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Crumbs organizes the transport machinery by regulating apical levels of PI(4,5)P2 in Drosophila

  1. Johanna Lattner
  2. Weihua Leng
  3. Elisabeth Knust
  4. Marko Brankatschk  Is a corresponding author
  5. David Flores-Benitez  Is a corresponding author
  1. Max-Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Germany
  2. The Biotechnological Center of the TU Dresden (BIOTEC), Germany
Research Article
Cite this article as: eLife 2019;8:e50900 doi: 10.7554/eLife.50900
15 figures, 9 videos, 3 tables and 1 additional file

Figures

Figure 1 with 2 supplements
Crb is required for efficient apical secretion in SG cells.

(A) Scheme indicating the anatomic location of the SG in the larval stage. (B-G) Localization of Crb (B,C), Sdt (B’,C’), Baz (D,E) and Dlg (F,G) in control (B,B’,D,F, fkh>/+) and Crb KD (C,C’,E,G, fkh >UAS crbRNAi) animals. H. Pupariation efficiency of controls (black and blue) and larvae with reduced levels of Crb (magenta) at 29 °C. Error bars indicate the standard error of the mean, n indicates number of traced individual larvae of the corresponding genotypes in three independent experiments. (I,J) Localization of the apical transmembrane protein Cadherin99C in SGs from control (I) and Crb KD (J) animals. (K,L) Localization of the secreted apical cargo SerpCBD-GFP in live SGs of control (K, fkh >UAS SerpCBD-GFP) and Crb KD (L, fkh >UAS crbRNAi; UAS-SerpCBD-GFP) animals. Arrows indicate the apical plasma membrane. Arrowheads mark the lateral plasma domain. Dotted lines indicate the basal membrane. Scale bar in A indicates10 µm applies to all panels. (M, M) Plotted is the fluorescence intensity (arbitrary units) of SerpCBD-GFP along the apical-to-basal direction in live SGs of control (black, fkh >UAS SerpCBD-GFP) and Crb KD (magenta, fkh >UAS crbRNAi; UAS-SerpCBD-GFP). Error bars indicate the standard error of the mean, n indicates number of glands from the corresponding genotypes.

Figure 1—source data 1

Dataset for tracking of larval development.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig1-data1-v2.xlsx
Figure 1—source data 2

Dataset for SerpCBD-GFP fluorescence intensity in control glands.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig1-data2-v2.xlsx
Figure 1—source data 3

Dataset for SerpCBD-GFP fluorescence intensity in Crb KD glands.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig1-data3-v2.xlsx
Figure 1—figure supplement 1
Knock-down of the Crb protein complex in larval SGs disrupts apical secretion.

(A,B) Maximal projections showing F-actin (phalloidin staining) of control (A, fkh>/+) and Crb KD (B, fkh >UAS crbRNAi) animals. (C-L) Localization of the polarity proteins DPatj (C,D), aPKC (E,F), Par-6 (G,H), Yrt (I,J) and Cora (K,L) in control (C,E,G,I,K, fkh>/+) and Crb KD (D,F,H,J,L, fkh >UAS crbRNAi) animals. (M,N) Localization of the apical membrane marker CD8-RFP in live SGs of control (M, fkh >UAS-CD8-RFP) and Crb KD (N, fkh >UAS crbRNAi; UAS-CD8-RFP) animals. (O,P) Localization of the glycoprotein reporter PNA-GFP in live SGs of control (O, fkh >UAS PNA-GFP) and Crb KD (P, fkh >UAS crbRNAi; UAS-PNA-GFP) animals. Q. Western blot probed for Crb (left) and Tubulin (right) loaded with control (fkh>/+, lanes with ‘crbRNAi -”) and Crb KD (fkh >UAS crbRNAi, lanes with ‘crbRNAi +”) SG protein lysates. Arrows point to the respective proteins. R. Plotted is the tube length of glands measured along the secretory segment in control (fkh>/+), Crb KD (fkh >UAS crbRNAi) and Sdt KD (fkh >UAS sdtRNAi) animals. Statistical significance was tested in a one-way analysis of variance (ANOVA) followed by a Dunnett’s multiple-comparison test. n = 18 glands for control, 20 for Crb KD and 11 for Sdt KD. (S,T) Maximal projections showing F-actin (phalloidin staining) of control (S fkh>/+) and Sdt KD (T, fkh >UAS sdtRNAi) animals. U-DD Localization of Crb (U,V), Sdt (U’,V’), DPatj (W,X), Par-6 (Y,Z), Dlg (AA,BB) and Cad99C (CC,DD) in control (U,U’,W,Y,AA,CC, fkh>/+) and Sdt KD (V,V’,X,Z,BB,DD, fkh >UAS sdtRNAi) animals. (EE,FF) Localization of apical membrane marker CD8-RFP in live SGs of control (EE, fkh >UAS-CD8-RFP) and Sdt KD (FF, fkh >UAS sdtRNAi; UAS-CD8-RFP) animals. (GG,HH) Localization of secreted apical cargo SerpCBD-GFP in live SGs of control (GG, fkh >UAS SerpCBD-GFP) and Sdt KD (HH, fkh >UAS sdtRNAi; UAS-SerpCBD-GFP) animals. (II,JJ) Localization of the glycoprotein reporter PNA-GFP in live SGs of control (II, fkh >UAS PNA-GFP) and Sdt KD (JJ, fkh >UAS sdtRNAi; UAS-PNA-GFP) animals. Arrows point to apical and arrowheads to the lateral domain. Dotted lines indicate the basal membrane. Scale bar in (A) indicates 50 µm (for A,B,S,T) and in (C) 10 µm (for C-P and U-JJ). KK. Plotted is the puparium formation efficiency of controls (black and blue) and larvae with reduced levels of Sdt (magenta) at 29 °C. Error bars indicate the standard error of the mean, n indicates number of traced individual larvae of the corresponding genotypes in three independent experiments. LL. Plotted is the fluorescence intensity (arbitrary units) of SerpCBD-GFP along the apical-to-basal direction in live SGs of control (black -same shown in Figure 1M, fkh >UAS SerpCBD-GFP) and Sdt KD (magenta, fkh >UAS sdtRNAi; UAS-SerpCBD-GFP) animals. Error bars indicate the standard error of the mean, n indicates number of glands of the corresponding genotypes.

Figure 1—figure supplement 1—source data 1

Dataset for salivary gland lengths.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig1-figsupp1-data1-v2.xlsx
Figure 1—figure supplement 1—source data 2

Dataset for tracking of larval development.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig1-figsupp1-data2-v2.xlsx
Figure 1—figure supplement 1—source data 3

Dataset for SerpCBD-GFP fluorescence intensity in control glands (note is the same dataset for Figure 1M control).

https://cdn.elifesciences.org/articles/50900/elife-50900-fig1-figsupp1-data3-v2.xlsx
Figure 1—figure supplement 1—source data 4

Dataset for SerpCBD-GFP fluorescence intensity in Sdt KD glands.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig1-figsupp1-data4-v2.xlsx
Figure 1—figure supplement 2
The Crb protein complex is dispensable for maintenance of cell-cell junctions in larval SGs.

(A,C) Transmission electron microscopic (TEM) images, visualizing the overview of the cell-cell junction close to the apical domain and ZA ultrastructure of SG cells of feeding larvae. Shown are cell-cell contacts (magenta), gaps along the cell-cell junctions (green), multivesicular bodies (blue) (A,C) and the ZA (yellow arrowhead, A’,C’) of control (A,A’ fkh>/+) and Crb KD (C,C’ fkh >UAS crbRNAi) animals. Scale bars in A,C indicate 1 μm and in A’,C’ indicate 100 nm. (B,D) TEM images, visualizing the SJ ultrastructure of SG cells of feeding larvae. Representative images of the SJs of control (B, fkh>/+) and Crb KD (D fkh >UAS crbRNAi) animals are shown. Scale bars in indicate 100 nm. (E-J) Confocal images of SGs probed for the SJ proteins Sinu (E,F), Kune (G,H) and Fas3 (I,J) of control (E,G,I, fkh>/+) and Crb KD (F,H,J, fkh >UAS crbRNAi) animals. Shown are single optical slices and maximal projections of half of the z-stack (half SG-tube). Arrows point to apical, and arrowheads to lateral localizations. Dotted lines indicate the basal membrane. Scale bar in E (applies to panels E-J) indicates10 µm. (K-M’) The barrier function of the SG epithelium was tested ex vivo in a Dextran-permeability assay. Localization of endogenously expressed Fas3-GFP (K,L,M) and Dextran-rhodamine 10 kDa (K’,L’,M’) in control (K,K’, Fas3-GFP, fkh>/+), Crb KD (L,L’, Fas3-GFP, fkh >UAS crbRNAi) and the positive control for leaky SGs, Fas3 KD (M,M’, Fas3-GFP, fkh >UAS gfpRNAi) in live SGs. Insert in (M) displays the corresponding image with brightness adjusted manually using Photoshop to reveal the SG. Dotted lines indicate the basal membrane. Asterisks mark the SG lumen. Scale bar in K indicates 10 µm and applies to panels (K-M’).

Figure 2 with 4 supplements
Crb is necessary to specifically maintain the apical cytoskeleton and the morphology of the apical membrane.

