Volume 11, Issue 3, Pages 363-374 (September 2006) The FERM Protein Yurt Is a Negative Regulatory Component of the Crumbs Complex that Controls Epithelial Polarity and Apical Membrane Size  Patrick Laprise, Slobodan Beronja, Nancy F. Silva-Gagliardi, Milena Pellikka, Abbie M. Jensen, C. Jane McGlade, Ulrich Tepass  Developmental Cell  Volume 11, Issue 3, Pages 363-374 (September 2006) DOI: 10.1016/j.devcel.2006.06.001 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Yurt Isoforms Expression and Localization (A) Overnight embryo lysate was treated with dual specificity λ phosphatase prior to immunoblot analysis with Yurt-specific antibody (GP7). (B) Immunoblot showing the developmental expression profile of Yurt isoforms. (C) Schematics of the yurt genomic region, yurt transcripts, and the Yurt protein. Blue color indicates open reading frame. PDB, PDZ binding site; VR, variable region. (D) Yurt expression in yurt M/Z embryos compared to wild-type (WT) embryos. (E) During cellularization (stage 5), Yurt (red) is found in the apical and lateral membrane and enriched at the furrow canals marked by PATJ (green). (F) At stage 9, Yurt staining (red) is found at the lateral membrane and enriched just basal to the ZA labeled by anti-Arm antibody (green). (G–H) At stage 14, Yurt is enriched in the upper third of the lateral membrane, overlaps with the ZA (Arm staining in G′), and is found in the marginal zone where it colocalizes with Crb (H″). (I) Schematic of adult PRCs. ZA, zonula adherens. (J–K) At 75% pd, Yurt (red) is found at the basolateral membrane of PRCs and is enriched at the base of the ZA (Arm, blue) but is not detected at the apical stalk membrane marked by PATJ (green). (L) At 85% pd, Yurt (red) is detected at the basolateral membrane but also extends apical (arrowhead) beyond the ZA (Arm, blue) and partially overlaps with PATJ (green) in the stalk membrane. (M) At 90% pd, Yurt (red) is predominantly detected at the apical stalk membrane (PATJ, green). (N) crb11A22 mutant adult PRCs are identified by the loss of PATJ staining (green; marked by asterisks), and failed to recruit Yurt (red) to the stalk membrane. Instead, Yurt is enriched at the basolateral membrane. Scale bars: (E)–(H), 10 μm; (J)–(N), 5 μm. Developmental Cell 2006 11, 363-374DOI: (10.1016/j.devcel.2006.06.001) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 Yurt Interacts with Crb through Its FERM Domain (A) ptc-Gal4 UAS-crb embryo. Overexpression of Crb (green) leads to an increase in the levels of Yurt (red). (B) Coimmunoprecipitation of Crb using anti-Yurt antibody and wild-type embryos stages 6–10, 12–17, and an overnight (O/N) collection. (C) Lysate from an overnight embryo collection was subjected to GST pull-down with glutathione-agarose beads loaded with either GST, GST fused with the FERM domain of Yurt (GST-Yurt FERM), GST fused to a C-terminal portion of Yurt (GST-Yurt C TERM), or GST fused to the FERM domain of Coracle (GST-Cor FERM). Crb was specifically pulled down in the presence of the FERM domain of Yurt. (D) Far-Western using the FERM domain of Yurt as bait and GST-Crbintra as a probe shows a direct interaction between these two polypeptides. Scale bar: (A), 50 μm. Developmental Cell 2006 11, 363-374DOI: (10.1016/j.devcel.2006.06.001) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 YMO1 Interacts with All Three Human CRB Proteins (A) Schematic of the human CRB proteins and YMO1 and the mutant versions used. TM, transmembrane; FDB, FERM domain binding site; PDB, PDZ domain binding site. (B and C) Adult mouse retina labeled for YMO1 (green, B) and CRB2 (green, C) and pan-Cadherin antibodies (Cad, red). (D–F) Co-IP from lysates of HEK293T cells that were cotransfected with pairs of cDNA or a vector control as indicated. The expression level of proteins from the transfected cDNAs was monitored with whole-cell lysates (Lysate). (G–I) HEK293T cells were transfected with cDNAs encoding different proteins as indicated and lysed 24–48 hr later. Lysates were subjected to pull-down experiments with GST fused to the cytoplasmic tail of human CRB proteins. GST protein alone or fused to the cytoplasmic tail of human syndecan 1 (GST-hSyn) were used as negative controls. The expression levels were monitored with whole-cell lysate (Lysate), and Coomassie staining was used to evaluate the amount of GST fusion protein. Developmental Cell 2006 11, 363-374DOI: (10.1016/j.devcel.2006.06.001) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Crb Is Mislocalized in yurt Mutant Embryos (A, B, E, and G) Crb distribution in a wild-type stage 11 (A and E) and stage 13 (B and G) embryo is restricted to the apical membrane. (C and F) Stage 12 yurt75 M/Z mutant stained for Crb showing ectopic localization of Crb in the basolateral membrane. (D