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First published online 21 May 2008
doi: 10.1242/jcs.031088

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Multiple domains of Stardust differentially mediate localisation of the Crumbs-Stardust complex during photoreceptor development in Drosophila

¹ Max-Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, Dresden, Germany
² Institut für Genetik, Heinrich-Heine Universität Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany

View larger version (32K): [in this window] [in a new window]   Fig. 1. Structure and expression of Sdt isoforms. (A) Two sdt transgenes encoding full-length Sdt proteins were used: SdtA [also known as Sdt-MAGUK1 (Bachmann et al., 2001)], and Sdt-B2 (Berger et al., 2007). (B) List of Sdt variants used in this study. All transgene-encoded proteins in A and B carry a FLAG-tag at their N-terminus. (C) Western blot analysis of Sdt-FLAG proteins from the eyes of flies that carry large clones mutant for sdtK85 and express the respective transgene using Rh1GAL4. Anti-FLAG antibody was used to detect Sdt-FLAG proteins. Eyes of flies with large clones mutant for sdtK85 but not expressing Sdt-transgenes were used as control.

View larger version (96K): [in this window] [in a new window]   Fig. 2. (A-D) Localisation of Sdt and Drosophila PATJ in sdtK85 mutant pupal photoreceptor cells upon expression of different Sdt transgenes. Optical cross sections of pupal eyes at 40-45% p.d., before formation of the stalk membrane. MARCM clones mutant for sdtK85 (A) and those mutant for sdtK85 and expressing different Sdt-encoding transgenes (B-D) were stained with anti-Sdt-PDZ (A,C), anti-SdtN (B), or anti-PATJ (D) (red), and anti-E-cadherin (A,C,D) or anti-Arm (B) to stain the zonula adherens (blue). Mutant cells expressing a transgene are marked by GFP (green). Depending on the optical section level, the green staining is sometimes weaker. Cells not expressing GFP are wild type and serve as a control to demonstrate that Sdt and PATJ localise apically in PRCs at this stage.

View larger version (125K): [in this window] [in a new window]   Fig. 3. (A-D'") Localisation of Drosophila Par-6 in sdtK85-mutant pupal photoreceptor cells upon expression of different Sdt transgenes. Optical cross sections of pupal eyes at 40-45% p.d., before formation of the stalk membrane. sdtK85 mutant MARCM clones expressing different sdt transgenes, stained with anti-Par-6 (red) and anti-E-cadherin to stain the zonula adherens (blue). Mutant cells expressing a transgene are visualised using expression of GFP (green). Cells not expressing GFP are wild-type and serve as a control to demonstrate that Par-6 localises apically in PRCs at this stage. In sdtK85 mutant PRCs (A-A'"), Par-6 is reduced in amount and delocalised from the apical membrane (arrows). In sdtK85 mutant PRCs that express Sdt-B2 (B-B'"), Par-6 localises apically. In most sdtK85-mutant PRCs that express Sdt-A (C-C'"), Par-6 localises apically but, occasionally, also basolateral (arrows). Expression of Sdt-PDZ in sdtK85 mutant PRCs does not restore apical localisation of Par-6 (D-D'").

View larger version (78K): [in this window] [in a new window]   Fig. 4. Localisation of Sdt proteins encoded by transgenes in sdtK85-mutant adult photoreceptor cells. Optical cross-sections of adult Drosophila eyes stained with antibodies against Sdt-PDZ (A,B,E) or with aSdt-N (C,D) (red), and phalloidin to highlight the F-actin-rich rhabdomeres (blue). (A) Sdt localises at the stalk membrane in wild-type PRCs. (B) sdtK85 eyes show morphological defects in the rhabdomeres and no Sdt protein can be detected. (C) Sdt-B2 localises at the stalk membrane when expressed in sdtK85-mutant PRCs. (D) Sdt-PDZ localises at the rhabdomere base when expressed in sdtK85-mutant PRCs. (E) Sdt-N is distributed throughout the cell when expressed in sdtK85-mutant PRCs. Note that the stalk of R7 is not labelled by anti-Sdt antibody in C-E, because Rh1-Gal4 is only expressed in the six outer PRCs. This makes it sometimes more difficult to detect the rhabdomere of R7.