(A-F) Localization and quantification of F-actin (phalloidin staining, A-C) and βH-Spec (D-F) in control (A,D, fkh>/+) and Crb KD (B,E, fkh >UAS crbRNAi) SGs. Violin graphs (C,F) show the fluorescence intensity (apical vs lateral ratio) indicating the mean and quartiles for F-actin (C, n = 36 cells for control and 28 cells for Crb KD) and βH-Spec (F, n = 44 cells for control and 40 cells for Crb KD). Statistical significance was analyzed in an unpaired two-tailed t-test. (G-H) Localization of phospho-Moe in control (G, Crb-GFP, fkh>/+) and Crb KD (H, Crb-GFP, fkh >UAS gfpRNAi) SGs. (I,J) Localization of the apical protein Stranded at second (Sas-YFP) in live SGs of control (I, fkh>/+) and Crb KD (J, Crb-GFP, fkh >UAS gfpRNAi) animals. Shown are single optical slices and maximal projections of half of the z-stack (half SG-tube). Arrows point to the apical domain of the cell. Dotted lines indicate the basal membrane. Scale bar in (A) displays 10 µm and applies to panes (A-J). (K-L’) TEM images of SGs prepared using the high-pressure freezing technique, visualizing the apical aspect of SG cells of control (K,K’, fkh>/+) and Crb KD (L,L’, fkh >UAS crbRNAi) animals. The brackets in K,L’ indicate the apical microvilli. Asterisks in (L’) mark large intracellular vesicles found in Crb-deficient glands. Arrowheads in L’ indicate microvilli found inside vesicles. Scale bars in (K,L) indicate 5 µm and in (K’,L’) indicate 1 µm. (M, M) Mean number of microvilli following along the apical membrane over a distance of 1 µm, adjacent to the membrane and 1 µm above the apical membrane in SG cells of control (fkh>/+) and Crb KD (fkh >UAS crbRNAi) animals. The heatmap indicates the scale bar for the number of microvilli/µm.

Figure 2—figure supplement 1
Crb is necessary to specifically maintain the apical membrane organization.

(A-F) Localization of endogenously tagged Crb-GFP (A,B), Sdt (C,D) and F-actin (phalloidin staining, E,F) in SGs of control (A,C,E, Crb-GFP, fkh>/+) and Crb KD (B,D,F, Crb-GFP, fkh >UAS gfpRNAi) animals. (G-J) Localization of αTubulin (G,H) and α-Spec (I,J) in SGs of control (G,I, fkh>/+) and Crb KD (H,J, fkh >UAS crbRNAi) animals. Shown are single optical slices. Arrows point to the apical and arrowheads to the lateral membrane domain. Scale bars (A,E,G) indicate 10 µm.

Figure 2—figure supplement 2
TEM images of intracellular extensions of apical membrane in Crb-deficient glands.

(A-C’) TEM images of SGs from Crb KD (fkh >UAS crbRNAi) animals prepared by high-pressure freezing technique. Asterisks mark large intracellular vesicles found in Crb-deficient glands. Arrowheads indicate microvilli found inside vesicles. Scale bars in (A-C) indicate 5 µm and in (A’-C’) indicate 1 µm.

Figure 2—figure supplement 3
Increased lysosomal activity in Crb and Sdt deficient glands.

(A-D) Maximal projections of SGs incubated ex vivo with Lysotracker for 30 min of control (A,C, fkh>/+), Crb KD (B, fkh >UAS crbRNAi) and Sdt KD (D, fkh >UAS sdtRNAi) animals. Lysotracker fluorescence intensity increases as pH in lysosomal compartments acidifies, that is become more active. Dotted lines indicate the basal membrane. Scale bar in A displays 10 µm.

Figure 2—figure supplement 4
KD of βH-Spec induces the formation of PAMS.

(A,B) Localization of βH-Spec in control (A, fkh>/+) and βH-Spec KD (B, fkh >UAS kstRNAi) animals. Insert (B) displays the corresponding image with brightness adjusted manually using Photoshop to visualize the SG. (C,D) Localization of phospho-Moe in SGs of control (C, fkh>/+) and βH-Spec KD (D, fkh >UAS kstRNAi) animals. (E,F) Localization of Crb in control (E, fkh>/+) and βH-Spec KD (F, fkh >UAS kstRNAi) SGs, respectively. Arrows point to the apical membrane domain. Scale bar (A) indicates 10 µm and applies to all panels.

Figure 3 with 2 supplements
MyoV KD induces the intracellular extension of the apical membrane and disrupts apical secretion.

(A-C) Single optical slices and maximal projection of half of the z-stack (half SG-tube) showing the localization of MyoV in fixed SGs of control (A, fkh>/+), Crb KD (B, fkh >UAS crbRNAi) and βH-Spec KD (C, fkh >UAS kstRNAi) animals. (D, D) Plotted is the intensity (arbitrary units) of MyoV detected by immunofluorescence along the apical-to-basal direction in SGs of control (black, fkh>/+), Crb KD (magenta, fkh >UAS crbRNAi) and βH-Spec (green, fkh >UAS kstRNAi) animals. Error bars indicate the standard error of the mean, n indicates number of glands from the corresponding genotypes. (E-J) Maximal projection of half of the z-stack (half SG-tube) showing the localization of Crb (E,F), Phospho-Moe (G,H) and Sas-YFP in SGs of control (E,G,I, fkh>/+) and MyoV KD (F,H,J, fkh >UAS didumRNAi) animals. (K,L) Localization of SerpCBD-GFP in live SGs of control (K, fkh >UAS SerpCBD-GFP) and MyoV KD (L, fkh >UAS didumRNAi; UAS-SerpCBD-GFP) animals. Arrows point to the apical and dotted lines indicate the basal membrane. Scale bars in (A,E,K) indicate 10 µm. (M, M) Plotted is the fluorescence intensity (arbitrary units) of SerpCBD-GFP along the apical-to-basal direction in live SGs of control (black, fkh >UAS SerpCBD-GFP), and MyoV KD (magenta, fkh >UAS didumRNAi; UAS-SerpCBD-GFP) animals. Error bars indicate the standard error of the mean, n indicates number of glands from the corresponding genotypes.

Figure 3—source data 1

Dataset for MyosinV fluorescence intensity in control glands.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig3-data1-v2.xlsx
Figure 3—source data 2

Dataset for MyosinV fluorescence intensity in Crb KD glands.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig3-data2-v2.xlsx
Figure 3—source data 3

Dataset for MyosinV fluorescence intensity in βH-Spec KD glands.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig3-data3-v2.xlsx
Figure 3—source data 4

Dataset for SerpCBD-GFP fluorescence intensity in control glands.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig3-data4-v2.xlsx
Figure 3—source data 5

Dataset for SerpCBD-GFP fluorescence intensity in MyoV KD glands.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig3-data5-v2.xlsx
Figure 3—figure supplement 1
Proper apical localization of MyoV requires Crb.

(A-C) Localization and quantification of MyoV-GFP fluorescence intensity detected along the apical-to-basal axis in live SGs of control (black, fkh >UAS MyosinV-GFP) and Crb KD (magenta, fkh >UAS crbRNAi, UAS-MyosinV-GFP) animals. Error bars indicate the standard error of the mean, n indicates number of glands from the corresponding genotypes. Dotted lines indicate the basal membrane. Scale bars in (A) indicate 10 µm.

Figure 3—figure supplement 1—source data 1

Dataset for MyosinV-GFP fluorescence intensity in control glands.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig3-figsupp1-data1-v2.xlsx
Figure 3—figure supplement 1—source data 2

Dataset for MyosinV-GFP fluorescence intensity in Crb KD glands.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig3-figsupp1-data2-v2.xlsx
Figure 3—figure supplement 2
βH-Spec is required for proper apical secretion.

(A-C) Localization and quantification of SerpCBD-GFP fluorescence intensity detected along the apical-to-basal axis in live SGs of control (black, fkh >UAS SerpCBD-GFP) and βH-Spec KD (magenta, fkh >UAS kstRNAi, UAS-SerpCBD-GFP) animals. Error bars indicate the standard error of the mean, n indicates number of glands from the corresponding genotypes. Dotted lines indicate the basal membrane. Scale bars in (A) indicate 10 µm.

Figure 3—figure supplement 2—source data 1

Dataset for SerpCBD-GFP fluorescence intensity in control glands.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig3-figsupp2-data1-v2.xlsx
Figure 3—figure supplement 2—source data 2

Dataset for SerpCBD-GFP fluorescence intensity in βH-Spec KD glands.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig3-figsupp2-data2-v2.xlsx
Figure 4 with 3 supplements
Crb organizes the apical Rab machinery in larval SG cells.

(A-H’) Confocal images of SGs to localize endogenously expressed Rab-YFP proteins. Rab6-YFP (A-B’), Rab11-YFP (C-D’), Rab30-YFP (E-F’) and Rab1-YFP (G-H’) in control (A,C,E,G, fkh>/+) and Crb KD (B,D,F,H, fkh >UAS crbRNAi) SGs. Dotted-line squares in A-H indicate the area blown-up to the right of the respective panel (A’-H’). Arrows point to the apical pool of Rab6-YFP (A’), Rab11-YFP (C’) and Rab30-YFP (E’). Arrowheads mark the intracellular vesicular localization of Rab6-YFP (A’,B’) and Rab1-YFP (G’,H’). Scale bar (A) indicates 10 µm. (I, I) Western blot of endogenously expressed Rab-YFP proteins. Rab1-YFP, Rab6-YFP, Rab11-YFP, and Rab30-YFP in control (fkh>/+) and Crb KD (fkh >UAS crbRNAi) SGs, indicated as crbRNAi – or +, respectively. Membranes were probed for tubulin (loading control) and for GFP; arrowheads point to Rab-YFP proteins.

