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First published online August 4, 2003
doi: 10.1242/10.1242/dev.00648

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Interaction of Par-6 and Crumbs complexes is essential for photoreceptor morphogenesis in Drosophila

¹ Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, TX 77030, USA
² Program in Developmental Biology, Baylor College of Medicine, One Baylor Plaza, TX 77030, USA
³ Department of Ophthalmology, Baylor College of Medicine, One Baylor Plaza, TX 77030, USA

View larger version (90K): [in a new window]   Fig. 1. Crb is required for Sdt localization at rhabdomere stalk. (A) A schematic representation of photoreceptor cell in pupal stage. Crb and Dlt colocalize specifically to the rhabdomere stalk between the rhabdomeres emerging from the apical surface and the AJs. Confocal images shown in B-E were sectioned as indicated by the broken line. (B-E) Co-localization of Dlt and Sdt in photoreceptors at 40% pd. Dlt (green) is localized in apical membrane in the most central region of a photoreceptor cluster whereas AJ (red) is immediately basal to the Dlt domain (B). Later, rhabdomeres form apical to the Dlt domain, which will become the rhabdomere stalk between rhabdomere and AJ. Dlt (C, red) and Sdt (D, green) are colocalized in the rhabdomere stalks. Both proteins show extensive colocalization (E). (F-N) Dlt (F,I,L; red) and Sdt (G,J,M; green) are mislocalized in the crb- photoreceptors cell autonomously. In crb- mutant cells identified by the absence of GFP staining (H,K,N; blue), Dlt and Sdt are co-mislocalized (H,K,N). Mislocalization of Dlt and Sdt in crb- mutant cells is relatively mild in the earlier pupal eyes (F-H; 25% pd), but becomes more severe in the later pupal eyes (I-K; 35% pd) and then completely diffused to basolateral membrane (L-N; 50% pd).

View larger version (107K): [in a new window]   Fig. 2. Crb is sufficient to recruit Dlt and Sdt. (A-C) Dlt (A, red) and Sdt (B, green) colocalize in the rhabdomere stalk (C). (D-F) Mislocalization of Dlt (D, red) in the photoreceptor was induced by overexpression of CrbPBM using GMR-Gal4. In this situation, Sdt (E, green) was co-mislocalized together with Dlt (F).

View larger version (95K): [in a new window]   Fig. 3. Sdt is required for correct localization of Crb and Dlt. (A-C) Dlt (B, green) is absent (arrowheads) in sdt- clones marked by the absence of GFP (C, blue) or Sdt (A, red) staining. Arrows show the presence of Dlt in Sdt+ cells in a mosaic photoreceptor cluster, indicating cell-autonomous function of Sdt. (D-F) Crb (D, red) is absent or strongly reduced in the stalk (arrows in F) of sdt- cells marked by the absence of Dlt (E, green) or GFP (F, blue). (G) Top view of a three-dimensional reconstruction of sdt- clones marked by the absence of Sdt staining (green). Arm staining indicates that AJ was expanded basolaterally and mispositioned in sdt- clones marked by the absence of Sdt (green). (H) DE-cad (red), another marker of AJs is expanded basolaterally. Mutant clones were marked by the absence of GFP staining (blue). (I) F-actins (phalloidin, red) are diffused (arrowheads) and/or displaced from the apical to the lateral position of the photoreceptors (arrows). sdt- mutants are marked by the absence of GFP (blue). (J) Longitudinal section of sdt- clones containing wild-type (right) and sdt- cells (left) marked by the absence of Sdt staining (green). Arm staining is fragmented and widely mispositioned to the lateral positions. (K,L) An oblique (K) and a longitudinal section (L) of w- sdt- adult eye clones marked by the absence of red pigments (labeled with brackets). The oblique section (K) shows arrays of ommatidia from the distal end to the proximal end at the floor of the retina. sdt- ommatidia in the distal region show rhabdomeres, whereas mutant ommatidia in the proximal region show severe disruption. The longitudinal section (L) shows continuous elongated rhabdomeres in sdt+ ommatidia and discontinuous disrupted rhabdomeres in sdt- ommatidia. (M,N) Transmission electron micrographs of a wild-type (M) and a sdt- photoreceptor cluster (N) from a mosaic adult eye. These tangential sections were made at the distal region of the eye. The rhabdomeres are bulky and fused in the sdt- photoreceptors (N) compared with the wild type (M).

