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First published online September 30, 2004
doi: 10.1242/10.1242/dev.01372

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Drosophila contactin, a homolog of vertebrate contactin, is required for septate junction organization and paracellular barrier function

Catherine Faivre-Sarrailh^1,*,, Swati Banerjee^2,*, Jingjun Li², Michael Hortsch³, Monique Laval¹ and Manzoor A. Bhat^2,

¹ Neurobiologie des Interactions Cellulaires et Neurophysiopathologie, UMR 6184 CNRS, Institut Jean-Roche, Boulevard Pierre Dramard, 13916 Marseille Cedex 20, France
² Department of Cell and Molecular Physiology, Neuroscience Center and Curriculum in Neurobiology, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7545, USA
³ Department of Cell and Developmental Biology, University of Michigan, Medical School, Ann Arbor, MI 48109-0616, USA

View larger version (41K): [in a new window]   Fig. 1. CONT represents the single Drosophila homolog of the vertebrate contactin family. CONT is encoded by the transcriptional unit CG1084 (FlyBase), which was predicted from the Drosophila genome sequence. (A) The translation of cont cDNA reveals an ORF of 1390 amino acids. The signal peptide and the GPI anchor signal are indicated in bold. The C-lectin domain is underlined. The predicted N-glycosylation sites are marked in bold and are underlined. These sequence data are available from GenBank under Accession Number AY229991. (B) The modular protein domain structure for the CONT protein is shown in comparison with vertebrate contactin. Except for the N-terminal C-lectin domain in CONT, both proteins display a similar domain organization. (C) The phylogenetic tree obtained after aligning the protein sequences encoding rat contactin, TAG-1, NB-2, NB-3, BIG-1 and BIG-2 with the Drosophila CONT sequence. For this phylogenetic tree analysis, the CONT C-lectin domain was not considered. (D) Immunoblots of lipid rafts and microsomes that were prepared from 18-hour-old embryos. (Left panel) A single protein band with an apparent molecular weight of 180 kDa can be detected in both fractions when probed with the guinea-pig anti-CONT antiserum, which is directed against the N-terminal C-lectin domain. As a control, Fas1 is highly concentrated in the lipid raft fraction. (Middle panel) The microsomes were incubated with PI-PLC in the presence or absence of ZnCl2 followed by high-speed centrifugation. Solubilized pellet (5 µl) (P) and 25 µl of supernatant (S) were analyzed. As shown in the middle panel, a significant proportion of CONT was released in the supernatant after PI-PLC treatment. This release of CONT from the microsomal pellet was completely inhibited by the presence of ZnCl2. The asterisk shows a degradation band that resulted from incubation at 37°C. As a control, the major fraction of Fas1 was cleaved by PI-PLC and recovered in the supernatant, this release was also inhibited by the presence of ZnCl2. (Right panel) N-glycosidase-F treatment of the microsomal NP-40 extracts reduces the size of CONT to 155 kDa, close to the size predicted from the cDNA sequence (158 kDa).

View larger version (145K): [in a new window]   Fig. 2. CONT is expressed in ectodermal epithelia and glial cells of the PNS in Drosophila embryos. (A-D) A confocal section of a whole-mount stage 16 wild-type embryo triple stained with anti-CONT (A, blue), anti-NRX IV (B, red) and anti-NRG (C, green). CONT is expressed in all the ectodermally derived epithelial cells, including epidermis, salivary glands, trachea, hindgut and peripheral glial cells. The focus is on the midline of the CNS and the exiting axon tracts and parts of the epidermis. The merged image of the three panels is shown in D. Scale bar: 50 µm for A-D. (E-H) Part of the CNS midline and peripheral glial cells at a higher magnification shows that CONT (E, blue) and NRG (G, green) are not expressed in midline glial cells where NRX IV is strongly expressed (F, red, see arrow pointing to the midline glial cells). The roots of the glial cells, which ensheath axonal commissures in the CNS, express all three proteins (E-H, arrowheads). In the peripheral nerves, the expression pattern of all three proteins overlap (asterisks). (I-L) A region of the epidermal epithelial cells at a higher magnification demonstrates that CONT (I, blue), NRX IV (J, red) and NRG (green) are co-expressed at SJs (L, merged color white). NRG expression extends more basolaterally (arrow in K and L), whereas CONT and NRX IV are restricted to SJ domains. The arrowheads in I-L indicate overlapping expression along the peripheral nerve. (M-P) Part of the hindgut at a higher magnification also shows that CONT (M, blue), NRX IV (N, red) and NRG (O, green) are concentrated at SJs, but that NRG extends more basolaterally (arrowheads in O and P). Scale bar: 16 µm for E-P.

View larger version (11K): [in a new window]   Fig. 3. Genomic structure of the cont locus and generation of cont mutants. (A) Based on the FlyBase genomic sequence information, the cont gene spans 5.7 kb. Comparison of the genomic (FlyBase) and cDNA (Accession Number AY229991) sequences revealed that the cont locus is composed of eight exons (black bars) with intronic sequences ranging from 53 to a maximum of 350 nucleotides. (B) Genomic map of 17 kb region that includes CG11739, cont and tube. The P element (KG9756) is inserted in the first intron of CG11739. cont is within 100 bp downstream of CG11739, followed by tube within 300 bp. These loci are unusually close to each other. A chromosomal deficiency Df(3R)XM3 that uncovers this region deletes cont and the neighboring loci. One of the imprecise excisions from KG9756 mobilization removes 5 kb of the genomic region resulting in the loss of CG11739 and cont. A 7 kb SpeI (Sp) genomic construct rescues the lethality of contex956 to adulthood.