Figure 4—figure supplement 1
Localization of Rab-YFP proteins after KD of Crb in larval SGs.

Shown are images from SGs (optical sections at the level of the gland lumen) stained for GFP (green), DAPI (blue) and Dlg (magenta). Six panels are shown for each Rab-YFP protein indicated, of controls (upper row) and Crb KD (lower row) in the respective Rab-YFP background. Asterisks indicate the SG lumen, scale bars indicate 10 µm. Note no DAPI staining is shown in the panels for Rab35.

Figure 4—figure supplement 2
The apical cytocortex is necessary for the organization of apical Rab6 and Rab11 trafficking machinery.

(A-H) Localization of endogenously expressed Rab-YFP proteins. Rab6-YFP (A,B), Rab11-YFP (C,D), Rab30-YFP (E,F) and Rab1-YFP (G,H) in control (A,C,E,Gfkh>/+) and βH-Spec KD (B,D,F,H, fkh >UAS kstRNAi). Dotted lines indicate basal membrane. Scale bar (A) indicates 10 µm and applies to all panels.

Figure 4—figure supplement 3
Loss of Rab11 in larval SG induces the formation of PAMS.

(A-D’’) Localization of heterologous protein CD8-RFP and endogenously expressed Rab6-YFP (A-B’’) and Rab11-YFP (C-D’’) in control (A,-A’’,C-C’’, fkh >UAS-CD8-RFP) and GFP KD (B-B’’,D-D’’, fkh >UAS gfpRNAi; UAS-CD8-RFP) animals. Arrows point to the PAMS found in Rab11-YFP KD (D’’). Asterisk (D’) marks the lumen. Dotted lines indicate basal membrane. Inserts (B,D) display the corresponding SGs with brightness intensified manually using Photoshop to visualize the SG. Scale bar (A) indicates 10 µm and applies to all panels.

Figure 5 with 2 supplements
Crb organizes the apical secretory machinery by negatively regulating Pten A.

(A) Simplified scheme of PI(4,5)P2 biosynthesis. (B-G) Maximal projection of half of the z-stack (half SG-tube) showing the localization of PI(4,5)P2 (PLCδ-PH-EGFP reporter) in live SGs of control (B, fkh >UAS-PLCδ-PH-EGFP), Crb KD (C, fkh >UAS crbRNAi; UAS-PLCδ-PH-EGFP), Pten KD (D, fkh >UAS ptenRNAi; UAS-PLCδ-PH-EGFP), double KD of Crb and Pten (E, fkh >UAS crbRNAi, UAS-ptenRNAi; UAS-PLCδ-PH-EGFP), Pi3K92E KD (F, fkh >UAS-pi3k92ERNAi; UAS-PLCδ-PH-EGFP) and double KD of Crb and Pi3K92E (G, fkh >UAS crbRNAi, UAS-pi3k92ERNAi; UAS-PLCδ-PH-EGFP) animals. (H, H) Plotted is the fluorescence intensity (arbitrary units) of PLCδ-PH-EGFP along the apical-to-basal axis in live SGs of the genotypes indicated in (B-G), respectively. Error bars indicate the standard error of the mean, n indicates number of glands for the corresponding genotype. (I-K) Localization and quantification of over-expressed Pten2-GFP in SGs of control (I, fkh >UAS-Pten2-GFP) and Crb KD (J, fkh >UAS crbRNAi; UAS-Pten2-GFP) animals. Violin graph (K) indicates the fluorescence intensity (apical vs lateral ratio) indicating the mean and quartiles (n = 28 cells for control and 36 cells for Crb KD). Statistical significance was analyzed in an unpaired two-tailed t-test. (L-N) Localization and quantification of Ocrl-RFP fluorescence intensity detected along the apical-to-basal axis in live SGs of control (black, fkh>/+) and Crb KD (magenta, fkh >UAS crbRNAi) animals. Error bars indicate the standard error of the mean, n indicates number of glands of the corresponding genotypes. (O-Q) Localization and quantification of PLCδ-PH-EGFP fluorescence intensity detected along the apical-to-basal axis in live SGs of control (black, fkh>/+) and Ocrl KD (orange, fkh >UAS ocrlRNAi) animals. Error bars indicate the standard error of the mean, n indicates the number of glands of the corresponding genotypes. Arrows point to the apical membrane domain. Arrowheads point to the lateral membrane. Dotted lines indicate the basal membrane. Scale bars in (B,I,L,O) indicate 10 µm.

Figure 5—source data 1

Dataset for PLCδ-PH-EGFP fluorescence intensity in control glands (corresponding to panel H).

https://cdn.elifesciences.org/articles/50900/elife-50900-fig5-data1-v2.xlsx
Figure 5—source data 2

Dataset for PLCδ-PH-EGFP fluorescence intensity in Crb KD glands (corresponding to panel H).

https://cdn.elifesciences.org/articles/50900/elife-50900-fig5-data2-v2.xlsx
Figure 5—source data 3

Dataset for PLCδ-PH-EGFP fluorescence intensity in Pten KD glands (corresponding to panel H).

https://cdn.elifesciences.org/articles/50900/elife-50900-fig5-data3-v2.xlsx
Figure 5—source data 4

Dataset for PLCδ-PH-EGFP fluorescence intensity in glands with double KD of Crb and Pten (corresponding to panel H).

https://cdn.elifesciences.org/articles/50900/elife-50900-fig5-data4-v2.xlsx
Figure 5—source data 5

Dataset for PLCδ-PH-EGFP fluorescence intensity in Pi3K92E KD glands (corresponding to panel H).

https://cdn.elifesciences.org/articles/50900/elife-50900-fig5-data5-v2.xlsx
Figure 5—source data 6

Dataset for PLCδ-PH-EGFP fluorescence intensity in glands with double KD of Crb and Pi3K92E (corresponding to panel H).

https://cdn.elifesciences.org/articles/50900/elife-50900-fig5-data6-v2.xlsx
Figure 5—source data 7

Dataset for Pten2-GFP fluorescence intensity (corresponding to panel K).

https://cdn.elifesciences.org/articles/50900/elife-50900-fig5-data7-v2.xlsx
Figure 5—source data 8

Dataset for Ocrl-RFP fluorescence intensity in control glands (corresponding to panel N).

https://cdn.elifesciences.org/articles/50900/elife-50900-fig5-data8-v2.xlsx
Figure 5—source data 9

Dataset for Ocrl-RFP fluorescence intensity in Crb KD glands (corresponding to panel N).

https://cdn.elifesciences.org/articles/50900/elife-50900-fig5-data9-v2.xlsx
Figure 5—source data 10

Dataset for PLCδ-PH-EGFP fluorescence intensity in control glands (corresponding to panel Q).

https://cdn.elifesciences.org/articles/50900/elife-50900-fig5-data10-v2.xlsx
Figure 5—source data 11

Dataset for PLCδ-PH-EGFP fluorescence intensity in Ocrl KD glands (corresponding to panel Q).

https://cdn.elifesciences.org/articles/50900/elife-50900-fig5-data11-v2.xlsx
Figure 5—source data 12

Dataset for number of PAMS and diameter of PAMS.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig5-data12-v2.xlsx
Figure 5—figure supplement 1
The Crb protein complex regulates apical levels of PI(4,5)P2 and the secretory activity of SGs.

