Resources and reagents.

The basement membrane (BM) is an essential structural element of tissues, and its diversification participates in organ morphogenesis. However, the traffic routes associated with BM formation and the mechanistic modulations explaining its diversification are still poorly understood. Drosophila melanogaster follicular epithelium relies on a BM composed of oriented BM fibrils and a more homogenous matrix. Here, we determined the specific molecular identity and cell exit sites of BM protein secretory routes. First, we found that Rab10 and Rab8 define two parallel routes for BM protein secretion. When both routes were abolished, BM production was fully blocked; however, genetic interactions revealed that these two routes competed. Rab10 promoted lateral and planar-polarized secretion, whereas Rab8 promoted basal secretion, leading to the formation of BM fibrils and homogenous BM, respectively. We also found that the dystrophin-associated protein complex (DAPC) and Rab10 were both present in a planar-polarized tubular compartment containing BM proteins. DAPC was essential for fibril formation and sufficient to reorient secretion towards the Rab10 route. Moreover, we identified a dual function for the exocyst complex in this context. First, the Exo70 subunit directly interacted with dystrophin to limit its planar polarization. Second, the exocyst complex was also required for the Rab8 route. Altogether, these results highlight important mechanistic aspects of BM protein secretion and illustrate how BM diversity can emerge from the spatial control of distinct traffic routes.</div

<i>Exocyst is required for the Rab8 dependent route</i>.

(A) Lateral (A-A’) and basal (A”) focal planes or color-coded z projection in function of the z axis of the 12 lateral and basal focal planes used for BM secretion quantification (A”’) in Exo70 null mutant individual single-cell clones that express Col IV-GFP (green, top), stained for F-actin (magenta) and immunostained for GFP without permeabilization (white). Scale bars, 5 μm. Dotty white line (A) highlights the cell periphery. Double dotty red lines (A’) and white surface delimited by dotty cyan line (A”) indicate the surface of the given z-section taken into account for lateral or basal secretion quantification respectively. Scale bars, 5 μm. (B) Quantification of the planar orientation of the lateral BM protein secretion in Exo70 null mutant single cells. (C) Quantification of the lateral fraction of BM secretion (%) in control and Exo70 null mutant single-cell clones. In order on the graph, n = 28 and 13 cells. (D) Quantification of the BM fibril fraction (%) in stage 8 follicles of the indicated genotypes. In order on the graph, n = 10, 10, 13, 16, 14 follicles. (E-G) Cross-sections of stage 8 follicles of the indicated genotypes: (E, E’) tj> Exo70 RNAi, (F, F’) tj> Rab10 RNAi, (G, G’) tj > Exo70 RNAi > Rab10 RNAi. Images show Col IV-GFP (white, left; green, right) and F-actin (magenta) localization. Scale bar, 10 μm. (H) Quantification of the ratio between apical and basal Col IV-GFP fluorescence intensity (%) in stage 8 follicles of the indicated genotypes. In order on the graph, n = 24, 23, 18, 30 follicles. (I) Quantification of Dys-GFP mean signal intensity at the trailing edge vs adjacent lateral front side in control and Rab8 KD follicle cells. In order on the graph, n = 53 and 63 follicle cells. J) Scheme representing the three different BM secretion routes with their cell exit site and their components. For all graphs, data are the mean ± SD; ns, not significant, *p <0.05, ****p <0.0001 (Unpaired t test for C, Ordinary one-way ANOVA with Tukey’s multiple comparisons test for D, H; Mann-Whitney test for I).</p

S4 Fig -

(A-A‘) Basal and (A”,A”’) suprabasal view of stage 8 follicles expressing Rab10-RFP (magenta) in WT cells (GFP cells, green) or in Dys null mutant cell clones (no GFP). (B) Rab10-RFP mean signal intensity at the trailing edge vs adjacent lateral front side of follicle cells of the indicated genotypes, on 0.45 μm projections starting 0.15 μm below the basal surface. Data are the mean ± SD. In order on graph, n = 73 and 56 follicle cells. (C) Quantification of the lateral fraction of BM secretion (%) in single-cell clones overexpressing Col IV-GFP in control and Dys null mutant follicles. Data are the mean ± SD. In order on graph, n = 21 and 18 cells. ns, not significant (unpaired t test). (D) Quantification of the planar orientation of lateral BM protein secretion from individual single-cell clones in control (red) and Dys null mutant (blue). (E-G) Sagittal view of follicles overexpressing Dg-GFP (E, E’) or expressing endogenous Dys-sfGFP in WT context (F, F’) or in Dg overexpression (G-G’). Dg-GFP is shown in white, Dys-sfGFP in green and F-actin in magenta. (H, I) basal and (H’, I’) suprabasal images of tj>Dg-GFP in (H) WT or (I) Dys mutant conditions. (J) Quantifications of BM fibril fraction (%) of stage 8 WT or tj> Dg follicles. Data represent mean ± SD. Unpaired t test, *p tj> Dg (L, L’)) follicles visualized with Col IV-GFP (green). Scale bars, 5 μm in (A, H-I’), 10μm in (E-G’ and K-L’) (TIFF)</p

<i>DAPC is sufficient to recruit the Rab10-dependent route</i>.

