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First published online 23 March 2004
doi: 10.1242/jcs.01029

Journal of Cell Science 117, 1899-1909 (2004)
Published by The Company of Biologists 2004
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Cell type-specific recruitment of Drosophila Lin-7 to distinct MAGUK-based protein complexes defines novel roles for Sdt and Dlg-S97

André Bachmann1, Marco Timmer2, Jimena Sierralta3, Grazia Pietrini4, Eckart D. Gundelfinger2, Elisabeth Knust1 and Ulrich Thomas2,*

1 Institut für Genetik, Heinrich-Heine-Universität Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany
2 Leibniz Institute for Neurobiology, Department of Neurochemistry, Brenneckestr. 6, 39118 Magdeburg, Germany
3 Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile and CENI, Millenium Nucleus for Integrative Neuroscience, Santiago, Chile 6530499
4 Department of Medical Pharmacology, Center of Excellence on Neurodegenerative Diseases, University of Milan, IN-CNR, Cellular and Molecular Pharmacology Section, Italy



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  		Fig. 1. DLin-7 encodes a single-PDZ domain protein. (A) Exon-intron structure of the DLin-7/CG7662 locus (black boxes, ORF) from the cytological interval 96B19 (genomic scaffold AE003750). (B) RT-PCR performed on poly(A)+ RNA of different developmental stages (staged embryos in hours, L1-L3, larval stages; pP, prepupa; P, pupa; F, female adult). (C) Northern blot of embryonic poly(A)+ RNA hybridized with a probe covering the L27 and PDZ domain of DLin-7. (D) Protein structure of DLin-7 and two putative orthologues from mammals (MALS3) and C. elegans (LIN-7). The green and lilac colours indicate the L27 and the PDZ domains, respectively. The percentages of amino acid identities of the two domains with respect to the corresponding domains of DLin-7 are shown. (E) Western blot analysis of wild-type embryos (1) and embryos overexpressing a Flag-tagged DLin-7 transgene (2) probed with anti-DLin-7 antibodies. Note the additional, slightly larger band in lane 2 that corresponds to the Flag-DLin-7 protein.

 


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  		Fig. 2. In vitro interaction of DLin-7 with members of different MAGUK subfamilies. (A) Proteins that were tested for interaction in a yeast two-hybrid assay. Subregions expressed as baits or preys are indicated as bars together with the positions of the first and last amino acid residues. The symbols used for the various protein domains are summarized in the box below. See Discussion for an interpretation of the cryptic L27 domain of Sdt. (B) Results of the yeast two-hybrid assay. The strength of each interaction was classified by the time period between the onset of the ß-Gal filter assay and the detection of a clearly visible color reaction; ++++, within 30 minutes or less; +++, within 1 hour; ++, within 3 hours; +, within 5 hours; –, more than 5 hours. The empty bait vector pGBKT7 served as a negative control. The weak interaction between DLin-7 and Dlg-S97 was also observed in the reverse bait-prey combination.

 


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  		Fig. 3. Modulation of DLin-7 protein levels in wing imaginal discs by means of the GAL4/UAS-system. (A) ptc-Gal4-driven overexpression of Flag-DLin-7 strikingly increases Flag-DLin-7 protein levels. (B) Upon overexpression of DLin-7-ds-RNA with ptc-Gal4 the amount of DLin-7 protein is significantly reduced.

 


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  		Fig. 4. Subcellular localization of DLin-7 in wing imaginal discs and its recruitment by Sdt. (A-F) In wing imaginal discs DLin-7 is localized apical to Dlg, which is restricted to the septate junction in epithelia (A-C), and colocalizes with Sdt in the SAR (D-F). (G-I) ptc-Gal4-driven overexpression of Sdt increases apical levels of DLin-7. (G'-I') Note that both overexpressed Sdt and, as a consequence, DLin-7 are targeted to the SAR (white arrowheads mark the borders of the overexpression domain). (J) DLin-7 and Sdt-GUK interact in vitro. Sepharose beads carrying a GST-DLin-7 fusion protein or GST alone were incubated with biotinylated Sdt-GUK. Bound protein was eluted and analysed by SDS-PAGE. Binding was observed between GST-DLin-7 and Sdt-GUK (right lane), but not between GST and Sdt-GUK (middle lane). The left lane shows labelled Sdt-GUK protein equivalent to 10% of the material analysed in the experimental lanes. (K) Anti-mLin-7 antibody (Perego et al., 2002Go) immunoprecipitates Sdt from embryos overexpressing Sdt-MAGUK protein (GAL4daG32/UAS-sdtMAGUK, left lane), whereas a control IgG (anti-ß-Gal) does not (middle lane). The western blot was then probed with anti-Sdt antibody. The left lane shows the corresponding input control with approximately 50 µg of total protein from embryonic extracts.

