(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 crb^RNAi) 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 crb^RNAi; 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 crb^RNAi; 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.
Figure 1—source data 2.Dataset for SerpCBD-GFP fluorescence intensity in control glands.
Figure 1—source data 3.Dataset for SerpCBD-GFP fluorescence intensity in Crb KD glands.

(A,B) Maximal projections showing F-actin (phalloidin staining) of control (A, fkh>/+) and Crb KD (B, fkh >UAS crb^RNAi) 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 crb^RNAi) 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 crb^RNAi; 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 crb^RNAi; UAS-PNA-GFP) animals. Q. Western blot probed for Crb (left) and Tubulin (right) loaded with control (fkh>/+, lanes with ‘crb^RNAi -”) and Crb KD (fkh >UAS crb^RNAi, lanes with ‘crb^RNAi +”) 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 crb^RNAi) and Sdt KD (fkh >UAS sdt^RNAi) 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 sdt^RNAi) 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 sdt^RNAi) 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 sdt^RNAi; 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 sdt^RNAi; 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 sdt^RNAi; 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 , fkh >UAS SerpCBD-GFP) and Sdt KD (magenta, fkh >UAS sdt^RNAi; 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.
Figure 1—figure supplement 1—source data 2.Dataset for tracking of larval development.
Figure 1—figure supplement 1—source data 3.Dataset for SerpCBD-GFP fluorescence intensity in control glands (note is the same dataset for control).
Figure 1—figure supplement 1—source data 4.Dataset for SerpCBD-GFP fluorescence intensity in Sdt KD glands.

(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 crb^RNAi) 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 crb^RNAi) 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 crb^RNAi) 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 crb^RNAi) and the positive control for leaky SGs, Fas3 KD (M,M’, Fas3-GFP, fkh >UAS gfp^RNAi) 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’).

(A-F) Localization and quantification of F-actin (phalloidin staining, A-C) and β_H-Spec (D-F) in control (A,D, fkh>/+) and Crb KD (B,E, fkh >UAS crb^RNAi) 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 β_H-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 gfp^RNAi) 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 gfp^RNAi) 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 crb^RNAi) 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 crb^RNAi) animals. The heatmap indicates the scale bar for the number of microvilli/µm.
Figure 2—source data 1.Dataset for phalloidin fluorescence intensity.
Figure 2—source data 2.Dataset for β_H-Spec fluorescence intensity.
Figure 2—source data 3.Dataset for microvilli quantifications.

(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 gfp^RNAi) 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 crb^RNAi) 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.

(A-C’) TEM images of SGs from Crb KD (fkh >UAS crb^RNAi) 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.

(A-D) Maximal projections of SGs incubated ex vivo with Lysotracker for 30 min of control (A,C, fkh>/+), Crb KD (B, fkh >UAS crb^RNAi) and Sdt KD (D, fkh >UAS sdt^RNAi) 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.

(A,B) Localization of β_H-Spec in control (A, fkh>/+) and β_H-Spec KD (B, fkh >UAS kst^RNAi) 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 β_H-Spec KD (D, fkh >UAS kst^RNAi) animals. (E,F) Localization of Crb in control (E, fkh>/+) and β_H-Spec KD (F, fkh >UAS kst^RNAi) SGs, respectively. Arrows point to the apical membrane domain. Scale bar (A) indicates 10 µm and applies to all panels.

(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 crb^RNAi) and β_H-Spec KD (C, fkh >UAS kst^RNAi) 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 crb^RNAi) and β_H-Spec (green, fkh >UAS kst^RNAi) 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 didum^RNAi) animals. (K,L) Localization of SerpCBD-GFP in live SGs of control (K, fkh >UAS SerpCBD-GFP) and MyoV KD (L, fkh >UAS didum^RNAi; 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 didum^RNAi; 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.
Figure 3—source data 2.Dataset for MyosinV fluorescence intensity in Crb KD glands.
Figure 3—source data 3.Dataset for MyosinV fluorescence intensity in βH-Spec KD glands.
Figure 3—source data 4.Dataset for SerpCBD-GFP fluorescence intensity in control glands.
Figure 3—source data 5.Dataset for SerpCBD-GFP fluorescence intensity in MyoV KD glands.

(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 crb^RNAi, 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.
Figure 3—figure supplement 1—source data 2.Dataset for MyosinV-GFP fluorescence intensity in Crb KD glands.

