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First published online 31 January 2007
doi: 10.1242/dev.02785

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Drosophila Varicose, a member of a new subgroup of basolateral MAGUKs, is required for septate junctions and tracheal morphogenesis

Victoria M. Wu^1,*, Marcus H. Yu^1,*, Raehum Paik², Swati Banerjee², Zhiguo Liang³, Sarah M. Paul¹, Manzoor A. Bhat^2,4 and Greg J. Beitel^1,

¹ Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208, USA.
² Department of Cell and Molecular Physiology, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA.
³ Department of Microbiology and Immunology, University of Illinois-Chicago, Chicago, IL 60612, USA.
⁴ UNC Neuroscience Center, Curriculum in Neurobiology, Neurodevelopmental Disorders Research Center, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA.

View larger version (43K): [in this window] [in a new window]   Fig. 1. vari/CG9326 encodes a MAGUK required for tracheal tube size control. (A,B,E,F,H,I) Compared to wild type, vari mutant trachea are long (B,F) and are missing lumenal segments of the ganglionic branches (bracket in I). (C,D) RNAi knockdown of CG9326 phenocopies vari mutations. (G,J) Ubiquitous expression of the short vari isoform rescues vari null mutant phenotypes. (K) The vari/CG9326 genomic locus, transcript organization, protein domains and mutations. , alternative splicing generates either a 35 or 14 amino acid linker between the PDZ and SH3 domains. Note the PDZ-SH3 linker in PALS2, a mouse homolog of Vari, is also alternatively spliced to produce linkers of different length (Kamberov et al., 2000); *, splicing at a GC splice donor site was confirmed by sequencing seven independent cDNAs and the genomic DNA of multiple wild-type and lab stocks. (L-N) Varicose (green) co-localizes with the SJ marker Nrx (red) in wild-type (variF033/+) surface epithelia (L), but is absent in genetically null mutants variF033 (M) and variR3 (N). Left and right panels show the same image, but left panels show guinea pig anti-Vari (green channel), whereas right panels show Vari and Nrx (composite red/green). Trachea visualized in A,B,F-J with monoclonal 2A12, in C,D with btl::gal4 UAS GFP. Genotype in G,J is variF033 da::gal4/variF033 UAS vari short. (A-J,L-N) Stage 16 animals. Dashed lines indicate location of basal cell surfaces; L and M are cropped from the same image of adjacent heterozygous and homozygous animals. Scale bars: in B, 25 µm for A,B; in J, 50 µm for C-J; in N, 1.5 µm for L-N. ORF, open reading frame; UTR, untranslated region; WT, wild type.

View larger version (71K): [in this window] [in a new window]   Fig. 2. Vari and its homologs define a new subgroup of epithelial MAGUKs. (A) A dendrogram showing the sequence similarities between Vari and other MAGUKs. Conserved subgroups are delineated by colored lines with subgroup names or members and representative domain structures adjacent. Colored lines show species representation. PALS2 is the mouse homolog of VAM-1 (Kamberov et al., 2000). Semitransparent lines beside the P55 (MPP1) subgroup indicate that while invertebrates lack distinct P55 genes, alternative splice forms of the LIN-2/Caki subgroup may be the functional equivalent.?, MAGUKs that cannot be assigned to conserved subgroups. Numbers at dendrogram node points show the percentage of dendrograms created in 1000 bootstrap iterations containing the displayed grouping. Human Carma3 was used as an outgroup to root the displayed dendrogram. (B) C-terminal alignments of the MAGUK subgroups. Residues conserved between more than 75% of shown subgroup members are marked in red. Potential PDZ-binding motifs for each subgroup are underlined in the consensus. , Dr_ZO-1 C-terminal sequence predicted from genomic sequence and is absent in current gene annotations; ***, end of coding sequence.

View larger version (98K): [in this window] [in a new window]   Fig. 3. Vari is an SJ component. (A-D') Vari localizes to epithelial SJs in the hindgut (A,C) and trachea (A',C') of wild-type animals where it co-localizes with the canonical SJ marker Cor (A,A',B,B'). (D) Schematic diagrams showing the organization of membrane domains and cell-cell junctions in the hindgut (D) and trachea (D'). (E-K) Vari is required for stable formation of SJs, as in variF033 null mutants the SJ components Nrx, Sinu and Dlg do not accumulate in a specific region, but instead are mislocalized along the basolateral membrane (F,F',I,I',K,K'). In vari3953b partial loss-of-function mutants, some SJ components such as Cor (G,G') are almost entirely mislocalized, whereas others such as Dlg only show reduced localization (L,L'). (M-N') Localization of Vari to the SJ depends on other SJ components, as Vari does not specifically localize to the SJ region in nrx (M,M') or nrv2 (N,N') mutants. (O-R) Vari is not required for apical-basal polarity, as localization of the adherens junction and apical markers Arm and Veli is not disrupted in variF033 null mutants. (S) Vari protein does not accumulate at SJ regions in animals homozygous for vari alleles that affect the HOOK domain (Table 1). All animals stage 16. Scale bar: in S, 5 µm for all images. WT, wild type.

View larger version (92K): [in this window] [in a new window]   Fig. 4. Vari is required for the assembly rather than stability of SJs. (A-F) In wild-type animals, Cor localizes predominantly to SJs during stage 14, and then continues to accumulate in SJs during stages 15 and 16 (A-C). In variF033 null mutants SJs do not assemble, as indicated by the failure of Cor to localize specifically to the SJ region (D-F). Instead, Cor shows diffuse basolateral and basal localization at all stages. Abnormal accumulations of Cor at the basal surface are visible during stages 15 and 16 (arrows in E and F). (G-L) In variF033 null homozygotes expressing either the short (G-I) or long (J-L) isoforms of Vari, SJ junction barrier function is restored (data not shown) and the Vari isoforms (green) and Cor (red) show their normal localization to SJs. G-L are stage 16 animals of genotype variF033 UAS::Vari-isoform/variF033 da-Gal4. Hindgut shown in all images. Blue lines, apical cell surface; white lines, basal cell surface. Scale bar: in L, 5 µm for A-L.

View larger version (73K): [in this window] [in a new window]   Fig. 5. vari is required for accumulation of Verm and Serp in the tracheal lumen. (A-E) Lumenal secretion and accumulation of the putative matrix modifying protein Verm is disrupted in vari (B-D) and other SJ mutants such as Lac (E). Notably, in some mutants such as vari3953b and Lack11012b, Verm accumulates in the cytoplasm (arrows in C and E). vari3953b mutants show variable expressivity of the Verm secretion defect, with some animals showing extensive cytoplasmic Verm accumulation (C) and others showing little (D). (F-H) Lumenal accumulation of the Verm-related protein Serp is defective in vari and Lac mutants, but in contrast to Verm, Serp is completely absent in vari and Lac mutants, including in the hypomorphic vari3953b allele. Scale bar: in H, 2.5 µm for A-H.

View larger version (18K): [in this window] [in a new window]   Fig. 6. Vari interacts with Nrx through the PDZ domain. (A) A purified C-terminal fragment of Nrx is co-precipitated by purified Glutathione S-transferase fused to the short isoform of Vari and the Vari PDZ domain, but not GST or GST fused to the Vari-SH3 domain. (B) GSTVari and GST-VPDZ co-precipitate Nrx but not Crb from 12-18-hour-old embryo lysates, despite the similarity of the Crb and Nrx C-terminal PDZ-binding motifs (ERLI vs EIFI). NaCl concentrations: 150, 350 and 550 mmol/l. GST, Glutathione S-transferase; IB, immunoblot antisera; VPDZ, Vari PDZ domain.