(A-D) Maximal projection of half of the z-stack (half SG-tube) showing the localization of PI(4,5)P2 (PLCδ-PH-EGFP reporter) in live SGs of control (A, fkh >UAS-PLCδ-PH-EGFP), Sdt KD (B, fkh >UAS sdtRNAi; UAS-PLCδ-PH-EGFP), βH-Spec KD (C, fkh >UAS kstRNAi; UAS-PLCδ-PH-EGFP) and MyoV KD (L, fkh >UAS didumRNAi; UAS-PLCδ-PH-EGFP) animals. (E) Violin graphs indicating the mean and quartiles for the apical surface quantification (area and volume) in live salivary glands of control (A, fkh >UAS-PLCδ-PH-EGFP, n = 33 cells), Crb KD (B, fkh >UAS crbRNAi; UAS-PLCδ-PH-EGFP, n = 25 cells), βH-Spec KD (C, fkh >UAS kstRNAi; UAS-PLCδ-PH-EGFP, n = 29 cells) and MyoV KD (L, fkh >UAS didumRNAi; UAS-PLCδ-PH-EGFP, n = 24 cells) animals. Statistical significance was analyzed in a one-way analysis of variance (ANOVA) followed by a Dunnett’s multiple-comparison test. (F,G) Localization of PLCδ-PH-EGFP is shown in live SGs of late 3rd instar wandering larvae of control (F, fkh >UAS-PLCδ-PH-EGFP) and Crb KD (G, fkh >UAS crbRNAi; UAS-PLCδ-PH-EGFP) animals. (H) Violin graphs of the tube length from live SGs of control (first column, n = 18), Crb KD (second column, n = 20), Pten KD (third column, n = 16), double KD of Crb and Pten (fourth column, n = 19), Pi3K92E KD (fifth column, n = 29), and double KD of Crb and Pi3K92E (sixth column, n = 22). Values for control and Crb KD are the same as shown in Figure 1—figure supplement 1R. (I-M) Localization of PI(4,5)P2 is shown in live SGs from control (I, fkh >UAS-PLCδ-PH-EGFP), Crb KD (J, fkh >UAS crbRNAi; UAS-PLCδ-PH-EGFP), Sktl KD (K, fkh >UAS sktlRNAi; UAS-PLCδ-PH-EGFP) and double KD of Crb and Sktl (L, fkh >UAS sktlRNAi, UAS-crbRNAi; UAS-PLCδ-PH-EGFP) animals. (M) Plotted is the fluorescence intensity (arbitrary units) of PLCδ-PH-EGFP along the apical-to-basal direction in live SGs of the indicated genotypes. Error bars indicate the standard error of the mean, n indicates number of glands from the corresponding genotype. (N-R) Localization of PI(4,5)P2 is shown in live SGs from control (N,P, fkh >UAS-PLCδ-PH-EGFP) and Crb KD (O,Q, fkh >UAS crbRNAi; UAS-PLCδ-PH-EGFP), animals. The SGs were incubated for 30 min in Grace’s medium complemented with DMSO (N,O, Vehicle) or an inhibitor of the lipid phosphatase Pten (P,Q, VO-OHpic 10 µM). (R) Plotted is the fluorescence intensity (arbitrary units) of PLCδ-PH-EGFP along the apical-to-basal axis in live SGs of the indicated genotypes. Error bars indicate the standard error of the mean, n indicates number of glands from the corresponding genotype. Arrows point to the apical membrane domain. Scale bars in (A,F,I,N) indicate 10 µm.

Figure 5—figure supplement 1—source data 1

Dataset for apical surface quantifications.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig5-figsupp1-data1-v2.xlsx
Figure 5—figure supplement 1—source data 2

Dataset for salivary gland lengths.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig5-figsupp1-data2-v2.xlsx
Figure 5—figure supplement 1—source data 3

Dataset for PLCδ-PH-EGFP fluorescence intensity in control glands.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig5-figsupp1-data3-v2.xlsx
Figure 5—figure supplement 1—source data 4

Dataset for PLCδ-PH-EGFP fluorescence intensity in Crb KD glands.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig5-figsupp1-data4-v2.xlsx
Figure 5—figure supplement 1—source data 5

Dataset for PLCδ-PH-EGFP fluorescence intensity in Sktl KD glands.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig5-figsupp1-data5-v2.xlsx
Figure 5—figure supplement 1—source data 6

Dataset for PLCδ-PH-EGFP fluorescence intensity in glands with double KD of Crb and Sktl.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig5-figsupp1-data6-v2.xlsx
Figure 5—figure supplement 1—source data 7

Dataset for PLCδ-PH-EGFP fluorescence intensity in control glands incubated with vehicle.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig5-figsupp1-data7-v2.xlsx
Figure 5—figure supplement 1—source data 8

Dataset for PLCδ-PH-EGFP fluorescence intensity in Crb KD glands incubated with vehicle.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig5-figsupp1-data8-v2.xlsx
Figure 5—figure supplement 1—source data 9

Dataset for PLCδ-PH-EGFP fluorescence intensity in control glands incubated with VO-OHpic.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig5-figsupp1-data9-v2.xlsx
Figure 5—figure supplement 1—source data 10

Dataset for PLCδ-PH-EGFP fluorescence intensity in Crb KD glands incubated with VO-OHpic.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig5-figsupp1-data10-v2.xlsx
Figure 5—figure supplement 2
Pten2 over-expression induces formation of PAMS.

(A–C) Localization of PI(4,5)P2 is shown for live SGs of control (A, fkh >UAS-PLCδ-PH-EGFP), Pten2 over-expression (B, fkh >UAS-Pten2; UAS-PLCδ-PH-EGFP), and Sktl over-expression (C, fkh >UAS Sktl; UAS-PLCδ-PH-EGFP). To avoid saturation, the images of SGs over-expressing Pten2 (B) were acquired at a lower laser power with respect to the control (0.2% vs 0.5%). The insert shows the image taken with the same laser power as the control. (D–F) Single optical section showing the localization of PI(3,4,5)P3 (GPR1-PH-EGFP reporter) in live SGs of control (B, tub::GPR1-PH-EGFP, fkh>/+) and Crb KD (C, tub::GPR1-PH-EGFP, fkh >UAS crbRNAi) animals. Violin graph (F) indicates the fluorescence intensity (apical vs lateral ratio) indicating the mean and quartiles (n = 57 cells control and 53 cells Crb KD). Statistical significance was analyzed in an unpaired two-tailed t-test. Arrows point to the apical membrane domain. Scale bars in (A,D) indicate 10 µm.

Figure 5—figure supplement 2—source data 1

Dataset for GPR1-PH-EGFP fluorescence intensity.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig5-figsupp2-data1-v2.xlsx
Figure 6 with 1 supplement
Control of apical secretion and localization of Rab11 and Rab30 by Crb requires Pten.

(A-F) Maximal projection of 6.7µm through the SG lumen showing the localization of SerpCBD-GFP in live SGs of control (A, fkh >UAS SerpCBD-GFP), Crb KD (B, fkh >UAS crbRNAi; UAS-SerpCBD-GFP), Pten KD (C, fkh >UAS ptenRNAi; UAS-SerpCBD-GFP), double KD of Crb and Pten KD (D, fkh >UAS crbRNAi, UAS-ptenRNAi; UAS-SerpCBD-GFP), Pi3K92E KD (E, fkh >UAS-pi3k92ERNAi; UAS-SerpCBD-GFP), and double KD of Crb and Pi3K92E (F, fkh >UAS crbRNAi, UAS-pi3k92ERNAi; UAS-SerpCBD-GFP), respectively. (H-M) Localization of endogenously expressed Rab11-YFP in live SGs. Shown are control (H, Rab11-YFP, fkh>/+), Crb KD (I, Rab11-YFP, fkh >UAS crbRNAi), Pten KD (J, Rab11-YFP, fkh >UAS ptenRNAi), double KD of Crb and Pten (K, Rab11-YFP, fkh >UAS crbRNAi, UAS-ptenRNAi), Pi3K92E KD (L, Rab11-YFP, fkh >UAS-pi3k92ERNAi), and double KD of Crb and Pi3K92E (M, Rab11-YFP, fkh >UAS crbRNAi, UAS-pi3k92ERNAi) animals, respectively. (O-T) Localization of endogenously expressed Rab30-YFP in live SGs. Shown are control (O, Rab30-YFP, fkh>/+), Crb KD (P, Rab30-YFP, fkh >UAS crbRNAi), Pten KD (Q, Rab30-YFP, fkh >UAS ptenRNAi), double KD of Crb and Pten (R, Rab30-YFP, fkh >UAS crbRNAi, UAS-ptenRNAi), Pi3K92E KD (S, Rab30-YFP, fkh >UAS-pi3k92ERNAi), and double KD of Crb and Pi3K92E (T, Rab30-YFP, fkh >UAS crbRNAi, UAS-pi3k92ERNAi) animals, respectively. Arrows point to the apical, and dotted lines to the basal membrane domain. Scale bar in (A) indicates 10 µm and applies to all panels. (G,N,U) Plotted is the fluorescence intensity (arbitrary units) of SerpCBD-GFP (G), Rab11-YFP (N) and Rab30-YFP (U), respectively, along the apical-to-basal axis in live SGs of the indicated genotypes. Error bars indicate the standard error of the mean, n indicates number of glands of the corresponding genotypes. (V) Violin graph of estimated food intake in control (first column), Crb KD (second column), Pten KD (third column), double KD of Crb and Pten (fourth column), Pi3K92E KD (fifth column), and double KD of Crb and Pi3K92E (sixth column) larvae. The dotted line indicates the mean value of the control. 60 larvae of the corresponding genotype were pooled in each biological replica. 10 biological replicas were analyzed distributed in three independent experiments. Statistical significance was tested in a one-way analysis of variance (ANOVA) followed by a Dunnett’s multiple-comparison test. (W) Pupariation efficiency of control (black, fkh>/+), Crb KD (magenta, fkh >UAS crbRNAi), Pten KD (green, fkh >UAS ptenRNAi), double KD of Crb and Pten KD (yellow, fkh >UAS crbRNAi, UAS-ptenRNAi), Pi3K92E KD (blue, fkh >UAS-pi3k92ERNAi), and double KD of Crb and Pi3K92E (, fkh >UAS crbRNAi, UAS-pi3k92ERNAi) animals. Error bars indicate the standard error of the mean, n indicates number of traced individual larvae of the corresponding genotypes in at least 15 independent experiments.