(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 β_H-Spec KD (magenta, fkh >UAS kst^RNAi, 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.
Figure 3—figure supplement 2—source data 2.Dataset for SerpCBD-GFP fluorescence intensity in βH-Spec KD glands.

(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 crb^RNAi) 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 crb^RNAi) SGs, indicated as crb^RNAi – or +, respectively. Membranes were probed for tubulin (loading control) and for GFP; arrowheads point to Rab-YFP proteins.

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.

(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,G, fkh>/+) and β_H-Spec KD (B,D,F,H, fkh >UAS kst^RNAi). Dotted lines indicate basal membrane. Scale bar (A) indicates 10 µm and applies to all panels.

(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 gfp^RNAi; 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.

(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 crb^RNAi; UAS-PLCδ-PH-EGFP), Pten KD (D, fkh >UAS pten^RNAi; UAS-PLCδ-PH-EGFP), double KD of Crb and Pten (E, fkh >UAS crb^RNAi, UAS-pten^RNAi; UAS-PLCδ-PH-EGFP), Pi3K92E KD (F, fkh >UAS-pi3k92E^RNAi; UAS-PLCδ-PH-EGFP) and double KD of Crb and Pi3K92E (G, fkh >UAS crb^RNAi, UAS-pi3k92E^RNAi; 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 crb^RNAi; 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 crb^RNAi) 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 ocrl^RNAi) 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).
Figure 5—source data 2.Dataset for PLCδ-PH-EGFP fluorescence intensity in Crb KD glands (corresponding to panel H).
Figure 5—source data 3.Dataset for PLCδ-PH-EGFP fluorescence intensity in Pten KD glands (corresponding to panel H).
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).
Figure 5—source data 5.Dataset for PLCδ-PH-EGFP fluorescence intensity in Pi3K92E KD glands (corresponding to panel H).
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).
Figure 5—source data 7.Dataset for Pten2-GFP fluorescence intensity (corresponding to panel K).
Figure 5—source data 8.Dataset for Ocrl-RFP fluorescence intensity in control glands (corresponding to panel N).
Figure 5—source data 9.Dataset for Ocrl-RFP fluorescence intensity in Crb KD glands (corresponding to panel N).
Figure 5—source data 10.Dataset for PLCδ-PH-EGFP fluorescence intensity in control glands (corresponding to panel Q).
Figure 5—source data 11.Dataset for PLCδ-PH-EGFP fluorescence intensity in Ocrl KD glands (corresponding to panel Q).
Figure 5—source data 12.Dataset for number of PAMS and diameter of PAMS.

(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 sdt^RNAi; UAS-PLCδ-PH-EGFP), β_H-Spec KD (C, fkh >UAS kst^RNAi; UAS-PLCδ-PH-EGFP) and MyoV KD (L, fkh >UAS didum^RNAi; 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 crb^RNAi; UAS-PLCδ-PH-EGFP, n = 25 cells), β_H-Spec KD (C, fkh >UAS kst^RNAi; UAS-PLCδ-PH-EGFP, n = 29 cells) and MyoV KD (L, fkh >UAS didum^RNAi; 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 3^rd instar wandering larvae of control (F, fkh >UAS-PLCδ-PH-EGFP) and Crb KD (G, fkh >UAS crb^RNAi; 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 . (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 crb^RNAi; UAS-PLCδ-PH-EGFP), Sktl KD (K, fkh >UAS sktl^RNAi; UAS-PLCδ-PH-EGFP) and double KD of Crb and Sktl (L, fkh >UAS sktl^RNAi, UAS-crb^RNAi; 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 crb^RNAi; 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.
Figure 5—figure supplement 1—source data 2.Dataset for salivary gland lengths.
Figure 5—figure supplement 1—source data 3.Dataset for PLCδ-PH-EGFP fluorescence intensity in control glands.
Figure 5—figure supplement 1—source data 4.Dataset for PLCδ-PH-EGFP fluorescence intensity in Crb KD glands.
Figure 5—figure supplement 1—source data 5.Dataset for PLCδ-PH-EGFP fluorescence intensity in Sktl KD glands.
Figure 5—figure supplement 1—source data 6.Dataset for PLCδ-PH-EGFP fluorescence intensity in glands with double KD of Crb and Sktl.
Figure 5—figure supplement 1—source data 7.Dataset for PLCδ-PH-EGFP fluorescence intensity in control glands incubated with vehicle.
Figure 5—figure supplement 1—source data 8.Dataset for PLCδ-PH-EGFP fluorescence intensity in Crb KD glands incubated with vehicle.
Figure 5—figure supplement 1—source data 9.Dataset for PLCδ-PH-EGFP fluorescence intensity in control glands incubated with VO-OHpic.
Figure 5—figure supplement 1—source data 10.Dataset for PLCδ-PH-EGFP fluorescence intensity in Crb KD glands incubated with VO-OHpic.

