QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online September 29, 2004
doi: 10.1242/10.1242/jcs.01394

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Minniti, A. N. Articles by Brandan, E. Search for Related Content PubMed PubMed Citation Articles by Minniti, A. N. Articles by Brandan, E.

Caenorhabditis elegans syndecan (SDN-1) is required for normal egg laying and associates with the nervous system and the vulva

Alicia N. Minniti^*, Mariana Labarca^*, Claudia Hurtado and Enrique Brandan

Centro de Regulación Celular y Patología, Departamento de Biología Celular y Molecular, Facultad de Ciencias Biológicas, MIFAB, P. Universidad Católica de Chile, Casilla 114-D, Santiago, Chile

View larger version (21K): [in a new window]   Fig. 1. Caenorhabditis elegans synthesize heparan sulfate proteoglycans. (A) Wild-type worms were incubated with radioactive sulfate and after solubilization in 4 M guanidine-HCl followed by dialysis against 8 M urea, were loaded onto a DEAE-Sephacel column of 1.0 ml bed volume, pre-equilibrated with the urea buffer. The column was washed and eluted using a linear gradient from 0.2 to 1.0 M NaCl. Aliquots of each fraction were counted (closed circles) and conductivity was determined (open circles). (B) Proteoglycans eluted from the DEAE indicated by the bar, were pooled, concentrated on a small DEAE column and fractionated on a Sepharose CL-6B column. The chromatographic profiles of control samples (closed circles), samples pre-incubated with proteinase K (open circles) and samples treated with alkaline borohydride to release the GAG chains (open triangles) are shown. (C) Proteoglycans eluted from the DEAE and concentrated as in B, were fractionated on a Sephadex G-50 column. The profiles shown correspond to untreated samples (closed diamonds) and samples previously incubated with either chondroitinase ABC (open triangles) or nitrous acid (open diamonds).

View larger version (36K): [in a new window]   Fig. 2. C. elegans synthesize at least three species of heparan sulfate proteoglycans. (A) Western blot analysis was carried out using post-DEAE samples obtained from wild-type worms, dialyzed against heparitinase buffer and incubated with heparitinase. Samples were separated by SDS-PAGE on a 4-15% gradient gel, transferred onto nitrocellulose, incubated with anti--heparan sulfate antibody (3G10 epitope) and subjected to chemiluminescence detection. Three different core proteins migrating with apparent molecular masses of 80, 50 and 30 kDa were detected. (B) The electrophoretic migration pattern of the core proteins of heparan sulfate proteoglycans, obtained from C2C12 myoblast extracts incubated with the same anti--heparan sulfate antibody, is shown as a comparison. The pattern shows the following previously characterized murine heparan sulfate core proteins and their corresponding molecular weights: syn-3, syndecan-3; syn-1, syndecan-1; gly, glypican; syn-2, syndecan-2 and syn-4, syndecan-4.

View larger version (72K): [in a new window]   Fig. 3. Heparan sulfate proteoglycans in C. elegans localize to the nerve ring, nerve cords and vulva. Whole worms were permeabilized, incubated with heparitinase and stained with anti--heparan sulfate antibody. (a) Staining shows that heparan sulfate proteoglycans are located at the nerve ring (arrowheads), ventral nerve cord (white arrows) and dorsal nerve cord (gray arrows). (b) Heparan sulfate proteoglycans are also located in the vulva as seen from a ventral view (gray arrowheads). The white arrows indicate the ventral nerve cord and the gray arrows the dorsal nerve cord. (c) The staining of L2-L3 larva showed the location of heparan sulfate proteoglycans at the nerve ring (white arrowhead) and at the ventral and dorsal nerve cords (white and gray arrows, respectively). (d) The nervous system of a transgenic strain expressing the pan-neural GFP was visualized. (e) Heparan sulfate proteoglycans as seen in panel a were also immunodetected in this reporter strain. The white arrows indicate ventral nerve cord. (f) Merged images of panels d and e. The insert shows an enlarged area, with details of the neural bodies (green) and their close localization to the heparan sulfate proteoglycans (red). Controls without heparitinase treatment and without primary antibody are shown in panels g and h respectively. All images are 3D reconstructions of image stacks, except for d, e and f, which correspond to a single optic slice. Bar, 20 µm.