(A) Quantifications of BM fibril fraction (%) in stage 8 follicles of the indicated genotypes (n = 12, 11, 15, 11, 10 follicles) (B-F) Basal view of the BM in stage 8 Col IV-GFP-expressing follicles of the following genotypes (B) tj> Rab10-RFP (Rab10 OE) (C) tj >Rab10-RFP; Dys (D) Dys (E) tj,>Rab8 RNAi (Rab8 KD), (F) tj, >Rab8 RNAi; Dys. Scale bars, 10 μm. (G) Apical fraction quantification (%) of Col IV-GFP fluorescence intensity in stage 8 follicles of the indicated genotypes (In order on graph, n = 32, 34, 37, 32, 26 follicles). (H-L’) Cross-sections of stage 8 Col IV-GFP-expressing follicles of the following genotypes: (H, H’) tj>Rab10 RNAi (Rab10 KD), (I, I’) tj>Rab10 RNAi; Dys, (J, J’) Dys, (K, K’) tj>Rab8 RNAi, (L,L’) tj>Rab8 RNAi; Dys. Images show Col IV-GFP (green, bottom; white, top) and F-actin (magenta). Scale bar, 10 μm. (L) Apical fraction quantification (%) of Col IV-GFP fluorescence intensity in stage 8 follicles of the indicated genotypes (In order on graph, n = 32, 34, 37, 32, 26 follicles). (M-P) Basal and (M’-P’) suprabasal views of stage 8 follicles of the following genotypes: (M, M’) tj> Dg-GFP (DG-GFP overexpression (OE), (N, N’) tj> Dg; Dys-sfGFP, (O, O’) tj> Dg-GFP > Rab10-RFP, (P, P’) tj> Dg-.5 μm. (Q-S) Quantification of (Q) Dg-GFP, (R) Dys-sfGFP and (S) Rab10-RFP mean signal intensity at the trailing edge vs adjacent lateral front side of follicle cells of the indicated genotypes, on 0.45 μm projections starting 0.15 μm below the basal surface; n = 46, 41, 42 follicle cells (Q), n = 46, 39, follicle cells (R), n = 41, 43, 42 follicle cells (S). ****p <0.0001, ***p <0.001, *p<0.05, ns: not significant (Ordinary one-way ANOVA with Tukey’s multiple comparisons test for A, Kruskal-Wallis with Dunn’s comparison test for Q, S, or with uncorrected Dunn’s comparisons test for G, and Mann-Whitney test for R). In all graphs, data are the mean ± SD. (T-U) Representative images of F-actin (magenta) and Col IV-GFP (green) in (T) control and (U) Dg-overexpressing follicle cells in a plane in the middle of the cells. Note the presence of large BM fibrils between cells (white arrows) and the aberrant shape of cells in U. Scale bars, 10 μm.</p

Numerical data and statistical analysis supporting figures.

Numerical data and statistical analysis supporting figures.</p

<i>BM proteins are secreted from two distinct subcellular domains</i>.

(A) Scheme of the genetic components used to generate single-cell clones that overexpress Col IV-GFP and the protein of interest. (B) Image of a whole stage 8 follicle after induction of Col IV-GFP expression (green) in single-cell clones stained for F-actin (red) and immunostained for GFP (yellow) without permeabilization to label secreted Col-IV-GFP. (C-E) lateral (C-E’) and basal (C”-E”) focal planes of individual single-cell clones of the indicated genotypes expressing Col IV-GFP (green, top), stained for F-actin (magenta) and immunostained for GFP without permeabilization (white). Dotty white line (C-E) highlights the cell periphery. Double dotty red lines (C’-E’) and white surface delimited by dotty cyan line (C”-E”) indicate the surface of the given z-section considered for lateral or basal secretion quantification respectively. Color-coded z projection in function of the z axis of the lateral and basal 12 focal planes used for BM secretion quantifications (C”’-E”’). Scale bars, 5 μm. (F) Scheme of how BM secretion was quantified: lateral secretion was assessed using a hollow cylinder (red) of 1.5μm thickness based on cell cortex segmentation. Basal secretion was assessed using a full cylinder (cyan) made by projecting the ROI drawn on the basal surface and shrunk to 0.75μm in diameter on the three slices beyond the cell surface. (G) Quantification of the lateral fraction of BM secretion (%) in single-cell clones of the indicated genotypes overexpressing Col IV-GFP. In order on the graph, n = 21, 29, and 18 cells. Data are the mean ± SD; **p <0.01 (Ordinary one-way ANOVA with Dunnett’s comparison test). (H-J) Quantification of the planar orientation of lateral BM protein secretion from individual single-cell clones of the following genotypes: control (red), Rab10 OE (green), Rab8 OE (blue). Each arrow represents the vector between the isobarycenter of the hollow cylinder and its center of mass for one cell, using the signal intensity as weight. Images were oriented in such a way that the cell trailing edge points toward 90°.</p