(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 crb^RNAi) 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.

(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 crb^RNAi; UAS-SerpCBD-GFP), Pten KD (C, fkh >UAS pten^RNAi; UAS-SerpCBD-GFP), double KD of Crb and Pten KD (D, fkh >UAS crb^RNAi, UAS-pten^RNAi; UAS-SerpCBD-GFP), Pi3K92E KD (E, fkh >UAS-pi3k92E^RNAi; UAS-SerpCBD-GFP), and double KD of Crb and Pi3K92E (F, fkh >UAS crb^RNAi, UAS-pi3k92E^RNAi; 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 crb^RNAi), Pten KD (J, Rab11-YFP, fkh >UAS pten^RNAi), double KD of Crb and Pten (K, Rab11-YFP, fkh >UAS crb^RNAi, UAS-pten^RNAi), Pi3K92E KD (L, Rab11-YFP, fkh >UAS-pi3k92E^RNAi), and double KD of Crb and Pi3K92E (M, Rab11-YFP, fkh >UAS crb^RNAi, UAS-pi3k92E^RNAi) 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 crb^RNAi), Pten KD (Q, Rab30-YFP, fkh >UAS pten^RNAi), double KD of Crb and Pten (R, Rab30-YFP, fkh >UAS crb^RNAi, UAS-pten^RNAi), Pi3K92E KD (S, Rab30-YFP, fkh >UAS-pi3k92E^RNAi), and double KD of Crb and Pi3K92E (T, Rab30-YFP, fkh >UAS crb^RNAi, UAS-pi3k92E^RNAi) 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 crb^RNAi), Pten KD (green, fkh >UAS pten^RNAi), double KD of Crb and Pten KD (yellow, fkh >UAS crb^RNAi, UAS-pten^RNAi), Pi3K92E KD (blue, fkh >UAS-pi3k92E^RNAi), and double KD of Crb and Pi3K92E (, fkh >UAS crb^RNAi, UAS-pi3k92E^RNAi) 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.
Figure 6—source data 2.Dataset for SerpCBD-GFP fluorescence intensity in Crb KD glands.
Figure 6—source data 3.Dataset for SerpCBD-GFP fluorescence intensity in Pten KD glands.
Figure 6—source data 4.Dataset for SerpCBD-GFP fluorescence intensity in glands with double KD of Crb and Pten.
Figure 6—source data 5.Dataset for SerpCBD-GFP fluorescence intensity in Pi3K92E KD glands.
Figure 6—source data 6.Dataset for SerpCBD-GFP fluorescence intensity in glands with double KD of Crb and Pi3K92E.
Figure 6—source data 7.Dataset for Rab11-YFP fluorescence intensity in control glands.
Figure 6—source data 8.Dataset for Rab11-YFP fluorescence intensity in Crb KD glands.
Figure 6—source data 9.Dataset for Rab11-YFP fluorescence intensity in Pten KD glands.
Figure 6—source data 10.Dataset for Rab11-YFP fluorescence intensity in glands with double KD of Crb and Pten.
Figure 6—source data 11.Dataset for Rab11-YFP fluorescence intensity in Pi3K92E KD glands.
Figure 6—source data 12.Dataset for Rab11-YFP fluorescence intensity in glands with double KD of Crb and Pi3K92E.
Figure 6—source data 13.Dataset for Rab30-YFP fluorescence intensity in control glands.
Figure 6—source data 14.Dataset for Rab30-YFP fluorescence intensity in Crb KD glands.
Figure 6—source data 15.Dataset for Rab30-YFP fluorescence intensity in Pten KD glands.
Figure 6—source data 16.Dataset for Rab30-YFP fluorescence intensity in glands with double KD of Crb and Pten.
Figure 6—source data 17.Dataset for Rab30-YFP fluorescence intensity in Pi3K92E KD glands.
Figure 6—source data 18.Dataset for Rab30-YFP fluorescence intensity in glands with double KD of Crb and Pi3K92E.
Figure 6—source data 19.Dataset for food intake estimations.
Figure 6—source data 20.Dataset for tracking of larval development.

(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.