Larval salivary gland as a model for regulated exocytosis.

Images of a wandering larva (A-A’) and a prepupa (B-B’) expressing Sgs3-dsRed and visualized under white light (A and B) or by epiflourescence (A’-B’). Sgs3-dsRed localizes in salivary glands of wandering larvae (A’) and outside of the puparium in prepupae (B’). Scale bar 1 mm. (C) Confocal images of unfixed salivary glands dissected from larvae at the indicated time intervals (h AEL= hours after egg laying). Sgs3-GFP is shown in red and the plasma membrane labelled with myr-Tomato (sgs3-GFP, *h-Gal4/ UAS-myr-Tomato) is shown in cyan. At 96 h AEL, Sgs3 synthesis can be detected in the distal cells of the gland. Thereafter, Sgs3 expression gradually expands proximally, and at 116 h AEL all salivary gland cells express Sgs3, with the exception of ductal cells. Exocytosis of SGs begins by this time point, and Sgs3 can be detected in the gland lumen. At 120 h AEL concerted exocytosis of SGs has ended. Scale bar 300 μm. (D) Confocal images of SGs labelled with Sgs3-GFP; SG diameter distribution at each time interval is displayed below. Based on its diameter, SGs are classified as immature (diameter < 3 μm) or mature (diameter ≥ 3μm). Only SGs from distal cells were used for diameter determination. Data points for this graph are shown in Supplementary Table 1. For all time intervals analyzed n = 3, except for the 108-112 h AEL interval for which n = 4. “n” represents the number of salivary glands analyzed. Scale bar 5 μm.

The exocyst is required for Sgs3 secretion.

(A-B) Images of a larva and a prepupa expressing Sgs3-GFP, and visualized with epifluorescence. Sgs3-GFP is inside the salivary glands in control wandering larvae (A), and outside the puparium (A’) in control prepupae (sgs3-GFP, fkh-Gal4/ UAS-cherryRNAi). Expression of sec3 RNAi in salivary glands (UAS-sec3RNAi; sgs3-GFP, fkh-Gal4) does not affect expression of Sgs3-GFP in larvae (B), but blocks Sgs3-GFP release outside the puparium, so the protein is retained inside the salivary glands (B’). This phenotypic manifestation is referred as “retention phenotype”. Scale bar 1 mm. (C) Quantification of the penetrance of the retention phenotype in prepupae expressing the indicated RNAis. RNAis were expressed using a fkh-Gal4 driver and larvae were cultured at 29°C. All RNAis tested against exocyst complex subunits display a retention phenotype, with a penetrance significantly different from the control RNAi (UAS-cherryRNAi) according to Likelihood ratio test followed by Tuckeýs test (“*”=p-value< 0.05). controlRNAi (cherryRNAi), n= 7; exo70RNAiV, n= 11; exo70RNAiBL, n= 8; sec3RNAi, n= 6; sec5RNAiBL, n= 17; sec5RNAiV n= 6; sec10RNAi, n= 9; sec6RNAi, n= 11; sec8RNAi, n= 5; exo84RNAiBL, n=5 ; exo84RNAiV, n=6; sec15RNAiBL, n=3 ; sec15RNAiV, n=4. “n” represents the number of vials containing 20 to 30 prepupae per vial. For those genotypes with 100% penetrance no statistical analysis was performed due to the lack of standard error. For exocyst subunits with more than one RNAi line available, “BL” indicates a Bloomington Stock Center allele and “V” a Vienna Stock Center allele (see Supplementary Table 2 for stock numbers).

Phenotypic manifestations of exocyst subunits silencing.