Basal view of the BM at stage 8 in <i>Col IV-GFP</i> and <i>tj> Cg25c-GFP</i> follicle cells.

Quantifications of BM fibril fraction (%) in stage 8 follicles of the indicated genotypes. Data are the mean ± SD; ns, not significant (unpaired t test). In order on the graph, n = 10, 9 follicles. (TIFF)</p

S3 Fig -

(A-C’) Basal (C, C’), suprabasal (i.e. 0.4 μm above the basal surface) (B, B’) and middle (i.e. 0.8 μm below basal surface) (A, A’) views of stage 8 follicles that overexpress RFP-tagged Rab10 (magenta) and endogenous Col IV-GFP (green). (D-D”’) Middle view of stage 8 follicles that overexpress RFP-tagged Rab10 (black in 1st and 3rd line, white in 4th line), express endogenous Col IV-GFP (green) and that are stained with anti-golgin-245 antibody (magenta) and F-actin (white in 2nd line). (E-E”) Basal view of stage 8 follicles treated with collagenase before fixation to visualize intracellular endogenous basal Col IV-GFP (green) and Rab10-RFP (magenta). (F-F”) Basal view of a photobleached window at the surface of stage 8 living follicles taken 20 minutes after photobleaching to visualize intracellular basal Rab10-RFP (magenta) and endogenous Col IV-GFP (green) expression in non-bleached cells that have migrated in the bleached BM area. Scale bars, 5 μm. (TIFF)</p

S1 Fig -

(A) Cross-sections of stage 8 ovarian follicles showing Col IV-GFP (green, top; white, bottom) and F-actin (magenta) localization in control line (left) or tj>Rab10 RNAi (Rab10 knock-down) indicated lines: JF02058, KK109210 or GD13414. (B) Apical fraction quantification (%) of Col IV-GFP fluorescence intensity in stage 8 follicles of the indicated genotypes. Data are the mean ± SD; ****p Rab8 KD, Rab10 KD and Rab8-Rab10 double KD. (D) Cross-sections of stage 8 follicles of the indicated genotypes (control, Rab8-Rab10 double KD, Rab11 KD, Rab8-Rab10-Rab11 triple KD, showing aPKC (magenta, 1st line; white, 2nd line), Dlg (yellow, 3rd line; white, 4th line), E-Cad (orange, 5th line; white, 6th line) and Col IV-GFP (green). (TIFF)</p

Genotypes and specific conditions.

The basement membrane (BM) is an essential structural element of tissues, and its diversification participates in organ morphogenesis. However, the traffic routes associated with BM formation and the mechanistic modulations explaining its diversification are still poorly understood. Drosophila melanogaster follicular epithelium relies on a BM composed of oriented BM fibrils and a more homogenous matrix. Here, we determined the specific molecular identity and cell exit sites of BM protein secretory routes. First, we found that Rab10 and Rab8 define two parallel routes for BM protein secretion. When both routes were abolished, BM production was fully blocked; however, genetic interactions revealed that these two routes competed. Rab10 promoted lateral and planar-polarized secretion, whereas Rab8 promoted basal secretion, leading to the formation of BM fibrils and homogenous BM, respectively. We also found that the dystrophin-associated protein complex (DAPC) and Rab10 were both present in a planar-polarized tubular compartment containing BM proteins. DAPC was essential for fibril formation and sufficient to reorient secretion towards the Rab10 route. Moreover, we identified a dual function for the exocyst complex in this context. First, the Exo70 subunit directly interacted with dystrophin to limit its planar polarization. Second, the exocyst complex was also required for the Rab8 route. Altogether, these results highlight important mechanistic aspects of BM protein secretion and illustrate how BM diversity can emerge from the spatial control of distinct traffic routes.</div