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First published online 14 August 2007
doi: 10.1242/jcs.004770

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The Drosophila homolog of the Exo84 exocyst subunit promotes apical epithelial identity

¹ Developmental Biology Program, Sloan-Kettering Institute, 1275 York Avenue, New York, NY 10021, USA
² Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305, USA

View larger version (66K): [in this window] [in a new window]   Fig. 1. Disruption of epithelial structure and adherens junctions in onion rings mutant embryos. (A-C) Neurotactin localizes to basolateral cell surfaces in wild-type (A) and onr mutant embryos (B,C). At stage 6, onr mutants establish columnar epithelial morphology normally (B), whereas stage 10 onr mutants display a severe epithelial disruption (C) compared to the wild type (A). (D-I) At stage 10, Armadillo (green D-F) and DE-cadherin (green G-I) localize apically in the wild type (D,G) and in an onr mutant carrying the P[Exo84] transgene (I). onr mutants accumulate Armadillo (E,F) and DE-cadherin (H) at various positions along the apical-basal axis. Neurotactin (red D-F) and filamentous actin (F-actin red G-I) are enriched at basolateral surfaces in wild-type and onr mutant embryos. (F) A 5 µm projection of multiple optical sections stained for Armadillo (green), superimposed on a single 1 µm slice of the cell outline marker Neurotactin (red). Anterior, left; ventral, up. Bar, 20 µm.

View larger version (81K): [in this window] [in a new window]   Fig. 2. Disruption of apical or basolateral polarity leads to distinct effects on adherens junction localization. (A,C,E,G,I) DE-cadherin (green) and F-actin (red) in the wild type (A) and Exo84 (C), crumbs (E), dlg (G) and lgl (I) mutant embryos at stage 10. (B,D,F,H,J) Armadillo (green) and Neurotactin (red) in the wild type (B) and Exo84 (D), crb (F), dlg (H) and lgl (J) mutant embryos at stage 10. (A,B) In wild-type embryos, DE-cadherin (green A) and Armadillo (green B) localize to the apical edge of lateral interfaces and form a continuous circumferential band. (C-F) In Exo84 (C,D) and crumbs (E,F) mutants, DE-cadherin (green C,E) and Armadillo (green D,F) form large isolated puncta at various locations along the apical-basal axis. (G-J) In dlg (G,H) and lgl (I,J) mutants, DE-cadherin (green G,I) and Armadillo (green H,J) are diffusely distributed along the cell cortex. Anterior left, ventral up. Bars, 20 µm (A), 5 µm (B).

View larger version (115K): [in this window] [in a new window]   Fig. 3. Mislocalization of apical proteins in Exo84 mutant embryos. (A-J) Bazooka (green A,C-E,G-J), Crumbs (green B,F), aPKC (blue C,G) and dPATJ (red D,H) localize to the apical margin of lateral cell surfaces in the wild type (A-D,I), but form ectopic aggregates in Exo84 mutants (E-H,J). Dlg (red I,J) is excluded from the apical surface in the wild type (I). (J) Exo84 mutants display disrupted Bazooka localization in all cells pictured, whereas apical Dlg exclusion is often maintained. Neurotactin (red A,C,E,G) delineates cell outlines and DAPI (purple B,F) labels nuclei. (K,L) Armadillo (green K,L) and Bazooka (red K,L) colocalize in wild-type adherens junctions (K) and in basolateral aggregates in Exo84 mutants (L). (M-P) In the wild type (M,N), Armadillo (red) and Bazooka (blue) localize to adherens junctions (M) and are not detected basally (N, 4 µm below M). In Exo84 (O) and crumbs mutants (P), Armadillo (red) and Bazooka (blue) are mislocalized along the apical-basal axis. All embryos are stage 10. Anterior, left; ventral, down. Bar, 20 µm (A); 5 µm (K).

View larger version (129K): [in this window] [in a new window]   Fig. 4. Crumbs mislocalization precedes the loss of Bazooka and Armadillo in Exo84 mutants. (A-D) Crumbs (green), Bazooka (blue) and Armadillo (red) in stage 9 wild-type (A), Exo84 (B), arm (C) and shg (D) mutant embryos. (A) In the wild type, Crumbs, Bazooka and Armadillo localize apically. (B) At the onset of epithelial disruption in Exo84 mutants, Armadillo localizes apically whereas Crumbs is absent from large areas. The degree of apical Bazooka localization is intermediate. (C,D) In arm and shg mutants, Crumbs and Bazooka are absent in regions lacking Armadillo. Anterior, left; ventral, down. Bar, 10 µm.