At the end of larval development (116-120 h AEL) salivary gland cells of control individuals (sgs3-GFP, fkh-Gal4/ UAS-cherryRNAi), (A) are filled with mature SGs (insets) of homogenous size within a single cell and among neighboring cells. In cells expressing exo84RNAiV (UAS-exo84RNAiV; sgs3-GFP, fkh-Gal4) (B), three different phenotypes can be visualized in a single salivary gland: cells with mature SGs (MSG), cells with immature SGs (ISG) and cells with no SG and cells with Sgs3 retained in a mesh (MLS). Scale bar 30 μm. (C) Quantification of the penetrance of each of the three phenotypes obtained upon downregulation of each of the exocyst subunits. Larvae were grown at four different temperatures (29, 25, 21 or 19°C) to achieve different levels of RNAi expression. “n” = Number of salivary glands analyzed; controlRNAi (cherryRNAi), n = 4 (29°C), n = 5 (25, 21 and 19°C); exo70RNAiBL, n = 11 (29°C), n = 7 (25, 21°C), n = 6 (19°C); sec3RNAi, n = 7 (29, 25°C), n = 4 (21°C), n = 9 (19°C); sec5RNAiBL, n = 4 (29°C), n = 12 (25°C), n = 9 (21°C), n = 6 (19°C) ; sec10RNAi, n = 8 (29°C), n = 6 (25, 19°C), n = 7 (21°C); sec6RNAi, n = 9 (29°C), n = 4 (25°C); sec8RNAi, n = 8 (29°C), n = 6 (25, 19°C), n = 7 (21°C); exo84RNAiV, n = 8 (29°C), n = 4 (25, 19°C), n = 5 (21°C) ; sec15RNAiV, n = 4 (29, 25°C), n = 6 (21°C), n = 5 (19°C). (D) Quantification of the penetrance of the Sgs3-GFP retention phenotype in salivary glands of prepupae of the indicated genotypes. Only a few control individuals (sgs3-GFP, fkh-Gal4/ UAS-cherryRNAi) display the retention phenotype. Downregulation of exocyst subunits provokes significant retention of Sgs3 inside the salivary glands irrespectively of the temperature (25, 21 or 19°C). Expression of sec6RNAi at 21 or 19°C resulted in no synthesis of Sgs3-GFP or Sgs3-dsRed, so the distribution of phenotypes was not assessed for this genotype at these temperatures. RNAis were expressed with a fkh-Gal4 driver. (D) “n” = Number of vials with 20-30 larvae. controlRNAi (cherryRNAi), n = 7 (25°C), n = 9 (21°C), n = 19 (19°C); exo70RNAiBL, n = 13 (25°C), n = 9 (21°C), n = 22 (19°C); sec3RNAi, n = 9 (25°C), n =6 (21, 19°C); sec5RNAiBL, n = 9 (25°C), n = 7 (21°C), n = 12 (19°C); sec10RNAi, n = 10 (25°C), n = 5 (21°C), n = 4 (19°C); sec6RNAi, n = 10 (25°C); sec8RNAi, n = 5 (25°C), n = 8 (21°C), n = 22 (19°C); exo84RNAiV, n = 8 (25, 19°C), n = 6 (21°C) ; sec15RNAiV, n = 5 (25°C), n = 9 (21°C), n = 8 (19°C). Statistical analysis was performed using a Likelihood ratio test followed by Tuckeýs test (“*”=p-value< 0.05). For those genotypes with 100% penetrance no statistical analysis was performed due to the lack of standard error. “ns”= not significant. Comparisons were made between RNAis for each of the temperatures analyzed.

The exocyst is required for Sgs3-GFP exit from ER and SG biogenesis.

Confocal images of unfixed salivary glands of the indicated genotypes. Larvae were grown at 29°C to achieve maximal activation of the Gal4-UAS system, and maximal downregulation of exocyst subunits. (A) In control salivary glands (sgs3-GFP, fkh-Gal4/ UAS-cherryRNAi) 116-120 AEL, Sgs3-GFP is packed in mature SGs. In salivary glands expressing RNAis against any of the exocyst subunits SGs are not formed, and Sgs3-GFP exhibits a reticular distribution. Scale bar 5 μm. The ER, labeled with GFP-KDEL (UAS-GFP-KDEL), (B and D) or Bip-sfGFP-HDEL (UAS-Bip-sfGFP-HDEL), (F and H) distributes in between SGs in control salivary glands (B and F); in Sec15RNAi salivary glands (fkh-Gal4/ UAS-sec15RNAiBL) SGs do not form and the Sgs3-dsRed signal overlaps with the ER markers (D and H). (C, E, G and I) Two-dimensional line scans of fluorescence intensity across the white lines in panels B, D, F and H of Sgs3-dsRed and the ER markers. In all cases transgenes were expressed using fkh-Gal4. Scale bar 10 μm.