Figure 6—source data 1

Dataset for SerpCBD-GFP fluorescence intensity in control glands.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data1-v2.xlsx
Figure 6—source data 2

Dataset for SerpCBD-GFP fluorescence intensity in Crb KD glands.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data2-v2.xlsx
Figure 6—source data 3

Dataset for SerpCBD-GFP fluorescence intensity in Pten KD glands.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data3-v2.xlsx
Figure 6—source data 4

Dataset for SerpCBD-GFP fluorescence intensity in glands with double KD of Crb and Pten.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data4-v2.xlsx
Figure 6—source data 5

Dataset for SerpCBD-GFP fluorescence intensity in Pi3K92E KD glands.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data5-v2.xlsx
Figure 6—source data 6

Dataset for SerpCBD-GFP fluorescence intensity in glands with double KD of Crb and Pi3K92E.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data6-v2.xlsx
Figure 6—source data 7

Dataset for Rab11-YFP fluorescence intensity in control glands.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data7-v2.xlsx
Figure 6—source data 8

Dataset for Rab11-YFP fluorescence intensity in Crb KD glands.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data8-v2.xlsx
Figure 6—source data 9

Dataset for Rab11-YFP fluorescence intensity in Pten KD glands.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data9-v2.xlsx
Figure 6—source data 10

Dataset for Rab11-YFP fluorescence intensity in glands with double KD of Crb and Pten.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data10-v2.xlsx
Figure 6—source data 11

Dataset for Rab11-YFP fluorescence intensity in Pi3K92E KD glands.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data11-v2.xlsx
Figure 6—source data 12

Dataset for Rab11-YFP fluorescence intensity in glands with double KD of Crb and Pi3K92E.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data12-v2.xlsx
Figure 6—source data 13

Dataset for Rab30-YFP fluorescence intensity in control glands.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data13-v2.xlsx
Figure 6—source data 14

Dataset for Rab30-YFP fluorescence intensity in Crb KD glands.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data14-v2.xlsx
Figure 6—source data 15

Dataset for Rab30-YFP fluorescence intensity in Pten KD glands.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data15-v2.xlsx
Figure 6—source data 16

Dataset for Rab30-YFP fluorescence intensity in glands with double KD of Crb and Pten.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data16-v2.xlsx
Figure 6—source data 17

Dataset for Rab30-YFP fluorescence intensity in Pi3K92E KD glands.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data17-v2.xlsx
Figure 6—source data 18

Dataset for Rab30-YFP fluorescence intensity in glands with double KD of Crb and Pi3K92E.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data18-v2.xlsx
Figure 6—source data 19

Dataset for food intake estimations.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data19-v2.xlsx
Figure 6—source data 20

Dataset for tracking of larval development.

https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data20-v2.xlsx
Figure 6—figure supplement 1
Overexpression of Pten leads to loss of Rab11 and Rab30 from the apical domain.

(A,B) Localization of endogenously expressed Rab11-YFP in live SGs of control (A, Rab11-YFP, fkh>/+) and Pten OE (B, Rab11-YFP, fkh >UAS-Pten2) animals. (C,D) Localization of endogenously expressed Rab30-YFP in live SGs of control (C, Rab30-YFP, fkh>/+) and Pten OE (D, Rab30-YFP, fkh >UAS-Pten2) animals. Arrows point to the apical membrane domain. Scale bar (A) indicates 10 µm and applies to all panels.

Crb-dependent regulation of apical secretion in SG cells Schematic representation of Crb-dependent regulation of apical secretion in SG cells.

Under physiological conditions (left image), Crb mediates the apical localization of Moesin and βH-Spec, which link the Crb protein (blue) to the apical F-actin cytoskeleton (black ribbon). This Crb-cytocortex complex is necessary for organization of the apical Rab-dependent traffic machinery (depicted as Rab vesicles in yellow). Under these conditions Crb negatively regulates the activity of Pten via βH-Spec and MyoV. The precise molecular interactions involved in the negative regulation of Pten are not defined (see Discussion for details). The absence of Crb in the SG cells disrupts the efficient apical secretion (right image). The defects in apical secretion are a consequence of the disruption of the apical cytocortex (actin, βH-Spec), the loss of MyoV and the excessive production of PI(4,5)P2 (red dots) which require the activity of Pten. The loss of Ocrl form the apical membrane could also contribute to the increase in PI(4,5)P2 apical levels. Another consequence is the formation of a novel apical membrane sac enriched in PI(4,5)P2 (PAMS), Moe (green rectangles) and apical transmembrane proteins (not depicted).

Schematic representation of the experimental setup.

Indicated in the workflow are the times and incubation temperatures, as well as the time for dissections.

Author response image 1
Author response image 2
Author response image 3
Author response image 4
Author response image 5
Author response image 6
Author response image 7

Videos

Video 1
Fusion of a glue vesicle followed by expulsion of the cargo Sgs3-GFP into the lumen SG lumen of control (fkh>+, top) and Crb KD (fkh >UAS crbRNAi, bottom) animals.
Video 2
Overview showing the fusion of glue vesicles followed by expulsion of Sgs3-GFP into the SG lumen of control (fkh>+, top) and Crb KD (fkh >UAS crbRNAi, bottom) animals.

Note that the increase of fluorescence in the vesicle occurs when they open to the lumen.

Video 3
3D rendering of a SG from a Crb KD animal (fkh >UAS crbRNAi) probed for phospho-Moesin.

The extraction focuses on one cell to appreciate the accumulation of phospho-Moesin at the apical membrane. Apical is up.

Video 4
Live imaging of endogenously expressed Rab6-YFP in SGs of control (left, Rab6-YFP, fkh>/+) and Crb KD (right, Rab6-YFP, fkh >UAS crbRNAi).

5 min recording, time lapse 5 s.

Video 5
Live imaging of endogenously expressed Rab11-YFP in SGs of control (left, Rab11-YFP, fkh>/+) and Crb KD (right, Rab11-YFP, fkh >UAS crbRNAi).

5 min recording, time lapse 5 s.

Video 6
Live imaging of endogenously expressed Rab30-YFP in SGs of control (left, Rab30-YFP, fkh>/+) and Crb KD (right, Rab30-YFP, fkh >UAS crbRNAi).

5 min recording, time lapse 5 s.

Video 7
Live imaging of endogenously expressed Rab1-YFP in SGs of control (left, Rab1-YFP, fkh>/+) and Crb KD (right, Rab1-YFP, fkh >UAS crbRNAi).

5 min recording, time lapse 5 s.

Video 8
3D rendering of a fixed SG of a Crb KD animal expressing the PI(4,5)P2 reporter PLCδ-PH-EGFP (green) and stained for phospho-Moesin (magenta).

It is possible to appreciate the phospho-Moe and PI(4,5)P2-enriched apical membrane sac (PAMS) below the apical membrane. Scale bar indicates 5 µm.

Video 9
Live imaging of a SG of a Crb KD animal expressing the PI(4,5)P2 reporter PLCδ-PH-EGFP (fkh >UAS crbRNAi; UAS-PLCδ-PH-EGFP).

A single optical section is shown on the left. On the right, the maximal projection of the stack showing the whole PI(4,5)P2-enriched apical membrane sac (PAMS). The arrowhead appearing at 660 s on the right panel points to an apparent opening of the sac to the lumen. It is worth noting that the PAMS are very stationary, as the movie shows 20 min recording, time lapse 20 s. Apical is up.