View larger version (102K): [in a new window]   Fig. 4. NRX IV, NRG and CONT are interdependent for their localization at the SJs. (A-D) This region of the wild-type epidermis at stage 16 shows the SJ localization of NRX IV (A, blue), CONT (B, red) and NRG (C, green), and the merged image (D). (E-H) Epidermis from a nrx IV4304/nrx IV4304 embryo at stage 16 with a loss of NRX IV protein (E, blue), a diffuse localization of CONT around the cell cortex (F, red) and a uniform basolateral localization of NRG (G, green). White arrowheads indicate the basolateral distribution of NRG (compare G with C). CONT is also seen in intracellular vesicles, as indicated by red arrowheads in F and H. (I-L) This part of the epidermis from a nrg1/Y embryo at stage 16 shows the loss of NRG staining (I, blue). White arrowheads indicate the basolateral distribution of both NRX IV (J, red) and CONT (K, green). Compare the merged image L with D. (M-P) Part of the epidermal epithelial layer from homozygous contex956 mutant embryo at stage 16, showing total loss of CONT (M, blue) and mislocalization of NRX IV (N, red) and NRG (O, green) along the basolateral area, as indicated with arrowheads (N-P). Merged image in P. (Q-V) Epidermis from hs-cont/hs-cont;contex956/contex956 homozygous embryos at stage 16 heat-shocked to induce the expression of CONT (Q and T, blue), show that NRX IV (R, green) or NRG (U, green) localization at SJs has been restored (compare arrowheads in R with N or in U with O). Merged images are shown in S and V. (W-Z) Epidermis from contex956/contex956 embryo rescued by SpeI genomic fragment at stage 16 shows normal expression and localization of CONT (W), and restoration of NRX IV (X) and NRG (Y) to SJs. A merged image of these proteins is shown in Z. Arrowheads indicate localization of these proteins at SJs. Scale bar: 16 µm.

View larger version (174K): [in a new window]   Fig. 5. Organization of SJs is altered in cont mutants. Ultrastructural analysis of the epidermal SJs in control heterozygous cont/GFP embryos (A,D), homozygous cont (B,E) and nrg1/Y mutant embryos (C) at stage 15. Adherens junctions at the apical side are indicated with asterisks. In control cont/GFP embryos, SJ strands (arrows) are observed at the level of a convoluted intercellular junction in the apicolateral region (A). Alignments of a small stretch of septa (arrows) are occasionally seen in cont (B) and nrg (C) mutants, and are often very basal to adherens junctions in these mutant embryos. High-magnification views of SJ strands in control (D) and cont mutant (E) embryos show that mutant SJ strands are narrower compared with control SJs. Intramembranous structures display a scalloped appearance in the heterozygous (arrowheads in D) but not in the cont mutant, whereas spacing between septa is normal in cont mutant. Scale bar: 200 nm for A-C; 100 nm for D,E.

View larger version (96K): [in a new window]   Fig. 6. The paracellular barrier is compromised in cont mutant embryos. (A) cont/GFP heterozygous late stage embryo injected with rhodamine-labeled dextran into the body cavity reveals an intact paracellular barrier as the dye fails to penetrate into the lumen of the salivary glands 20 minutes after injection (arrow). (B) cont mutant embryos injected with the dye into the body cavity reveals breakdown of the paracellular barrier as the dye penetrates into the lumen of the salivary glands within 18 minutes (arrow). (C) cont mutant embryos injected with the dye into the body cavity reveals breakdown of the paracellular barrier in the tracheal lumen (arrowhead). The tracheal tube is convoluted in the cont mutant embryo. The salivary gland filled with the dye is indicated by an asterisk. Scale bar: 20 µm for A,B; 60 µm for C.

View larger version (30K): [in a new window]   Fig. 7. CONT, NRX IV and NRG form a biochemical complex. Co-immunoprecipitation experiments reveal a tripartite complex between CONT, NRX IV and NRG. NP-40 embryonic lysates were immunoprecipitated with anti-NRX IV, anti-CONT, anti-NRG (1B7) antibodies. Controls were performed with anti-CONT pre-immune serum (PI). Immunoblots were probed with anti-NRX IV (A), rat anti-CONT (B) or anti-NRG 3C1 (C) antibodies. Molecular weight standards in kDa are indicated on the left. NRX IV is co-immunoprecipitated with CONT and reciprocally CONT is co-precipitated with anti-NRX IV antibodies. It was not possible to detect CONT in the anti-NRG immunoprecipitate. By contrast, NRG167 (asterisk) was detected in the immunoprecipitate with anti-CONT antibodies. A doublet of both 167 and 180 kDa NRG isoforms is detected in the NP-40 lysate (arrowheads). (D) COS cells were co-transfected with NRX IV and CONT. NP-40 lysate was immunoprecipitated with anti-NRX IV antibody. CONT is detected in the anti-NRX IV immunoprecipitate.

View larger version (52K): [in a new window]   Fig. 8. Cell-surface expression of CONT depends on NRX IV. Neuroblastoma N2a cells were transfected with NRX IV or CONT, or co-transfected with both constructs. (A) NRX IV is efficiently transported to the cell surface in the absence of CONT. (B-D) When transfected alone, CONT is not expressed at the plasma membrane. Double-immunostaining of CONT (B, red) and the ER marker BiP (C, green). The merged image (D) shows that CONT is retained in the ER (arrowheads). (E-H) Co-transfection of CONT (E) and NRX IV (F) in N2a cells results in expression of CONT protein at the plasma membrane. (G,H) Cell-surface expression of CONT in N2a cells co-transfected with CONT and NRX IV. Cells were fixed, permeabilized with Triton X-100 and processed for immunofluorescence staining in A-F. Immunostaining for CONT was carried out on living cells in G and then cells were fixed, permeabilized and processed for NRX IV immunostaining (H). Scale bar: 10 µm.