LRR domain of Scrib interacts with Tkv.

<p>(A) Diagram of different fragments of Scrib. Intensities of interaction between Scrib fragments and Tkv are shown at the right. (B, C) Co-IP of Scrib (full length or fragments) and Tkv. Scrib-MYC and Tkv-GFP were expressed in S2 cells, and cell lysates were immunopreciated by anti-GFP. Cell lysates (input) and immunoprecipitated proteins (IP: GFP) were analysed by Western blots probed with anti-GFP and anti-MYC antibodies. (D) Co-IP of LRR, Tkv and Mad. LRR-RFP, caTkv-GFP and Mad-FLAG were expressed in S2 cells, and cell lysates were immunopreciated by anti-RFP. Cell lysates (input) and immunoprecipitated proteins (IP: RFP) were analysed by Western blots probed with anti-GFP, anti-FLAG and anti-pMad antibodies. (E) Expression of Scrib (full length), LRR domain or Scrib without LRR domain in <i>scrib</i> mutant clones labeled by GFP. pMad staining in PCV region at 24 h AP. Dashed lines delineate the clone boundaries. (F) Quantification of pMad-positive cells in E. (G) Effects of <i>scrib</i>^<i>5</i> mutant clones on pMad (red) at 24 h AP in the PCV region. Mutant GFP-labeled cells (green) were generated using MARCM. Results shown are representative of one of three independent experiments (B-D).</p

Scrib regulates spatial distribution of Tkv in the PCV region.

<p>(A) A schematic of different planes (1 and 2) of PCV cells along the apicobasal axis in B. (B) Loss of Scrib affects Tkv distribution. <i>scrib</i> mutant clones are marked by mRFP and absence of Scrib staining. Tkv-YFP staining in the PCV region at 24 h AP. (C) Optical cross sections of B. Note that Tkv is more enriched basally in control but restricted more apically in <i>scrib</i> mutant cells. (D, E) Wild-type pupal wing. Tkv-YFP, Scrib and Rab5 staining in the PCV region at 24 h AP (D). Tkv-YFP, pMad and Scrib staining in the PCV region at 24 h AP (E). Arrows indicate that Tkv-YFP puncta co-localize with Scrib and Rab5 (D), or pMad and Scrib (E). (F) Tkv-YFP and Rab5 staining at the basolateral plane in control (<i>nub</i>^<i>ts</i>) and <i>scrib</i> RNAi (<i>nub</i>^<i>ts</i> <i>> scrib RNAi</i>) in the PCV region at 24 h AP. Arrows indicate that Tkv-YFP puncta co-localize with Rab5. (G) Quantification of co-localization of Tkv-YFP with Rab5. n = 20 cells for each sample. (H) Quantification of Tkv-YFP or Rab5 puncta. n = 20 cells for each sample. Error bar, SEM. **<i>P</i><0.01 in f, two-paired <i>t</i>-test. Images are the composite of sections with 2 μm thickness in D-F. Images have been taken by a 25X objective for B, C, and 63X objective for D-F.</p

Clathrin dependent endocytosis is required for BMP/Dpp signaling during PCV formation.

<p>(A-C) Adult wings of <i>ada</i>^<i>3</i><i>/+</i> (A), <i>dpp</i>^<i>hr4</i><i>/+</i> (B) and <i>ada</i>^<i>3</i><i>/dpp</i>^<i>hr4</i> (C). The PCV regions are indicated by arrows. (D) Quantification of adult wing phenotypes in A-C. (E, F) Adult wings of control (<i>nub</i>^<i>ts</i>) (E) and <i>AP-2u</i> RNAi <i>(nub</i>^<i>ts</i> <i>> AP-2u</i> RNAi) (F). The PCV regions are indicated by arrows. (G) Quantification of adult wing phenotypes in E, F and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006424#pgen.1006424.s003" target="_blank">S3A and S3B Fig</a>. (H) pMad staining in the PCV region at 24 h AP in control (upper panel) or <i>AP-2u</i> RNAi (lower panel). (I) Pupal wing at 24 h AP showing aPKC and DLG1 staining in <i>AP-2u</i> RNAi RNAi <i>(nub</i>^<i>ts</i> <i>> AP-2u</i> RNAi). Optical cross section focused on the PCV region (lower panels). (J) Effects of clonal overexpression of Rab5.WT (upper panel) or Rab5.DN (lower panel) on pMad staining (red) in the PCV region at 24 h AP. Rab5-expressing clones are marked by GFP (green). Nuclei are marked by DAPI (blue) staining. Dashed lines depict overexpression clones.</p

BMP signal regulates <i>scrib</i> transcription in the pupal wing.

<p>(A) Scrib-GFP (green), pMad staining (red), and DAPI (blue) at 18 to 24 h AP. Nuclei are marked by DAPI (blue) staining. (B-E) Plot profile analysis of Scrib-GFP (green) and pMad (red) in ROIs in A, corresponding to 18h AP, 20h AP, 22h AP and 24h AP, respectively. (F) Quantitative PCR analysis of mRNA levels in pupal wings at 24 h AP. mRNA levels in <i>dpp</i>^<i>shv</i> <i>> ca-tkv</i> flies were compared to control <i>yw</i> flies. Relative mRNA expression level of genes of interest was calculated with normalization to <i>gapdh</i> mRNA level. <i>ventral veinless</i> (<i>vvl</i>) was used as a positive control. Error bar, SEM. ****<i>P</i><0.001, **<i>P</i><0.01, two-paired <i>t</i>-test. (G, H) In situ hybridization analysis of <i>scrib</i> (G) and <i>dlg1</i> (H) in pupal wings at 24 h AP (upper panels). caTkv expression clones were generated using MARCM. Clones were marked by GFP (lower panels). Up-regulated <i>scrib</i> or <i>dlg1</i> corresponding to caTkv clones are indicated by arrows. Sense probe was applied to the wings expressing caTkv clones as a negative control.</p

The Scrib complex is required for PCV formation through regulating BMP/Dpp signaling.