Tables

Table 1
List of fly stocks used in this study.
DesignationGenotype (as reported in FlyBase when available)Description
Balancerw[1118]; In(2LR)Gla, wg[Gla-1]/CyO, P{w[+mC]=GAL4 twi.G}2.2, P{w[+mC]=UAS-2xEGFP}AH2.2Balancer for 2nd chromosome; BSC 6662
Balancerw[1118]; Dr[Mio]/TM3, P{w[+mC]=GAL4 twi.G}2.3, P{UAS-2xEGFP}AH2.3, Sb[1] Ser[1]Balancer for 3rd chromosome; BSC 6663
Balancerw[*]; ry[506] Dr[1]/TM6B, P{w[+mC]=Dfd-EYFP}3, Sb[1] Tb[1] ca[1]Balancer for 3rd chromosome; BSC 8704
crbRNAiw[1118]; P{GD14463}v39177Expresses the RNAi against crb under the control of UAS sequences; VDRC 39177
sdtRNAiw[1118]; P{GD9163}v23822Expresses the RNAi against sdt under the control of UAS sequences; VDRC 23822
sdtRNAiy[1] sc[*] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.HMS01652}attP40Expresses dsRNA for RNAi of sdt (FBgn0261873) under UAS control. BSC 37510
gfpRNAiy[1] sc[*] v[1]; P{y[+t7.7] v[+t1.8]=VALIUM20 EGFP.shRNA.3}attP40Expresses small hairpin RNA under the control of UAS for RNAi of EGFP and EYFP as well as fusion proteins containing these fluors, BSC 41559
gfpRNAiy[1] sc[*] v[1]; P{y[+t7.7] v[+t1.8]=VALIUM20 EGFP.shRNA.3}attP2Expresses small hairpin RNA under the control of UAS for RNAi of EGFP and EYFP as well as fusion proteins containing these fluors, BSC 41560
moeRNAiw[1118]; P{GD5211}v37917Expresses the RNAi against moe under the control of UAS sequences; VDRC 37917
 kstRNAiy[1] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.GLC01654}attP40Expresses dsRNA for RNAi of kst (FBgn0004167) under UAS control, BSC 50536
 ocrlRNAiy[1] sc[*] v[1] sev[21]; P{y[+t7.7] v[+t1.8]=TRiP.HMS01201}attP2/TM3, Sb[1]Expresses dsRNA for RNAi of Ocrl (FBgn0023508) under UAS control in the VALIUM20 vector. BSC 34722
 GAL80tsw[*]; P{w[+mC]=tubP-GAL80[ts]}7Expresses temperature-sensitive GAL80 under the control of the alphaTub84B promoter; outcrossed from BSC 7018
Dicerw[1118]; P{w[+mC]=UAS-Dcr-2.D}2Expresses Dicer-2 under UAS control, BSC 24650
myoVRNAiy[1] sc[*] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.HMC03900}attP40Expresses dsRNA for RNAi of didum (FBgn0261397) under UAS control; BSC 55740
ptenRNAiy[1] w[1118]; P{w[+mC]=UAS Pten.dsRNA.Exel}2Expresses a snapback transcript for RNAi of Pten under the control of UAS. BSC 8549
ptenRNAiw[1118]; P{w[+mC]=UAS Pten.dsRNA.Exel}3Expresses a snapback transcript for RNAi of Pten under the control of UAS. BSC 8550
pi3k92ERNAiy[1] sc[*] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.HMC05152}attP40Expresses dsRNA for RNAi of Pi3K92E (FBgn0015279) under UAS control. BSC 61182
pi3k92ERNAiy[1] sc[*] v[1]; P{y[+t7.7] v[+t1.8]=TRiP .GL00311}attP2Expresses dsRNA for RNAi of Pi3K92E (FBgn0015279) under UAS control. BSC 35798
sktlRNAiy[1] sc[*] v[1]; P{y[+t7.7] v[+t1.8]=TRiP .GL00072}attP2Expresses dsRNA for RNAi of sktl (FBgn0016984) under UAS control. BSC 35198
SerpCBD-GFPw[*];; UAS-SerpCBD-GFPExpresses the N-terminus of Serp including the signal peptide and chitin binding domain (CBD) fused to GFP (Luschnig et al., 2006), kindly provided by S. Luschning
MyosinV-GFPw[*];; UAS-didum-GFPExpresses full length didum (amino acids 1–1792) tagged at the C-terminal end with EGFP (Krauss et al., 2009), kindly provided by A. Ephrussi
Sas-Venusw[*];; tub::Sas-VenusStranded at Second fused with Venus under tubulin promoter on 3rd chromosome (Firmino et al., 2013)
PNA-GFPw[*]; M{w[+mC]=UAS PNA.GFP}ZH-86FbExpresses GFP-tagged peanut agglutinin under UAS control. BSC 55247
CD8-RFPw[*]; P{y[+t7.7] w[+mC]=10XUAS-IVS-mCD8::RFP}attP2Expresses mCD8-tagged RFP under the control of 10 UAS sequences. BSC 32218
PI(4,5)P2 sensory[1] w[*]; P{w[+mC]=UAS-PLCdelta-PH-EGFP}3Expresses GFP-tagged pleckstrin homology domain from human PLCδ. BSC 39693
PI(3,4,5)P3 sensorw[*];; tub::GPR1-PH-EGFPExpresses GFP-tagged pleckstrin homology domain from cytohesin/GRP1 (Pinal et al., 2006), kindly provided by F. Pichaud
Pten2-GFPw[*]; UAS-Pten2-GFPExpresses Pten2 isoform GFP-tagged under the control of UAS sequences (Pinal et al., 2006), kindly provided by F. Pichaud
Pten2w[*]; UAS-Pten2Expresses the Pten2 isoform under the control of UAS sequences (von Stein et al., 2005), kindly provided by A. Wodarz
fkhGAL4w[*]; fkh-GAL4On 3rd chromosome, expresses GAL4 under the control of the fkh promoter (Henderson and Andrew, 2000), kindly provided by K. Röpper
Fas3-GFPw[*]; P{w[+mC]=PTT-GA}Fas3[G00258]Fas3 fused with GFP protein trap. BSC 50841
DE-cad-GFPw*;DE-cad::GFPDE-cadherin fused with GFP knock-in allele; homozygous viable (Huang et al., 2009), kindly provided by Y. Hong
DE-cad-mTomatow*;DE-cad::mTomatoDE-cadherin fused with mTomato knock-in allele; homozygous viable (Huang et al., 2009), kindly provided by Y. Hong
Crb-GFPw*;;crb::GFP-ACrumbs fused with GFP knock-in allele; homozygous viable (Huang et al., 2009), kindly provided by Y. Hong
Lac-GFPw*; lac::GFPProtein trap line: lachesin fused with GFP under endogenous promoter on 2nd chromosome; homozygous viable (kindly provided by the Klämbt Protein trap consortium)
Nrv2-GFPw*; nrv2::GFPProtein trap line: nervana2 fused with GFP under endogenous promoter on 2nd chromosome; homozygous viable (kindly provided by the Klämbt Protein trap consortium)
Ocrl-RFPTI{T-STEP.TagRFP-T}Ocrl[KI] w[*]A T-STEP cassette was knocked into Ocrl to tag the endogenous protein with TagRFP-T. BSC 66529
Dlg-mTagRFPDlg-mTagRFPOn X chromosome, expresses Dlg-mTagRFP under the control of a ubiquitous promoter (Pinheiro et al., 2017), kindly provided by Y. Bellaïche
Rab-YFPRab-YFPendogenously YFP::tagged Rab protein library generated in Dunst et al. (2015)
BSC - Bloomington Drosophila stock Center
  1. VDRC - Vienna Drosophila Resource Center.