and H) Crb distribution is apolar, and the epidermis and trachea are multilayered in a stage 13 yurt75 M/Z embryo. (I and J) Face-on view of Crb distribution in the ventral epidermis of a stage 11 wild-type (I) and yurt75 M/Z mutant (J) embryo. Crb distribution is irregular when Yurt is absent. (K–N) Crb distribution in the ventral (K and L) or lateral (M and N) epidermis of a stage 13 wild-type (K and M) and a yurt75 M/Z (L and N) embryo. Disruption of Crb distribution and tissue structure is largely confined to the ventral epidermis in a yurt75 M/Z embryo. Scale bars: (A)–(D), 100 μm; (E)–(H), 10 μm; (I)–(N), 10 μm. Developmental Cell 2006 11, 363-374DOI: (10.1016/j.devcel.2006.06.001) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 Epithelial Polarity Defects and Enlarged Apical Membranes in yurt Mutants (A and B) PATJ distribution in the ventral epidermis of a stage 13 wild-type (A) and yurt75 M/Z (B) embryo. (C, F, and I) PATJ (C), βH-Spectrin (F), and Armadillo (I) distribution in the ventral epidermis of a stage 13 embryo overexpressing Crb (da-GAL4, UAS-crb). (D and E) Distribution of βH-Spectrin in the ventral epidermis of stage a 13 wild-type (D) and yurt75 M/Z (E) embryo. (G and H) Arm distribution in the ventral epidermis of a stage 13 wild-type (G) and yurt75 M/Z (H) embryo. (J) Cuticle of a wild-type embryo. (K–M) Cuticle of a zygotic yurt75 mutant (K) and a yurt75 M/Z mutant (L and M). Arrows point to convoluted ventral cuticle. (N) Transmission EM of a stage 17 wild-type embryo. Arrow points to cuticle. (O) Transmission EM showing that the cuticle is highly infolded and extended in stage 17 yurt75 M/Z mutant embryos. Arrows point to cuticle. Scale bars: (A)–(I), 10 μm; (J)–(L), 100 μm; (M), 50 μm; (N) and (O), 5 μm. Developmental Cell 2006 11, 363-374DOI: (10.1016/j.devcel.2006.06.001) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 Stalk Membrane Length Is Increased in yurt Mutant PRCs (A and B) Transmission EM of wild-type PRCs. Arrowheads point to ZAs and arrows to stalk membrane in (B), (D), (F), and (H). (C and D) yurt75 mutant PRCs show abnormal rhabdomere morphology and extended stalks. (E) yurt75 mutant PRCs show light-stress-dependent degeneration. (F) crb11A22 mutant PRCs have shortened stalks. (G and H) yurt75 crb11A22 double mutant PRCs show a crb-like phenotype with shortened stalk membranes. (I) Histogram showing the progressive increase in stalk length in yurt75 mutant PRCs at 1 day (d), 7 days, and 14 days after eclosure. Student's t test showed significant differences between wild-type and mutants at p < 0.0001; yurt 1 day versus yurt 7 days, p < 0.05; yurt 7 days versus yurt 14 days, p < 0.0001. No significant difference was found between crb11A22 and yurt75 crb11A22 double mutants. Error bars represent standard deviation. (J) Basolaterally displaced adherens junctions (arrowheads; Arm, blue) are seen in yurt75 mutant PRCs marked by the absence of Yurt (red). (K) Crb (blue) and βH-Spectrin (βH-Spec; green) localize to the stalk membrane in yurt75 mutant PRCs. Arrowhead points to apical abnormalities occasionally detected in yurt75 mutant cells. Scale bars: (A), (C), (E), and (G), 2 μm; (B), (D), (F), and (H), 1 μm; (J) and (K), 10 μm. Developmental Cell 2006 11, 363-374DOI: (10.1016/j.devcel.2006.06.001) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 7 Genetic Interaction between yurt and crb (A) Cuticle of a crb11A22 mutant embryo. (B) Cuticle of a crb11A22 yurt75 double M/Z mutant embryo. The crb mutant phenotype is epistatic over the yurt mutant phenotype. (C) Cuticle defects observed in a crb11A22 mutant embryo are ameliorated in a crb11A22 yurt75 zygotic double mutant embryo. (D and E) Cuticle defects observed in the crbS87-2 mutant embryo (D) are suppressed in a crbS87-2 yurt75 zygotic double mutant embryo (E). (F) Cuticle of a dlgm52 yurt75 zygotic double mutant embryo. Removal of zygotic dlg does not modify the yurt mutant phenotype. (G–I) Face-on view of Arm distribution in the ventral epidermis of a stage 13 wild-type (G), crb11A22 (H), and yurt75 crb11A22 (I) zygotic mutant embryo. (J–L) The defects in Cor distribution observed in a crbS87-2 mutant embryo (K) are suppressed in a crbS87-2 yurt75 zygotic double mutant embryo (L). (M) Model illustrating the functional interactions between Yurt and other polarity factors. Global regulatory feedback between apical and basolateral determinants controls apical-basal polarity, whereas local regulatory feedback between Yurt and other components of the Crb complex contributes to apical-basal polarity and to the regulation of apical membrane size. Scale bars: (A)–(F), 100 μm; (G)–(L), 10 μm. Developmental Cell 2006 11, 363-374DOI: (10.1016/j.devcel.2006.06.001) Copyright © 2006 Elsevier Inc. Terms and Conditions