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First published online December 12, 2006
doi: 10.1242/10.1242/dev.02710

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A pump-independent function of the Na,K-ATPase is required for epithelial junction function and tracheal tube-size control

¹ Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208, USA.
² Laboratory of Genetics, University of Wisconsin, Madison, WI 53706, USA.
³ Department of Pharmacology and Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260, USA.

View larger version (73K): [in this window] [in a new window]   Fig. 1. Drosophila Na,K-ATPase ß-subunits have distinct subcellular localizations. (A-C) Nrv1 (red) is expressed in many epithelial tissues, including the epidermis (A1,2), salivary gland (at low levels; B1,2), and trachea (C1,2). Nrv1 localizes to regions basal to and distinct from the SJ as marked by Nrv2 (green; A1,B1,C1). No Nrv1 protein is detectable in the trans-heterozygote of two small deficiencies [Df(ED)7007 and DF(ED)6569] that delete both nrv1 and nrv2 (A3,B3,C3). Levels of Nrv1 staining are unaffected by a nrv2-null mutation but the domain of Nrv1 localization is extended apically into the region normally occupied by SJs (A4,B4,C4). (D) Nrv3 is expressed in the chordotonal organ (D1) and the central nervous system (D2). In A1,B1,C1, the apical surface is denoted by dashed blue lines and the basal surface by dashed white lines. Scale bars: in A1, 5 µm for A1-B4,D1; in C1, 5 µm for C1-C4; in D2, 5 µm.

View larger version (103K): [in this window] [in a new window]   Fig. 2. The Nrv2 extracellular domain mediates SJ organization and tracheal tube morphogenesis. (A,B) When compared with wild-type (WT) animals (A1-5), nrv2[23B]-null mutants have tracheal defects including dorsal trunks (DTs) of increased length and irregular diameter (B1 versus A1) and lumenal staining gaps in ganglionic branches (GBs) (asterisks in B2 versus brackets in A2). nrv2 mutants have also lost the paracellular barrier function of the SJs which allows a dye to penetrate the epithelium and accumulate in the lumen (red tracheal tube in B3 versus A3 where the trachea exclude the dye). Dashed lines delineate tracheal lumen. nrv2 mutants also have disorganized SJs at the cellular level with reduced and partially mislocalized Coracle (B4 versus A4,A5). Lateral mislocalization of Coracle in the nrv2 mutant (B6, arrowhead) is apparent in overexposed images that saturate signal levels for Coracle in WT animals (B5); B5 and B6 are higher gain images of A5 and B4, respectively. (C-E) The mutant phenotypes are rescued by expression of Nrv2 (C1-4) but not by expression of Nrv1 (D1-4) or Nrv3 (E1-4) using a da-Gal4 driver. Neither Nrv1 nor Nrv3 are incorporated into an established SJ in nrv2 heterozygotes (D5,E5), whereas Nrv2 is incorporated (C5). (F,G) A chimera with the intracellular (I) and transmembrane (T) domains of Nrv3 and the extracellular (E) domain of Nrv2 rescued the nrv2 defects (F1-4), whereas a chimera with the Nrv2 IT and Nrv3 E domains did not (G1-4). Both chimeras could be incorporated into established SJs (F5,G5). Coracle is green and Nrv protein is red (anti-Nrv2.1 in A4-C5 and G4,5; anti-Nrv1 in D4,5; anti-Nrv3 in E4-F5). All animals are stage 16. Scale bars: in G2, 10 µm for A-G images 1,2; in G3, 10 µm for A-G image 3; in A5, 5 µm for A-G images 4,5 and B6.

View larger version (112K): [in this window] [in a new window]   Fig. 3. Multiple regions of the Nrv2 extracellular domain are required for function. (A) A chimera containing the entire Nrv2 extracellular domain and the Nrv3IT domains (Nrv2ABCD) rescues tracheal tube size and paracellular barrier function (A1 and see Fig. 2F1), is targeted to the lateral membrane and SJ (A2), and restores proper Coracle localization (green) and levels (A3,4). (B-G) By contrast, chimeras missing any region fail to restore tracheal tube size (data not shown) and barrier function (B-G, image 1). All chimeras were expressed (B2' and C-G image 2), with Nrv2A (C2), Nrv2CD (D2) and Nrv2BCD (E2) being targeted to the SJ and supporting SJ localization of Coracle at WT or near-WT levels (C3,D3,E3 and C4,D4,E4) indicating these chimeras nonetheless have some SJ-organizing activity. An anti-Nrv3 antibody recognizing the Nrv3I domain (red) was used to detect the chimeras because there is no expression of Nrv3 in nrv2/+ salivary glands [compare B2 (no chimera) and B2' (UAS Nrv2AB)]. Scale bars: in A1, 10 µm for A-G image 1; in A4, 5 µm for A-G images 2-4 and B2'.

View larger version (89K): [in this window] [in a new window]   Fig. 4. Rat 1 and some Drosophila ATP isoforms have SJ full activity. (A,B) The Na,K-ATPase -subunit (A) is encoded by one major locus at 93A that generates numerous isoforms by alternative splicing (B). Splice forms differ in the N-terminal 39 aa (green) and in a mutually exclusive exon (blue). The catalytic residue D369 (D394 in the fly) is denoted by a red dot. (C-H) Compared with the WT (C1-5), Atp [DTS1R2] mutants have long DTs with diameter defects (D1) and missing lumens in the GBs (D2). The mutant fails to exclude dye from the trachea (D3), has disorganized SJs (D4; Coracle, green; ATP, red), and no longer accumulates Verm in the tracheal lumen [D5 and Wang et al. (Wang et al., 2006)]. DT and GB morphology, barrier junction, Verm accumulation defects and Coracle localization are fully rescued when Long C (F1,4,5), Long C DN (G1,4,5), or rat 1 (H1,4,5) are expressed using a da-Gal4 driver. Expression of Short C gives only slight DT rescue (E1), no Coracle rescue (E4) and no Verm rescue (E5). Scale bars: in H2, 10 µm for C-H images 1,2; in H3,10 µm for C-H image 3; in H5, 5 µm for C-H images 4,5.