Table 2
List of detailed genotypes analyzed in each figure.
Figure 1
B,B'w*; UAS-crb[RNAi]/+
C,C'w*; UAS-crb[RNAi]/+; fkh-GAL4/+
Dw*; Rab30-YFP, UAS-crb[RNAi]/+
Ew*; Rab30-YFP, UAS-crb[RNAi]/+; fkh-GAL4/+
Fw*; UAS-crb[RNAi]/+; Rab11-YFP/+
Gw*; UAS-crb[RNAi]/+; Rab11-YFP/fkh-GAL4
Iw*;; fkhGAL4, ubiGAL80[ts]
Jw*; UAS-crb[RNAi]; fkhGAL4, ubiGAL80[ts]
Kw*;; fkhGAL4, UAS-SerpCBD-GFP-GFP/+
Lw*; UAS-crb[RNAi]/+; fkhGAL4, UAS-SerpCBD-GFP-GFP/+
Figure 1—figure supplement 1
A,C,E,G,I,Kw*; UAS-crb[RNAi]/+
B,D,F,H,J,Lw*; UAS-crb[RNAi]/+; fkh-GAL4/+
Mw*;; UAS-CD8-RFP/fkhGAL4
Nw*; UAS-crb[RNAi]/+; UAS-CD8-RFP/fkhGAL4
Ow*;; UAS-PNA-GFP/fkhGAL4 ubiGAL80[ts]
Pw*;; UAS-crb[RNAi]/+; UAS-PNA-GFP/fkhGAL4 ubiGAL80[ts]
Q: Controlw*; UAS-crb[RNAi]/+; Rab11-YFP/+
Q: Crb KDw*; UAS-crb[RNAi]/+; Rab11-YFP/fkh-GAL4
S,U,U’,W,Yw*;; UAS-std[RNAi]/+
T,V,V’,X,Zw*; UAS-sdt[RNAi]/fkh-GAL4
AAw*;; Rab11-YFP, UAS-sdt[RNAi]/Rab11-YFP
BBw*;; Rab11-YFP, UAS-sdtRNAi/Rab11-YFP, fkhGAL4
CCw*;; fkhGAL4, ubiGAL80[ts]/+
DDw*; UAS-sdt[RNAi]; fkhGAL4, ubiGAL80[ts]/+
EEw*;; fkhGAL4, UAS-CD8-RFP/+
FFw*;; fkhGAL4, UAS-CD8-RFP/UAS-sdt[RNAi]
GGw*;; fkhGAL4, UAS-SerpCBD-GFP/+
HHw*;; fkhGAL4, UAS-SerpCBD-GFP/UAS-sdt[RNAi]
IIw*;; fkhGAL4, UAS-PNA-GFP/+
JJw*;; fkhGAL4, UAS-PNA-GFP/UAS-sdt[RNAi]
Figure 1—figure supplement 2
A,A',Bw*; UAS-crb[RNAi]/+; Rab11-YFP/+
C,C’,D'w*; UAS-crb[RNAi]/+; Rab11-YFP/fkh-GAL4
E,G,Iw*; UAS-crb[RNAi]/+
F,H,Jw*; UAS-crb[RNAi]/+; fkh-GAL4/+
K,K'w*; Fas3-GFP/Fas3-GFP; fkhGAL4/+
L,L'w*; Fas3-GFP/Fas3-GFP, UAS-crb[RNAi]; fkhGAL4/+
M,M'w*; Fas3-GFP/Fas3-GFP; fkhGAL4/UAS-gfp[RNAi]
Figure 2
A,Dw*; UAS-crb[RNAi]/+
B,Ew*; UAS-crb[RNAi]/+; fkh-GAL4/+
Gw*; UAS-gfp[RNAi]/+; crb-GFP-A/crb-GFP-A
Hw*; UAS-gfp[RNAi]/+; crb-GFP-A/crb-GFP-A, fkh-GAL4
Iw*;; fkhGAL4, ubiGAL80[ts]/tub::Sas-Venus
Jw*; UAS-crb[RNAi]; fkhGAL4, ubiGAL80[ts]/tub::Sas-Venus
K,K'w*;; fkhGAL4, UAS-PLCdelta-PH-EGFP/+
L,L'w*; UAS-crb[RNAi]/+; fkhGAL4, UAS-PLCdelta-PH-EGFP/+
Figure 2—figure supplement 1
A,C,Ew*; UAS-gfp[RNAi]/+; crb-GFP-A/crb-GFP-A
B,D,Fw*; UAS-gfp[RNAi]/+; crb-GFP-A/crb-GFP-A, fkh-GAL4
G,Iw*; UAS-crb[RNAi]/+
H,Jw*; UAS-crb[RNAi]/+; fkh-GAL4/+
Figure 2—figure supplement 2
A-C'w*; UAS-crb[RNAi]/+; fkhGAL4, UAS-PLCdelta-PH-EGFP/+
Figure 2—figure supplement 3
Aw*; UAS-crb[RNAi]/+
Bw*; UAS-crb[RNAi]/+; fkh-GAL4/+
Cw*; UAS-sdt[RNAi]/+
Dw*; UAS-sdt[RNAi]/+; fkh-GAL4/+
Figure 2—figure supplement 4
A,C,Ew*;; fkhGAL4, ubiGAL80[ts]
B,D,Fw*; UAS-kst[RNAi]; fkhGAL4, ubiGAL80[ts]
Figure 3
Aw*;; fkhGAL4, ubiGAL80[ts]
Bw*; UAS-crb[RNAi]; fkhGAL4, ubiGAL80[ts]
Cw*; UAS-kst[RNAi]; fkhGAL4, ubiGAL80[ts]
E,Gw*;; fkhGAL4, ubiGAL80[ts]/+
F,Hw*; UAS-didum[RNAi]/+; fkhGAL4, ubiGAL80[ts]/+
Iw*;; fkhGAL4, ubiGAL80[ts]/tub::Sas-Venus
Jw*; UAS-didum[RNAi]/+; fkhGAL4, ubiGAL80[ts]/tub::Sas-Venus
Kw*;; fkhGAL4, UAS-SerpCBD-GFP/+
Lw*; UAS-didum[RNAi]/+; fkhGAL4, UAS-SerpCBD-GFP/+
Figure 3—figure supplement 1
Aw*;; fkhGAL4, ubiGAL80[ts]/UAS-MyoV-GFP
Bw*; UAS-crb[RNAi]/+; fkhGAL4, ubiGAL80[ts]/UAS-MyoV-GFP
Figure 3—figure supplement 2
Aw*;; fkhGAL4, ubiGAL80[ts]/UAS-SerpCBD-GFP
Bw*; UAS-kst[RNAi]; fkhGAL4, ubiGAL80[ts]/UAS-SerpCBD-GFP
Figure 4
Aw*; Rab6-YFP, UAS-crb[RNAi]/+
Bw*; Rab6-YFP, UAS-crb[RNAi]/+; fkh-GAL4/+
Cw*;; Rab11-YFP, fkhGAL4, ubiGAL80[ts]/Rab11-YFP
Dw*; UAS-crb[RNAi]/+; Rab11-YFP, fkhGAL4, ubiGAL80[ts]/Rab11-YFP
Ew*; Rab30-YFP/Rab30-YFP; fkhGAL4, ubiGAL80[ts]/+
Fw*; UAS-crb[RNAi], Rab30-YFP/Rab30-YFP; fkhGAL4, ubiGAL80[ts]/+
Gw*; UAS-crb[RNAi]/+; Rab1-YFP/+
Hw*; UAS-crb[RNAi]/+; Rab1-YFP/fkh-GAL4
I: Rab1 Controlw*; UAS-crb[RNAi]/+; Rab1-YFP/+
I: Rab1 Crb KDw*; UAS-crb[RNAi]/+; Rab1-YFP/fkh-GAL4
I: Rab6 Controlw*; Rab6-YFP, UAS-crb[RNAi]/+
I: Rab6 Crb KDw*; Rab6-YFP, UAS-crb[RNAi]/+; fkh-GAL4/+
I: Rab11 Controlw*; UAS-crb[RNAi]/+; Rab11-YFP/+
I: Rab11 Crb KDw*; UAS-crb[RNAi]/+; Rab11-YFP/fkh-GAL4
I: Rab30 Controlw*; UAS-crb[RNAi], Rab30-YFP/+;
I: Rab30 Crb KDw*; UAS-crb[RNAi], Rab30-YFP/+; fkhGAL4/+
Figure 4—figure supplement 1
Rab1 Controlw*;; Rab1-YFP/fkhGAL4
Rab1 Crb KDw*;UAS-crb[RNAi]/+; Rab1-YFP/fkhGAL4
Rab2 Controlw*; Rab2-YFP/+; fkhGAL4/+
Rab2 Crb KDw*; Rab2-YFP, UAS-crb[RNAi]/+; fkhGAL4/+
Rab4 Controlw*; Rab4-YFP/+; fkhGAL4/+
Rab4 Crb KDw*; Rab4-YFP, UAS-crb[RNAi]/+; fkhGAL4/+
Rab5 Controlw*; Rab5-YFP/+; fkhGAL4/+
Rab5 Crb KDw*; Rab5-YFP, UAS-crb[RNAi]/+; fkhGAL4/+
Rab6 Controlw*; Rab6-YFP/+; fkhGAL4/+
Rab6 Crb KDw*; Rab6-YFP, UAS-crb[RNAi]/+; fkhGAL4/+
Rab7 Controlw*;; Rab7-YFP/fkhGAL4
Rab7 Crb KDw*;UAS-crb[RNAi]/+; Rab7-YFP/fkhGAL4
Rab8 Controlw*;; Rab8-YFP/fkhGAL4
Rab8 Crb KDw*;UAS-crb[RNAi]/+; Rab8-YFP/fkhGAL4
Rab10 Controlw* Rab10-YFP/+;; fkhGAL4/+
Rab10 Crb KDw* Rab10-YFP/+; UAS-crb[RNAi]/+; fkhGAL4/+
Rab11 Controlw*;; Rab11-YFP/fkhGAL4
Rab11 Crb KDw*;UAS-crb[RNAi]/+; Rab11-YFP/fkhGAL4
Rab18 Controlw* Rab18-YFP/+;; fkhGAL4/+
Rab18 Crb KDw* Rab18-YFP/+; UAS-crb[RNAi]/+; fkhGAL4/+
Rab21 Controlw* Rab21-YFP/+;; fkhGAL4/+
Rab21 Crb KDw* Rab21-YFP/+; UAS-crb[RNAi]/+; fkhGAL4/+
Rab35 Controlw* Rab35-YFP/+;; fkhGAL4/+
Rab35 Crb KDw* Rab35-YFP/+; UAS-crb[RNAi]/+; fkhGAL4/+
Rab39 Controlw* Rab39-YFP/+;; fkhGAL4/+
Rab39 Crb KDw* Rab39-YFP/+; UAS-crb[RNAi]/+; fkhGAL4/+
Rab40 Controlw* Rab40-YFP/+;; fkhGAL4/+
Rab40 Crb KDw* Rab40-YFP/+; UAS-crb[RNAi]/+; fkhGAL4/+
Figure 4—figure supplement 2
Aw*; Rab6-YFP/Rab6-YFP; fhkGAL4/+
Bw*; Rab6-YFP, UAS-kst[RNAi]/Rab6-YFP; fhkGAL4/+
Cw*;; Rab11-YFP, fkhGAL4, ubiGAL80[ts]/Rab11-YFP
Dw*; UAS-kst[RNAi]/+; Rab11-YFP, fkhGAL4, ubiGAL80[ts]/Rab11-YFP
Ew*; Rab30-YFP/Rab30-YFP; fhkGAL4/+
Fw*; Rab30-YFP, UAS-kst[RNAi]/Rab30-YFP; fhkGAL4/+
Gw*;; Rab1-YFP/fkhGAL4, ubiGAL80[ts]