View larger version (45K): [in a new window]   Fig. 4. The 50 kDa core protein bearing heparan sulfate GAGs corresponds to the single C. elegans orthologue of vertebrate syndecans. (A) mRNA was isolated from wild-type and sdn-1 (ok449) mutant strains and RT-PCR was performed using primers in the positions indicated in Fig. 5A. The migration of the RTPCR products, by agarose gel electrophoresis, is shown. wt, wild-type worms; sdn-1, sdn-1 (ok449) mutant worms. (B) Northern blot analysis carried out using mRNA (30 µg samples) obtained as described from wild-type and sdn-1 (ok449) mutant worms. Blots were probed with specific 32P-labeled 632 bp hybridization probes. The migration of 18S and 28S ribosomal sub-units is shown as a standard. (C) Western blot analysis of wild-type and sdn-1 (ok449) mutant worms. Samples were obtained by sonication in PBS and protease inhibitors, treated directly with heparitinase, separated on a 10% polyacrylamide slab gel and visualized with anti--heparan sulfate antibody. Total absence of the 50 kDa core protein was observed in the sdn-1 mutants (low and high exposure), in contrast to its prominence in wild-type samples, whereas the other two core proteins (80 and 30 kDa) remained almost unaffected (high exposure).

View larger version (54K): [in a new window]   Fig. 5. Schematic representation of C. elegans SDN-1 and sequence comparison of its transmembrane and cytoplasmic domains with those of other species. (A) Model of SDN-1 showing its type I transmembrane protein nature. The locations of the GAG attachment sites in the ectodomain (white arrowheads), including a putative site closer to the transmembrane region, are shown. The postulated phosphorylated serine residue of the cytoplasmic domain (SerP) and the location of primer used to analyze wild-type and sdn-1 (ok449) worm strains (black arrows). The lower model shows the most likely form of the deleted SDN-1 protein, as deduced from sdn-1 (ok449) cDNA sequencing. (B) SDN-1 sequence for the conserved transmembrane and cytoplasmic domains is shown aligned to other syndecan sequences. A consensus sequence arose when the SDN-1 sequence was compared with syndecan-1, -2, -3 and -4 from different species.

View larger version (104K): [in a new window]   Fig. 6. C. elegans sdn-1 (ok449) mutants show loss of heparan sulfate proteoglycan immunoreactivity mainly in the nerve cords and vulva. Immunolocalization of heparan sulfate proteoglycans was carried out in whole worms as described for Fig. 3, for both wild-type (panels a and b) and sdn-1 (ok449) mutants (panels c and d). In sdn-1 (ok449) mutants, some immunoreactivity remained in the nerve ring shown in c (white arrowheads) but no staining was observed along the neural cords and the vulva (d). White arrows indicate the ventral nerve cord, gray arrows the dorsal nerve cord. All images are 3D reconstructions of image stacks. Bar, 20 µm.

View larger version (65K): [in a new window]   Fig. 7. SDN-1 located at the C. elegans nerve ring is specifically phosphorylated at its cytoplasmic domain. (A) Amino acid sequences of the cytoplasmic domain of SDN-1 from C. elegans (C.e) and syndecan-4 from M. musculus (M.m). The synthetic peptide used to generate the specific anti-phosphoserine syndecan-4 antibody (anti-P-syndecan-4) is boxed and the phosphorylated serine indicated. (B) (a) Anti--heparan sulfate immunoreactivity as shown in Fig. 3a. (b) Anti-P-syndecan 4 immunoreactivity in wild-type worms. The inserts show immunostaining with preincubated antibody with phosphorylated (upper insert) and non-phosphorylated peptide (lower insert). (c) Merged image of a and b. The insert shows anti-P-syndecan-4 immunoreactivity in the pan-neural GFP transgenic strain. The anti-P-syndecan-4 antibody stained specific neurons at the nerve ring and some anterior structures, but no immunoreactivity was detected in either the nerve cords or the vulva. (d) An enlarged area of the neurons stained in the nerve ring. In all images, white arrowheads indicate the nerve ring. All images are 3D reconstructions of image stacks. Bar, 20 µm.

View larger version (82K): [in a new window]   Fig. 8. C. elegans sdn-1 (ok449) mutants express a protein recognized by antibodies against the phosphorylated syndecan-4 epitope, with immunoreactivity localizing to the nerve ring. (a) Anti-P-syndecan-4 immunoreactivity in sdn-1 (ok449) mutants. (b,c) Double immunostaining of anti--heparan sulfate and anti-P-syndecan-4 immunoreactivity in sdn-1 (ok449) mutants respectively. (d) Merged images of b and c. The insert shows an enlarged area, with detail of the heparan sulfate proteoglycans (green) and their close localization to the phosphorylated SDN-1 (red). White arrowheads indicate the immunoreaction at the nerve ring. The image in panel a corresponds to a 3D reconstruction of an image stack, whereas the rest represent two optic slices. Bar, 20 µm.

View larger version (104K): [in a new window]   Fig. 9. C. elegans sdn-1 mutants show accumulation of embryos in the uterus and weak morphological defects of the invaginating vulva. (A,B) DIC images of in uterus embryos in wild-type worms and sdn-1 (ok449) mutants respectively. (C,D) DIC micrographs of the vulvae of mid-L4 larvae from both strains. In sdn-1 (ok449), the space between the vulva cells and the cuticle is slightly smaller than in wild-type samples. Bar, 10 µm.