<p>(A-C) Adult wings of control (<i>nub-GAL4</i>, <i>GAL80</i>^<i>ts</i> <i>(nub</i>^<i>ts</i><i>)</i>) (A), <i>scrib</i> RNAi (<i>nub</i>^<i>ts</i> <i>> scrib</i> RNAi) (B) and <i>dlg1</i> RNAi (<i>nub</i>^<i>ts</i> <i>> dlg1</i> RNAi) (C). RNAi flies were cultured at 25 (A), 25 (B), and 27 (C) °C, respectively. The PCV regions are indicated by arrows. (D) Quantification of adult wing phenotypes in A-C. (E, F) Pupal wings at 24 h AP showing aPKC and DLG1 staining in control (<i>nub</i>^<i>ts</i>) (E) and <i>scrib</i> RNAi (<i>nub</i>^<i>ts</i> <i>> scrib</i> RNAi) (F). Optical cross sections focused on the PCV region are shown at the lower panel. aPKC localizes apically and DLG1 basolaterally in both control and <i>scrib</i> RNAi wings. Note that the lumen of the wing vein is formed in control but not in <i>scrib</i> RNAi wings. (G, H) pMad staining in the PCV region in control (<i>nub</i>^<i>ts</i>) (G) and <i>scrib</i> RNAi (<i>nub</i>^<i>ts</i> > <i>scrib</i> RNAi) (H) at 24 h AP. (I) Effects of <i>scrib</i> mutant clones on pMad (red) at 24 h AP in the PCV region. Mutant GFP-labeled cells (green) were generated using MARCM system. Dashed box in left panel depicts the region of interest (ROI). Higher magnification pictures of the ROI are shown in the right panels.</p

Rab5 and LRR domain of Scrib form a complex with Tkv.

<p>(A) Co-IP of Rab5, Tkv and LRR domain of Scrib. Rab5-RFP, Tkv-GFP and LRR-MYC were expressed in S2 cells, and cell lysates were immunoprecipitated by anti-RFP. Cell lysates (input) and immunoprecipitated proteins (IP-RFP) were analyzed by Western blot probed with anti-RFP, anti-GFP and anti-MYC antibodies. (B, C) Co-IP of Rab5, caTkv and Mad. Rab5-RFP, caTkv-GFP and Mad-FLAG were expressed in S2 cells, and cell lysates were immunoprecipitated by anti-GFP or anti-RFP. Cell lysates (input) and immunoprecipitated proteins (IP: GFP in B, or IP: RFP in C) were analyzed by Western blot probed with anti-RFP, anti-GFP and anti-pMad antibodies. Results are representative of one of three independent experiments (A-C). (D) Expression of constitutively active Rab5 in <i>scrib</i> mutant clones labeled by GFP. pMad staining in PCV region at 24 h AP. Dashed lines delineate the clone boundaries. Nuclei are marked by DAPI (blue) staining. (E) Quantification of pMad positive cells in <i>scrib</i> mutant cells of D. Results are collected from 3 independent experiments.</p

Supplemental Material for Matamoro-Vidal et al., 2018

File S1. Data table with genotype, sex, area, stage, area, centroid size and landmark coordinates for each wing used in this study. <br><br>File S2. Movie showing size and shape changes among developmental stages (all genotypes pooled). <br><br>File S3. Movie showing relative shape changes among developmental stages (all genotypes pooled). This visualization shows shapes having equal sizes

DataSheet1_Scribble and α-Catenin cooperatively regulate epithelial homeostasis and growth.PDF

Epithelial homeostasis is an emergent property of both physical and biochemical signals emanating from neighboring cells and across tissue. A recent study reveals that Scribble, an apico-basal polarity determinant, cooperates with α-Catenin, an adherens junction component, to regulate tissue homeostasis in the Drosophila wing imaginal disc. However, it remains to be addressed whether similar mechanisms are utilized in vertebrates. In this study, we first address how α-Catenin cooperates with Scribble to regulate epithelial homeostasis and growth in mammalian cells. Our data show that α-Catenin and Scribble interact physically in mammalian cells. We then found that both α-Catenin and Scribble are required for regulating nuclear translocation of YAP, an effector of the Hippo signaling pathway. Furthermore, ectopic Scribble suffices to suppress YAP in an α-Catenin-dependent manner. Then, to test our hypothesis that Scribble amounts impact epithelial growth, we use the Drosophila wing imaginal disc. We show that Scribble expression is complementary to Yorkie signal, the Drosophila ortholog of YAP. Ectopic expression of full-length Scribble or Scribble Leucine Rich Region (LRR):α-Catenin chimera sufficiently down-regulates Yorkie signal, leading to smaller wing size. Moreover, Scribble LRR:α-Catenin chimera rescues scribble mutant clones in the wing imaginal disc to maintain tissue homeostasis. Taken together, our studies suggest that the association of cell polarity component Scribble with α-Catenin plays a conserved role in epithelial homeostasis and growth.</p