View larger version (42K): [in this window] [in a new window]   Fig. 5. The divergent L27 domain of Sdt interacts with the L27 domain of PATJ in yeast-two-hybrid assays. (A) Comparison of the two L27 domains of Pals1 with those of Sdt. The yellow boxes indicate conserved hydrophic amino acids required to form helices characteristic for L27 domains. (B) Only Sdt fragments containing the N-terminal L27 domain can bind a portion of PATJ that includes the L27 domain, in the yeast-two-hybrid system. The results of the interactions are tabulated on the right: +, interaction; –, no interaction.

View larger version (60K): [in this window] [in a new window]   Fig. 6. Expression of PATJ, Lin-7 and Crb. Western blots of extracts from wild-type eyes (w), sdtK85-mutant eyes, and sdtK85-mutant eyes expressing different sdt transgenes (four eyes per lane), probed with anti-PATJ and anti-Lin-7 (A), and anti-Crb (B) antibodies. Eyes used for this experiment contained large mutant clones with hardly any wild-type ommatidia (1%). antibody against -tubulin was used as a loading control.

View larger version (125K): [in this window] [in a new window]   Fig. 7. Localisation of PATJ and Lin-7 proteins in sdtK85-mutant adult photoreceptor cells upon expression of Sdt-encoding transgenes. Optical cross-sections of adult Drosophila eyes stained with anti-Sdt-N (A,B,C) or with anti-FLAG (D) (red), and with anti-PATJ (A,B) or anti-Lin-7 (C,D) (green). Phalloidin highlights the F-actin-rich rhabdomeres (blue). When Sdt-B2 is expressed in sdtK85 mutant PRCs (A-A"), both Sdt (A) and PATJ (A') localise at the stalk membrane. Upon Sdt-PDZ expression in sdtK85 mutant PRCs (B-B"), Sdt (B) and PATJ (B') co-localise at the rhabdomere base. When Sdt-L27C is expressed in sdtK85 mutant PRCs (C-C"), Sdt (C), but not Lin-7 (B') localises at the stalk membrane. When Sdt-N is expressed in sdtK85 mutant PRCs (D-D"), Sdt (D) and Lin-7 (D') are distributed throughout the cell.

View larger version (116K): [in this window] [in a new window]   Fig. 8. Localisation of Crb in sdtK85 mutant adult photoreceptor cells upon Sdt-encoding transgene expression. Optical cross-sections of adult Drosophila eyes stained with anti-Sdt-PDZ (red) and anti-Crb (green). Phalloidin highlights the F-actin-rich rhabdomeres (blue). (A) In sdtK85-mutant eyes, which develop rhabdomeres with morphological defects, no Sdt (A) or Crb (A') was detected. (B) Sdt-B2 localises at the stalk membrane when expressed in sdtK85-mutant PRCs (B) and restores localisation of Crb at the stalk membrane (B'). (C) Sdt-N expressed in sdtK85 mutant PRCs is spread throughout the cells and is only occasionally slightly enriched near the stalk membrane (C), where it then colocalises with Crb (C', arrows). (D) Sdt-SH3 localises at the rhabdomere base when expressed in sdtK85 mutant PRCs (D), and no Crb is detected (D').

View larger version (20K): [in this window] [in a new window]   Fig. 9. Rescue of stalk-membrane length by Sdt-encoding transgenes. Stalk-membrane length (mean ± s.e.m.) in sdtK85 (n=115), sdtK85 + Sdt-SH3 (n=123), sdtK85 + Sdt-PDZ (n=107), sdtK85 + Sdt-GUK (n=118), sdtK85 + Sdt-L27C (n=127), sdtK85 + Sdt-B2 (n=118), sdtK85 + Sdt-A (n=124) and wild-type (n=117) PRCs. One unit represents 1 µm. *P<0.0001 or +P<0.0001 (two-tailed test), significantly different compared with wild type or sdtK85, respectively.

View larger version (14K): [in this window] [in a new window]   Fig. 10. Summary of results. Apical localisation of the Sdt protein in the pupae only depends on the PDZ domain, whereas its correct localisation to the stalk membrane in the adult depends on different domains. The various Sdt domains are required differentially to localise other proteins in the pupae and the adult.

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