Neuroglian, Gliotactin, and the Na+/K+ ATPase are essential for septate junction function in Drosophila

Abstract

One essential function of epithelia is to form a barrier between the apical and basolateral surfaces of the epithelium. In vertebrate epithelia, the tight junction is the primary barrier to paracellular flow across epithelia, whereas in invertebrate epithelia, the septate junction (SJ) provides this function. In this study, we identify new proteins that are required for a functional paracellular barrier in Drosophila. In addition to the previously known components Coracle (COR) and Neurexin (NRX), we show that four other proteins, Gliotactin, Neuroglian (NRG), and both the alpha and beta subunits of the Na+/K+ ATPase, are required for formation of the paracellular barrier. In contrast to previous reports, we demonstrate that the Na pump is not localized basolaterally in epithelial cells, but instead is concentrated at the SJ. Data from immunoprecipitation and somatic mosaic studies suggest that COR, NRX, NRG, and the Na+/K+ ATPase form an interdependent complex. Furthermore, the observation that NRG, a Drosophila homologue of vertebrate neurofascin, is an SJ component is consistent with the notion that the invertebrate SJ is homologous to the vertebrate paranodal SJ. These findings have implications not only for invertebrate epithelia and barrier functions, but also for understanding of neuron-glial interactions in the mammalian nervous system.

Figure 1.

Identification of genes essential for formation of the paracellular barrier. Confocal sections of the salivary glands of live, late stage, embryos. Arrows indicate the lumen of the salivary glands in all panels. Embryos were injected posteriorly with a fluo-rescently labeled dextran that is restricted from entering the lumen of the salivary gland by the paracellular diffusion barrier in wild-type embryos (A). However, dye enters into the lumen of the salivary gland, indicating that this barrier is lost in embryos homozygous mutant for Nrg ¹⁴ (B), Gli ¹ (C), Atpα^01453a (D), and l(2)k13315 (E). The dye diffusion phenotype of P element mutation l(2)k13315 is rescued when UAS–Flag Nrv2.2 is expressed under the e22C-GAL4 driver in embryos (F). cor¹/cor² mutant embryos have an effective diffusion barrier (G) that is lost when one copy of Nrx is removed (H). Bar, 25 μm.

Figure 2.

The Nrv2 locus encodes two proteins that localize to the SJ and are disrupted in the l(2)k13315 mutant. (A) The Nrv2 locus encodes two transcripts, Nrv2.1 and Nrv2.2. The proteins encoded by these transcripts are type 2 transmembrane proteins, which differ only in their cytoplasmic domains. Also indicated is Nrv1, a gene located ∼3 kb downstream that also encodes an Na⁺/K⁺ ATPase β subunit. The schematic of the Nrv2 locus includes the P element insertion l(2)k13315 as well as two deletions created by imprecise excision of the P element. Nrv2 ^11A is a deletion of 1,589 bp, which removes the Nrv2.1 start site, and Nrv2 ^23B is a deletion of 2,176 bp, which removes all of the Nrv2 common exons. (B) The proteins encoded by the Nrv2 and Nrv1 loci were 5′ tagged with Flag and then expressed in the wing imaginal disc using the Apterous-GAL4 driver and UAS promoter. F-NRV2.2 (a, red in c), F-NRV2.1 (d, red in f), and F-NRV1 (g, red in i) all localize to the SJ where they colocalize with COR (b, e, and h; green in c, f, and i). Bar, 10 μm. (C) The anti-NRV antibody, mAb 5F7, which recognizes NRV2.1, NRV2.2, and NRV1, stains two protein bands from untransfected S2 cells (lane 4). The Flag-tagged Nrv transgenes were expressed in S2 cells using the Ubiquitin-GAL4 driver, and an immunoblot of these cells was probed using anti-Flag (top panel, red in bottom panel) and mAb 5F7 (middle panel, green in bottom panel). The banding pattern observed using the Flag-tagged NRV proteins indicates that NRV2.1 and NRV2.2 (lanes 3 and 4) run slower than NRV1 (lane 1). Overlaying these two antibodies shows which NRV protein corresponds to each band (bottom). (D) Protein from late stage embryos was examined for NRV protein by probing with mAb 5F7. Wild-type embryos have both NRV2 and NRV1 proteins; however the NRV2 protein band is completely absent in Nrv2 ^k13315 and Nrv2 ^23B homozygous embryos. The NRV2 band is still present, although greatly reduced, in embryos homozygous for Nrv2 ^11A.