Hw*; UAS-kst[RNAi]/+; Rab1-YFP/fkhGAL4, ubiGAL80[ts]
Figure 4—figure supplement 3
A-A’’w*; Rab6-YFP/Rab6-YFP; fhkGAL4, UAS-CD8-RFP/+
B-B’’w*; Rab6-YFP/Rab6-YFP, UAS-gfp[RNAi]; fhkGAL4, UAS-CD8-RFP/+
C-C’’w*;; Rab11-YFP, fkhGAL4, UAS-CD8-RFP/Rab11-YFP
D-D’’w*; UAS-gfp[RNAi]/+; Rab11-YFP, fkhGAL4, UAS-CD8-RFP/Rab11-YFP
Figure 5
Bw*;; fkhGAL4, UAS-PLCdelta-PH-EGFP/+
Cw*; UAS-crb[RNAi]/+; fkhGAL4, UAS-PLCdelta-PH-EGFP/+
Dw*;; fkhGAL4, UAS-PLCdelta-PH-EGFP/UAS-pten[RNAi]
Ew*; UAS-crb[RNAi]/+; fkhGAL4, UAS-PLCdelta-PH-EGFP/UAS-pten[RNAi]
Fw*;; fkhGAL4, UAS-PLCdelta-PH-EGFP/UAS-pi3k92E[RNAi]
Gw*; UAS-crb[RNAi]/+; fkhGAL4, UAS-PLCdelta-PH-EGFP/UAS-pi3k92E[RNAi]
Iw*;; UAS-pten2-GFP/fkh-GAL4, ubiGAL80[ts]
Jw*; UAS-crb[RNAi]/+; UAS-pten2-GFP/fkh-GAL4, ubiGAL80[ts]
LOcrl-RFP, w*/+;; fkh-GAL4, ubiGAL80[ts]/+
MOcrl-RFP, w*; UAS-crb[RNAi]/+; fkh-GAL4, ubiGAL80[ts]/+
Ow*;; fkhGAL4, UAS-PLCdelta-PH-EGFP/+
Pw*;; fkhGAL4, UAS-PLCdelta-PH-EGFP/UAS-ocrl[RNAi]
Figure 5—figure supplement 1
Aw*; DE-cad-mTomato/+; UAS-PLCdelta-PH-EGFP/fkhGAL4, ubiGAL80[ts]
Bw*;; fkhGAL4, UAS-PLCdelta-PH-EGFP/UAS-sdt[RNAi]
Cw*; UAS-kst[RNAi]/DE-cad-mTomato; fkhGAL4, ubiGAL80[ts]/UAS-PLCdelta-PH-EGFP
Dw*; UAS-didum[RNAi]/+; fkhGAL4 UAS-PLCdelta-PH-EGFP/+
Fw*;; UAS-PLCdelta-PH-EGFP/fkh-GAL4, ubiGAL80[ts]
Gw*; UAS-crb[RNAi]/+; UAS-PLCdelta-PH-EGFP/fkh-GAL4, ubiGAL80[ts]
Iw*;; fkhGAL4, UAS-PLCdelta-PH-EGFP/+
Jw*; UAS-crb[RNAi]/+; fkhGAL4, UAS-PLCdelta-PH-EGFP/+
Kw*;; fkhGAL4, UAS-PLCdelta-PH-EGFP/UAS-sktl[RNAi]
Lw*; UAS-crb[RNAi]/+; fkhGAL4, UAS-PLCdelta-PH-EGFP/UAS-sktl[RNAi]
N,Pw*;; fkhGAL4, ubiGAL80[ts]/UAS-PLCdelta-PH-EGFP
O,Qw*; UAS-crb[RNAi]/+; fkhGAL4, ubiGAL80[ts]/UAS-PLCdelta-PH-EGFP
Figure 5—figure supplement 2
Aw*;; fkhGAL4, UAS-PLCdelta-PH-EGFP/+
Bw*;; fkhGAL4, UAS-PLCdelta-PH-EGFP/UAS-pten2
CUAS-Sktl w*/+;; fkhGAL4, UAS-PLCdelta-PH-EGFP/+
Dw*;; fkhGAL4, ubiGAL80[ts]/tub::GPR1-PH-EGFP
Ew*; UAS-crb[RNAi]/+; fkhGAL4, ubiGAL80[ts]/tub::GPR1-PH-EGFP
Figure 6
Aw*;; fkhGAL4, UAS-SerpCBD-GFP/+
Bw*; UAS-crb[RNAi]/+; fkhGAL4, UAS-SerpCBD-GFP/+
Cw*;; fkhGAL4, UAS-SerpCBD-GFP/UAS-pten[RNAi]
Dw*; UAS-crb[RNAi]/+; fkhGAL4, UAS-SerpCBD-GFP/UAS-pten[RNAi]
Ew*;; fkhGAL4, UAS-SerpCBD-GFP/UAS-pi3k92E[RNAi]
Fw*; UAS-crb[RNAi]/+; fkhGAL4, UAS-SerpCBD-GFP/UAS-pi3k92E[RNAi]
Hw*;; Rab11-YFP, fkhGAL4, ubiGAL80[ts]/Rab11-YFP
Iw*; UAS-crb[RNAi]/+; Rab11-YFP, fkhGAL4, ubiGAL80[ts]/Rab11-YFP
Jw*; UAS-pten[RNAi]/+; Rab11-YFP, fkhGAL4, ubiGAL80[ts]/Rab11-YFP
Kw*; UAS-crb[RNAi]/UAS-pten[RNAi]; Rab11-YFP, fkhGAL4, ubiGAL80[ts]/Rab11-YFP
Lw*; UAS-pi3k92E[RNAi]/+; Rab11-YFP, fkhGAL4, ubiGAL80[ts]/Rab11-YFP
Mw*; UAS-crb[RNAi]/UAS-pi3k92E[RNAi]; Rab11-YFP, fkhGAL4, ubiGAL80[ts]/Rab11-YFP
Ow*; Rab30-YFP/Rab30-YFP; fkhGAL4, ubiGAL80[ts]/+
Pw*; UAS-crb[RNAi], Rab30-YFP/Rab30-YFP; fkhGAL4, ubiGAL80[ts]/+
Qw*; Rab30-YFP/Rab30-YFP; fkhGAL4, ubiGAL80[ts]/UAS-pten[RNAi]
Rw*; UAS-crb[RNAi], Rab30-YFP/Rab30-YFP; fkhGAL4, ubiGAL80[ts]/UAS-pten[RNAi]
Sw*; Rab30-YFP/Rab30-YFP; fkhGAL4, ubiGAL80[ts]/UAS-pi3k92E[RNAi]
Tw*; UAS-crb[RNAi], Rab30-YFP/Rab30-YFP; fkhGAL4, ubiGAL80[ts]/UAS-pi3k92E[RNAi]
Figure 6—figure supplement 1
Aw*;; Rab11-YFP, fkhGAL4, ubiGAL80[ts]/Rab11-YFP
Bw*;; Rab11-YFP, fkhGAL4, ubiGAL80[ts]/Rab11-YFP, UAS-pten2
Cw*; Rab30-YFP/Rab30-YFP; fkhGAL4, ubiGAL80[ts]/+
Dw*; Rab30-YFP/Rab30-YFP; fkhGAL4, ubiGAL80[ts]/UAS-pten2
Table 3
List of antibodies and probes employed.
DilutionFixationSource
DAPI1:200000FAInvitrogen Cat. D1306
Phalloidin Alexa Flour 488, 5551:2000FAInvitrogen Cat. A12379, A34055
Alexa Flour 488-, 568- and 647 -conjugated1:1000 - 1:2000Invitrogen
Mouse antibodies
Anti-α-Spectrin1:100MeOHDSHB 3A9
Anti-Coracle1:200MeOHDSHB C566.9
Anti-Disc large1:500MeOHDSHB 4F3
Anti-FasIII1:4MeOHDSHB 7G10
Anti-αTubulin1:2000MeOH/AcetoneMPI-CBG Antibody facility, P. Keller
Rabbit antibodies
Anti-aPKC (C-20)1:500MeOHSanta Cruz Biotechnology Cat. sc-216-G
Anti-Bazooka1:200MeOHkindly provided by A. Wodarz (Wodarz et al., 1999)
Anti-Stardust1:2000MeOH(Berger et al., 2007)
Anti-Cadherin99C1:250FAkindly provided by D. Godt (Glowinski et al., 2014)
Anti-GFP1:1000FAInvitrogen A-11122
Anti-Sinuous1:8000MeOHkindly provided by G.J. Beitel (Wu et al., 2004)
Anti-βHSpectrin1:5000MeOHkindly provided by G. Thomas (Thomas and Williams, 1999)
Anti-KuneKune1:5000MeOHkindly provided by M. Furuse (Nelson et al., 2010)
Anti-Phospho-Ezrin (Moesin)1:500FACell Signaling Technology Cat. 3141
Anti-Moesin (Q480)1:400FACell Signaling Technology Cat. 3150
Anti-MyosinV1:2000MeOH(Pocha et al., 2011a)
Anti-DPatj1:1000FA(Richard et al., 2006a)
Rat antibodies
Anti-Yurt1:500MeOHkindly provided by U. Tepass (Laprise et al., 2006)
Anti-Stardust1:2000FA(Berger et al., 2007)
Chicken antibodies
Anti-GFP1:100FAAbcam Cat. Ab13970
Guinea pig antibodies
Anti-Crumbs 2.81:500MeOH(Richard et al., 2006a)
Anti-Par61:500FAkindly provided by A. Wodarz (Shahab et al., 2015)
DSHB - Developmental Studies Hybridoma Bank (Iowa city, Iowa, USA)
Invitrogen, Molecular Probes (Eugene, Oregon, USA)
Santa Cruz Biotechnology, Inc (Dallas, Texas, USA)
Cell Signaling Technology (Danvers, Massachusetts, USA)
Abcam plc (Cambridge, United Kingdom)

Data availability

We provide as source data files all the data used for statistical analyses and generation of all graphs. These files are sorted according to the figure and the corresponding figure supplements, which correspond to Figure 1 and its supplements, Figure 2, Figure 3 and its supplements, Figure 5 and its supplements, and Figure 6.

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