizes to polarized sites in epithelial tissues. (A) Immunoblots of BSA-coupled LAD-1 peptides containing phosphorylated or nonphosphorylated FIGQY-tyrosine (+P and −P, respectively): i, LAD-1P antibody; ii, LAD-1NP antibody. (B) Immunoblots of total C. elegans lysates blotted with antibodies against the LAD-1 cytoplasmic tail (lane 2), LAD-1P (lane 4), LAD-1NP (lane 5), and 125I-labeled protein A (lane 3). Lane 5 shows that the 185 and 65 kD LAD-1 polypeptides contain the FIGQY epitope; only the 185-kD product is phosphorylated (lane 4, arrow). Lane 1 shows a Coomassie blue–stained C. elegans lysate and relative molecular weight markers. (C) Control nDf41 homozygous embryos, which contains the lad-1 gene, show LAD-1P (i) and LAD-1NP (ii) staining (green) unlike embryos homozygous for stDf7, which lacks the lad-1 gene; positive staining of JAM-1 is in red. (D) Preincubation of the LAD-1P antibody with the phosphorylated FIGQY peptide eliminated LAD-1P signal in stained animals; only JAM-1 immunostaining in red was detected (i). LAD-1P signal (green) is not displaced by preincubation of the LAD-1P antibody with the nonphosphorylated FIGQY peptide (ii). (E) Immunodetection of LAD-1P is localized in the pharynx and intestine (i, long and short arrow, respectively) and in the hypodermis (ii, arrow). LAD-1P (green) colocalized with JAM-1 (red) at the adherens junctions of hypodermal and intestinal cells. This colocalization is seen in the overlay panels as a yellow signal. Bar: (C and D) 10 μm; (E) 20 μm.

Mentions: Both antibodies were specific against LAD-1, since no LAD-1 immunofluorescence signal could be detected with either antibody in arrested stDf7 homozygous embryos, which lack the lad-1 gene (Fig. 4 C); only immunodetection of JAM-1 by the control antibody, MH27, was seen. On the other hand, embryos homozygous for the nDf41 deficiency, which does not remove the lad-1 gene, showed wild- type levels and localization of LAD-1. Moreover, the FIGQY sequence against which the antibodies were generated is not duplicated in the C. elegans genome. Taken together, these results indicated that these two peptide antibodies are specific for LAD-1. We next confirmed that the phosphorylated FIGQY-tyrosine LAD-1 antibody was indeed phosphotyrosine specific. Preincubation of the antibody with the tyrosine-phosphorylated FIGQY peptide before addition to methanol-fixed animals eliminated immunostaining but left the JAM-1 signal by the control antibody, MH27, intact (Fig. 4, D, i, red signal). On the contrary, preincubation of the antibody with the nonphosphorylated peptide did not eliminate the phosphorylated LAD-1 signal (Fig. 4, D, ii, green signal).


LAD-1, the Caenorhabditis elegans L1CAM homologue, participates in embryonic and gonadal morphogenesis and is a substrate for fibroblast growth factor receptor pathway-dependent phosphotyrosine-based signaling.

Chen L, Ong B, Bennett V - J. Cell Biol. (2001)

Phosphorylation of LAD-1 at the conserved tyrosine residue of the ankyrin-binding motif, FIGQY, occurs in vivo, and LAD-1P localizes to polarized sites in epithelial tissues. (A) Immunoblots of BSA-coupled LAD-1 peptides containing phosphorylated or nonphosphorylated FIGQY-tyrosine (+P and −P, respectively): i, LAD-1P antibody; ii, LAD-1NP antibody. (B) Immunoblots of total C. elegans lysates blotted with antibodies against the LAD-1 cytoplasmic tail (lane 2), LAD-1P (lane 4), LAD-1NP (lane 5), and 125I-labeled protein A (lane 3). Lane 5 shows that the 185 and 65 kD LAD-1 polypeptides contain the FIGQY epitope; only the 185-kD product is phosphorylated (lane 4, arrow). Lane 1 shows a Coomassie blue–stained C. elegans lysate and relative molecular weight markers. (C) Control nDf41 homozygous embryos, which contains the lad-1 gene, show LAD-1P (i) and LAD-1NP (ii) staining (green) unlike embryos homozygous for stDf7, which lacks the lad-1 gene; positive staining of JAM-1 is in red. (D) Preincubation of the LAD-1P antibody with the phosphorylated FIGQY peptide eliminated LAD-1P signal in stained animals; only JAM-1 immunostaining in red was detected (i). LAD-1P signal (green) is not displaced by preincubation of the LAD-1P antibody with the nonphosphorylated FIGQY peptide (ii). (E) Immunodetection of LAD-1P is localized in the pharynx and intestine (i, long and short arrow, respectively) and in the hypodermis (ii, arrow). LAD-1P (green) colocalized with JAM-1 (red) at the adherens junctions of hypodermal and intestinal cells. This colocalization is seen in the overlay panels as a yellow signal. Bar: (C and D) 10 μm; (E) 20 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196473&req=5