Figure 3.

The transverse septae are reduced or absent from the epidermal SJ of co r, Nrv2, Atpα, Nrg, and Gli mutant embryos. Wild-type embryos have distinct junctional structures in the apical-lateral domain of polarized epithelial cells, including the adherens junction (A, arrowhead) and the SJ (A, between arrows). The inset in A demonstrates the alignment of septae between uniformly spaced membranes of adjacent cells (inset corresponds to region basal to the upper arrow). In cor ⁵, Nrv2 ^k13315, Atpα^01453a, Nrg ¹⁴, and Gli ¹ embryos, the adherens junction remains intact (B–F, arrowheads); however, the septae are disrupted.

Figure 4.

NRV, ATPα, and NRG colocalize with COR at the SJ. Anti-COR staining indicates the region of the SJ (A, E, and I). Antibodies recognizing NRV (B), ATPα (F), and NRG (J) localize to the region of the SJ and colocalize with COR (yellow indicates colocalization in C, G, and K). Gene trap lines (Morin et al., 2001) express GFP fused in frame with NRV2 (line G74) (D), ATPα (line G109) (H, M, and N), and NRG (line G305) (L). The GFP gene traps demonstrate SJ localization of these proteins in living wing imaginal disc (D,H, and L), embryonic salivary gland (M), and embryonic epidermal (N) cells. The apical (a) and basal (b) surfaces of the embryonic epithelia are indicated. Bars, 10 μm.

Figure 5.

Somatic mosaic analysis reveals interdependent relationships between some, but not all, SJ components. (A–X) Cross sections of somatic mosaic clones in wing imaginal epithelia. Arrows indicate the clone boundary, with homozygous mutant cells found between the arrows. (A–D) Nrv2 ^k11315 mutant cells were marked by an absence of GFP (A, green in D). Nrv mutant cells stained for COR (B, red in D) and NRX (C, blue in D) show these proteins reduced or absent. (E–P) cor ⁵ mutant cells are identified by lack of staining for COR between the arrows (E, I, and M; green in H, L, and P). In cor ⁻ cells, ATPα (F, red in H), NRG (J, red in L), and NRV (N, red in P) protein levels are reduced or absent compared with expression in the surrounding wild-type cells. However, the apical marker DLT is unaffected (G, K, and O; blue in H, L, and P). Additional SJ markers, DLG (R, red in T) and FASIII (V, red in X), are not disrupted in cor ⁵ or Nrg ¹⁴ mutant cells, respectively, nor is the adherens junction marker DCAD2 (S and W; blue in T and X). (Y and Z) Tangential section of the apical region of the imaginal epithelium containing a cor ⁵ clone. Both COR (Y) and NRX (Z) are reduced in wild-type cells where they contact mutant cells (indicated by arrows). Bars: (A–X) 20 μm; (Y and Z) 5 μm.

Figure 6.

COR, NRX, NRV, ATPα, and NRG interact in vivo to form a protein complex. (A) NRX and NRG coimmunoprecipitate with COR in lysates from wild-type embryos, suggesting that these proteins are present in a complex. (B) COR and NRX precipitate with NRG, confirming this result. (C) NRV is a part of this complex because it precipitates both COR and NRX. (D) Embryo lysates from cor ⁵, Nrx ¹⁴, Nrv2 ^k13315, Atpα^01453a, Nrg ¹⁴, and Gli ¹ mutant embryos were probed with antibodies that detect COR, NRX, NRV, ATPα, and NRG. In most cases, depletion of one component of the complex did not affect stability of other components. Anti-Moesin was used as a loading control.