View larger version (92K): [in this window] [in a new window]   Fig. 5. Exo84 genetically interacts with apical and basolateral components. (A-F) Wild-type cuticle at the end of embryogenesis (A). In Exo84 at 25°C (B) and crumbs (C) small scraps of cuticle form, whereas dlg (D) and lgl (E) produce a continuous, malformed cuticle. Exo84 mutants at 20°C exhibit a weak defect in cuticle formation resulting in small ventral holes (F). (G-I) In strongly defective embryos, little cuticle is present (G). In moderately defective embryos, defects range from large ventral holes (H) to embryos with patches of cuticle. A weak classification indicates small ventral holes (I). (J) Cuticle defects in combinations of Exo84 with mutations in epithelial polarity genes. Exo84 embryos at 20°C that carry mutations in crumbs, sec5 or sec6 exhibit a stronger defect in cuticle integrity than Exo84 alone. By contrast, a decrease in the dosage of the basolateral determinants dlg or lgl partially restored cuticle formation in Exo84 mutants at 25°C. Overexpression of crumbs in Exo84 embryos allowed partial cuticle formation, whereas overexpression of lgl in Exo84 enhanced the cuticular defects. Defects in Exo84 mutants at 25°C were not suppressed by crumbs overexpression. For mutant combinations of Exo84 and crumbs, all Exo84 mutant embryos received half the maternal dosage of crumbs and one half were predicted to be homozygous for crumbs. For mutant combinations of Exo84 and sec5, sec6, dlg or lgl, all embryos received half the maternal dosage of sec5, sec6, lgl or dlg and one quarter were predicted to be homozygous for sec5, sec6, lgl or dlg. For experiments in which crumbs or lgl were overexpressed in an Exo84 background, one quarter of the embryos were predicted to receive both the Gal4 driver and the UAS transgene (see Materials and Methods). Percentages represent the fraction of embryos that did not hatch. Anterior, left; ventral, down. Bar, 100 µm.

View larger version (106K): [in this window] [in a new window]   Fig. 6. The recycling endosome compartment is disrupted in Exo84 mutants. (A) Schematic of results depicting the distribution of Golgi (Lva), early endosome (Rab5), late endosome (Hrs) and recycling endosome (Rab11) compartments in cross section in wild-type and Exo84 mutant embryos. (B-J) Localization of Lva (red B,C), Rab11 (green D-F), Rab5-GFP (green G,H), Hrs (green I,J), Armadillo (green B,C) and Bazooka (blue B,C,G-J, red D-F) in stage 10 wild-type (B,D,G,I), Exo84 (C,E,H,J) and crumbs mutant (F) embryos. (B,C) The Golgi compartment (Lva, red) appears normal in size and distribution in the wild type (B) and Exo84 mutant (C). (D-F) Recycling endosomes (Rab11, green) are diffusely distributed in the apical cytoplasm in wild type (D), but form large aggregates in Exo84 mutants (E). Despite disruption of epithelial polarity and Bazooka localization (red) in crumbs mutants (F), recycling endosomes are comparable in appearance to the wild type (D). (G-J) Early endosomes (Rab5-GFP, green G,H) and late endosomes (Hrs, green I,J) are distinct compartments from recycling endosomes (Rab11, red) in the wild type (G,I) and Exo84 mutant (H,J). Bazooka aggregates (blue) colocalize with recycling endosome aggregates. Anterior, left; ventral, down. Bar, 10 µm (B); 5 µm (G,I).

View larger version (121K): [in this window] [in a new window]   Fig. 7. Aggregation of polarity proteins in Exo84 mutant embryos. (A) Schematic depicting the distribution of Crumbs (Crb), Bazooka (Baz), Armadillo (Arm), and Rab11 in cross section in wild-type and Exo84 mutant embryos. In Exo84 mutants, these proteins colocalize in large aggregates. (B,C) Stage 10 Exo84 mutant embryos stained for Crumbs (green), Armadillo (red) and Bazooka (blue). In severely disrupted cells in which Crumbs, Armadillo and Bazooka proteins are absent from the apical surface (panel B, lower right), ectopic aggregates of these proteins occur basolaterally (C, shown 2 µm below B). Note that cells adjacent to the affected region lack apical Crumbs but maintain junctional Armadillo (B). (D) Aggregation of DE-cadherin (green) and Bazooka (red) in Exo84 mutant embryos, shown 4 µm below the apical surface. (E) Colocalization of Crumbs (red) with recycling endosome aggregates (Rab11, green) in Exo84 embryos. (F-I) An Exo84 embryo imaged at three positions along the apical-basal axis. In regions where Bazooka (blue) and Armadillo (red) are absent from the apical surface (F), large accumulations of Bazooka and Armadillo colocalized with recycling endosomes in z-planes 2 µm (G) and 4 µm (H) below the apical plane in (F). (I) An enlarged view of the boxed area in G. (J-M) In the wild type (J,L) and shg;Exo84 (K,M), aggregation of Crumbs (red L,M) and Bazooka (blue) occurs despite strongly reduced levels of Armadillo (green) and E-cadherin (red J,K). Anterior, left; ventral, down. Bars, 10 µm.

View larger version (8K): [in this window] [in a new window]   Fig. 8. A model for exocyst function in epithelial polarity of the Drosophila embryo. Epithelial polarity is established correctly in Exo84 mutant embryos (A). Crumbs (red) and adherens junction proteins (blue) localize to the apicolateral cell surface, and recycling endosomes (green) are distributed throughout the apical cytoplasm. (B) Failure to traffic Crumbs to the apical surface, first apparent at stage 9, is accompanied by a loss of apical identity and a mislocalization of adherens junction proteins along the cell surface in a manner that resembles crumbs mutants. (C) At later stages, a defect in trafficking from recycling endosomes to the cell surface causes apical and adherens junction proteins to accumulate in an enlarged recycling endosome compartment.