fig4: Phosphorylation of LAD-1 at the conserved tyrosine residue of the ankyrin-binding motif, FIGQY, occurs in vivo, and LAD-1P localizes to polarized sites in epithelial tissues. (A) Immunoblots of BSA-coupled LAD-1 peptides containing phosphorylated or nonphosphorylated FIGQY-tyrosine (+P and −P, respectively): i, LAD-1P antibody; ii, LAD-1NP antibody. (B) Immunoblots of total C. elegans lysates blotted with antibodies against the LAD-1 cytoplasmic tail (lane 2), LAD-1P (lane 4), LAD-1NP (lane 5), and 125I-labeled protein A (lane 3). Lane 5 shows that the 185 and 65 kD LAD-1 polypeptides contain the FIGQY epitope; only the 185-kD product is phosphorylated (lane 4, arrow). Lane 1 shows a Coomassie blue–stained C. elegans lysate and relative molecular weight markers. (C) Control nDf41 homozygous embryos, which contains the lad-1 gene, show LAD-1P (i) and LAD-1NP (ii) staining (green) unlike embryos homozygous for stDf7, which lacks the lad-1 gene; positive staining of JAM-1 is in red. (D) Preincubation of the LAD-1P antibody with the phosphorylated FIGQY peptide eliminated LAD-1P signal in stained animals; only JAM-1 immunostaining in red was detected (i). LAD-1P signal (green) is not displaced by preincubation of the LAD-1P antibody with the nonphosphorylated FIGQY peptide (ii). (E) Immunodetection of LAD-1P is localized in the pharynx and intestine (i, long and short arrow, respectively) and in the hypodermis (ii, arrow). LAD-1P (green) colocalized with JAM-1 (red) at the adherens junctions of hypodermal and intestinal cells. This colocalization is seen in the overlay panels as a yellow signal. Bar: (C and D) 10 μm; (E) 20 μm.
Mentions: Both antibodies were specific against LAD-1, since no LAD-1 immunofluorescence signal could be detected with either antibody in arrested stDf7 homozygous embryos, which lack the lad-1 gene (Fig. 4 C); only immunodetection of JAM-1 by the control antibody, MH27, was seen. On the other hand, embryos homozygous for the nDf41 deficiency, which does not remove the lad-1 gene, showed wild- type levels and localization of LAD-1. Moreover, the FIGQY sequence against which the antibodies were generated is not duplicated in the C. elegans genome. Taken together, these results indicated that these two peptide antibodies are specific for LAD-1. We next confirmed that the phosphorylated FIGQY-tyrosine LAD-1 antibody was indeed phosphotyrosine specific. Preincubation of the antibody with the tyrosine-phosphorylated FIGQY peptide before addition to methanol-fixed animals eliminated immunostaining but left the JAM-1 signal by the control antibody, MH27, intact (Fig. 4, D, i, red signal). On the contrary, preincubation of the antibody with the nonphosphorylated peptide did not eliminate the phosphorylated LAD-1 signal (Fig. 4, D, ii, green signal).

Bottom Line: In addition, the ubiquitously expressed LAD-1, which binds to ankyrin-G, colocalizes with the C. elegans ankyrin, UNC-44, in multiple tissues at sites of cell-cell contact.Immunofluorescence studies revealed that FIGQY-tyrosine-phosphorylated LAD-1 does not colocalize with nonphosphorylated LAD-1 or UNC-44 ankyrin but instead is localized to sites that undergo mechanical stress in polarized epithelia and axon-body wall muscle junctions.These findings suggest a novel ankyrin-independent role for LAD-1 related to FGFR signaling.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Cell Biology, and Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA. l.chen@cellbio.duke.edu

ABSTRACT
This study shows that L1-like adhesion (LAD-1), the sole Caenorhabditis elegans homologue of the L1 family of neuronal adhesion molecules, is required for proper development of the germline and the early embryo and embryonic and gonadal morphogenesis. In addition, the ubiquitously expressed LAD-1, which binds to ankyrin-G, colocalizes with the C. elegans ankyrin, UNC-44, in multiple tissues at sites of cell-cell contact. Finally, we show that LAD-1 is phosphorylated in a fibroblast growth factor receptor (FGFR) pathway-dependent manner on a tyrosine residue in the highly conserved ankyrin-binding motif, FIGQY, which was shown previously to abolish the L1 family of cell adhesion molecule (L1CAM) binding to ankyrin in cultured cells. Immunofluorescence studies revealed that FIGQY-tyrosine-phosphorylated LAD-1 does not colocalize with nonphosphorylated LAD-1 or UNC-44 ankyrin but instead is localized to sites that undergo mechanical stress in polarized epithelia and axon-body wall muscle junctions. These findings suggest a novel ankyrin-independent role for LAD-1 related to FGFR signaling. Taken together, these results indicate that L1CAMs constitute a family of ubiquitous adhesion molecules, which participate in tissue morphogenesis and maintaining tissue integrity in metazoans.

Show MeSH
Related in: MedlinePlus