View larger version (52K): [in a new window]   Fig. 4. Colocalization of Par-6 complex with Crb complex and direct binding of Dlt to Par-6. (A-D) Both Par-6 (A, green) and aPKC (B, green) colocalize with Dlt (red, A-C) at the apical domain of photoreceptor cluster. aPKC not only colocalizes with Dlt in the apical membrane but also overlaps with AJs (B). Baz does not co-localize with Dlt (C). Baz colocalizes with Arm (D) at the AJ of photoreceptors. (E-G) In eye discs of the third instar larvae, Par-6 colocalizes with Dlt (E) and Baz colocalizes with Arm (G), but not with Dlt (F). (H) Direct binding of Dlt to Par-6, but not to aPKC or Baz. MBP-Dlt fusion protein was attached to amylose bead and used for the binding to GST-Par-6, GST-aPKC or GST-Baz. The blot was probed with anti-GST antibody. (I,J) Direct binding of Dlt by its PDZ3 region with Par-6. Each of GST constructs fused with Dlt deletion mutant proteins was attached to glutathione-agarose bead and used for the binding test with MBP-Par-6. The blots were probed with anti-MBP antibody. (K,L) Deletion of C-terminal PBM motif of Par-6 did not affect the binding of Par-6 to Dlt (K). GST or GST-Dlt was attached to beads and used for binding of MBP-Par-6 or MBP-Par-6C. The blot was probed with anti-MBP antibody (K). The N-terminal region of Par-6 is required for the binding to Dlt, but the PDZ domain is not (L). Each deletion mutant protein of Par-6 was attached to beads and used for binding with MBP-Dlt. The blot was probed with anti-MBP (L). (M,N) Schematic of molecular interaction between Dlt and Par-6. Binding is indicated by two-headed arrow. AID and CRIB are atypical PKC interaction domain and Cdc42-Rac1 interaction domain, respectively.

View larger version (119K): [in a new window]   Fig. 5. Mislocalization of Par-6, aPKC, and Baz in crb- photoreceptors. Localization of Par-6 complex proteins was examined in crb- mutant clones (No GFP colored in blue). (A-C) Dlt (A, red) is greatly reduced or mislocalized in crb- mutants. Par-6 (B, green) is co-mislocalized with Dlt (C). (D-F) aPKC (E, green) is also reduced and co-mislocalized with Dlt (D, red). (G-I) Baz (H, green) is expanded basolaterally and remains basal to Dlt (G, red). (J-L) Baz (K, green) is co-mislocalized with Arm in the basolateral membrane (J, red). Defects in crb- cells are cell-autonomous based on the phenotypes specifically associated with crb- cells in mosaic photoreceptor clusters.

View larger version (125K): [in a new window]   Fig. 6. Mislocalization of Par-6, aPKC and Baz in sdt- and loss of Sdt in dltMY10. The localization of Par-6, aPKC, and Baz was examined in sdt- mutants (no GFP in blue). (A-C) Par-6 (B, green) is mislocalized whereas Dlt (A, red) is undetectable in sdt- mutants. (D-F) aPKC (E, green) is mislocalized in sdt- mutants. (G-I) Baz (H, green) is basolaterally expanded and colocalized with Arm (G, red) in sdt- mutants. (J-L) Sdt (K, green) is lost in dltMY10 mutant photoreceptor cluster (arrowhead) indicated by the absence of Dlt (J, red). The arrow indicates cell-autonomous loss of Sdt in dltMY10 cells in a mosaic cluster.

View larger version (101K): [in a new window]   Fig. 7. Crbintra is sufficient to recruit Par-6 complex and AJs. (A,B) Overexpression of CrbPBM causes ectopic co-localization of Dlt (red), Par-6 (A; green) and aPKC (B; green). (C) Arm (red) is displaced by the overexpression of CrbJM, but Par-6 (green) is not. (D-F) The localization of Baz was examined using CrbPBM (D) or CrbJM (E,F) overexpression. Dlt (red) was ectopically mislocalized by the CrbPBM expression (D), but Baz (green) was not. Baz (E,F) was ectopically displaced by the CrbJM overexpression. In this situation, Arm (E; red) was mispositioned with Baz, but Dlt (F; red) was not. A-C are tangential sections; D-F are longitudinal sections.

View larger version (105K): [in a new window]   Fig. 8. Par-6 complex is required for localization of Crb complex and AJ. (A,B) Dlt (arrowhead; A, red) and Arm (arrows; B, red) are mislocalized in par-6- mutant cells marked by loss of Par-6 (green) staining. (C,D) Dlt (arrowheads; C, red) is diffused away from apical membrane, and Arm (arrows; D, red) is displaced in aPKC- mutants marked by the absence of aPKC (green) staining. (E,F) In baz- clones indicated by loss of Baz (green) staining, Dlt (arrowheads; E, red) is lost and Arm (arrows; F, red) is mislocalized at proximal section. But Arm is not detected at distal section (data not shown).