 


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  		Fig. 5. DLin-7 is expressed in the CNS and at NMJs. (A) Ventral nerve cord of a 3rd instar larva. DLin-7 is enriched in the neuropil area. Arrows mark the periphery of the cortical area. (B-D). Muscle 12 at abdominal segment A3 is innervated by four different motornerve terminals (Ib, Is, II, III) as revealed by anti-HRP immunoreactivity (B), but only type I boutons exhibit clear DLin-7 specific immunoreactivity (C,D). (E-G) Confocal section of a branch of the NMJ at muscle 6. Note that the presynaptic marker HRP (E) is largely surrounded by DLin-7 specific immunoreactivity (F,G). (H-J) Confocal section of a NMJ at muscle 12, double-labeled with anti-Dlg (I) and anti-DLin-7 (J) antibodies. Arrowhead marks a site of reduced immunoreactivity. Bar, 20 µm (A); 10 µm (B-J).

 


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  		Fig. 6. Mutations in dlg affect the localization of DLin-7 at larval NMJs. NMJs at muscle 12 of w1118 control larvae (A,B) and dlg-mutant larvae (C-H) were double-labeled with anti-HRP antibodies (A,C,E,G) and antibodies against DLin-7 (B,D,F,H). Each image represents a stack of 15 to 17 optical sections taken at 0.5 µm steps.

 


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  		Fig. 7. Dlg-dependent localization of transgenically expressed Flag-DLin-7 at NMJs. (A) Western blot analysis on body wall muscle extracts using anti-DLin-7. A Coomassie staining was used as a loading control to ensure that equal amounts of body wall extracts were analyzed. M, molecular weight marker; lane 1, w1118; lane 2, dlg+;;UAS-Flag-DLin-7/Gal4-C57; lane 3, dlgXI-2/Y;;UAS-Flag-DLin-7/Gal4-C57. (B-G) Confocal sections of synaptic boutons at muscle 12 double-labeled with anti-HRP (B,D,F) and anti-Flag (C,E,G). (B,C) w1118; (D,E) dlg+;;UAS-Flag-DLin-7/Gal4-C57; (F,G) dlgXI-2/Y;;UAS-Flag-DLin-7/Gal4-C57.

 


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  		Fig. 8. Isoform-specific interaction between Dlg-S97 and DLin-7. (A-F) Rescue of DLin-7 at dlg-mutant NMJs on muscle 12 upon postsynaptic expression of EGFP-Dlg-A versus EGFP-Dlg-S97. DLin-7-specific immunofluorescences of w1118 (A) and dlgXI-2 (B) are shown for reference. (C) Gal4-C57-driven EGFP-Dlg-A was targeted to NMJs and to nuclei (arrow), whereas co-staining reveals no obvious enrichment for DLin-7 at either compartment (D). Postsynaptic targeting of EGFP-Dlg-S97 (E) was accompanied by effective restoration of DLin-7 (F). Each image represents a stack of 17 optical sections taken at 0.5 µm steps. (G) Co-immunoprecipitation assay performed on body wall muscle extracts from w1118- (lanes 1, 3) and UAS-Flag-DLin-7/Gal4-C57 (lanes 2, 4) 3rd instar larvae using anti-Flag antibody. Input control samples (lanes 1, 2) and immunoprecipitated protein samples (lanes 3, 4) were analyzed by western blotting using anti-DlgPDZ1+2. Note the enrichment of the 120 kDa isoform in lane 4.

 


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  		Fig. 9. Ectopic expression of EGFP-Dlg-S97 and Dlg-S97N-EGFP. (A-I) Confocal sections of wing imaginal disc epithelia expressing EGFP-Dlg-S97 (A-C), EGFP-Dlg-S97 and Flag-DLin-7 (D-F) or Dlg-S97N-EGFP (G-I) under the control of ptc-Gal4, co-stained with anti-DLin-7 antibodies (B,E,H). Areas flanking the ptc-expression domain served as an internal control to assess possible effects on the subcellular distribution of DLin-7. Merged images are shown in C, F and I. No striking redistribution of DLin-7 was caused by either EGFP-Dlg-S97, which was enriched basolaterally (depicted by arrowheads in C,F), or Dlg-S97N-EGFP, which exhibited strong nuclear localization (G). (J-L) Mislocalization of DLin-7 in nuclei of muscles 6 and 7 upon Gal4-C57 driven expression of Dlg-S97N-EGFP. Each image represents a stack of 20 optical sections taken at 0.5 µm steps. Nuclear enrichment of Dlg-S97N-EGFP (G) was paralleled by partial targeting of DLin-7 to nuclei (H), which was not observed in w1118 (I). Arrows mark selected nuclei. Bars, 10 µm (in I for panels A-I; in L, for panels J-L).

 

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© The Company of Biologists Ltd 2004