The exocyst is required to maintain normal Golgi complex morphology.

Confocal images of unfixed salivary glands of the indicated genotypes. Larvae were grown at 29°C to achieve maximal activation of the Gal4-UAS system and maximal downregulation of exocyst subunits. In control larvae (fkh-Gal4/ UAS-whiteRNAi), Sgs3-dsRed (A) or Sgs3-GFP (C) are in mature SGs. In Sec15RNAi salivary glands (fkh-Gal4/ UAS-sec15RNAiBL) Sgs3 is retained in a mesh-like structure (B and D), also shown in Figure 4. The cis-Golgi Complex was labelled with Grasp65-GFP (UAS-Grasp65-GFP), (A and B), and the trans-Golgi with RFP-Golgi (UAS-RFP-Golgi), (C and D). The morphology of the cis- and trans-Golgi complexes changes dramatically in Sec15-knockdown cells (B’’ and D”), in comparison to controls (A” and C”). Transgenes were expressed with fkh-Gal4. Scale bar 10 μm. Model of Sgs3 transit from the ER through the cis-Golgi complex to forming SGs, and sprouting of SGs from the trans-Golgi complex (E). The exocyst is needed for tethering the ER to the cis-Golgi complex. In the absence of the exocyst (F) the ER and cis-Golgi disconnect, resulting in Sgs3 retention in the ER. Normal morphology of the Golgi complex (CG) is severely affected.

The exocyst is required for secretory granule homotypic fusion.

(A) Confocal images of unfixed salivary glands of the indicated genotypes. In control salivary glands (sgs3-GFP, fkh-Gal4/ UAS-cherryRNAi), Sgs3-GFP is in mature SGs. RNAis targeting any of the subunits of the exocyst were expressed at the indicated temperatures, resulting in salivary cells with immature granules. Scale bar 5 μm. (B-D) Sec15-GFP (cyan) was expressed at 18°C and SGs containing Sgs3-dsRed can be visualized (red) (sgs3-dsRed/ UAS-sec15-GFP; fkh-Gal4). Sec15-GFP can be observed as dots on the periphery of SGs of 1, 3 or 5 μm in diameter, and in some cases in between adjacent SGs (arrows). Scale bar 5 μm. (E) Expression of EGFP (cyan) at 25°C (sgs3-dsRed; UAS-EGFP, fkh-Gal4) does not affect SG size. (F) Expression of Sec15-GFP (cyan dots) at 25°C (sgs3-dsRed/ UAS-sec15-GFP; fkh-Gal4) generates giant SGs (asterisks). Scale bar 10 μm. (G) Quantification of the percentage of salivary gland cells with at least one SG larger than 8 μm diameter; (Wald Test, p-value < 0.05). “n” represents the number of salivary glands analyzed; scale bar 10 μm. (H) Still panels of Supplementary movie 1 showing that during a homotypic fusion event, Sec15-GFP accumulates precisely at the contact site between two neighboring SGs (dotted circle). Scale bar 5 μm. Transgenes were expressed using fkh-Gal4.

The exocyst mediates the acquisition of secretory granule maturation factors.

Confocal images of unfixed salivary glands of the indicated genotypes. Larvae were grown at 21°C to reduce the activity of the Gal4-UAS system, and generate a maximal proportion of cells with immature SGs. (A-B) Recruitment of Syt1-GFP (UAS-Syt1-GFP); of (D-E) CD63-GFP (UAS-CD63-GFP); of (G-H) YFP-Rab1 and of (J-K) YFP-Rab11 was analyzed in control salivary glands expressing whiteRNAi (fkh-Gal4/ UAS-whiteRNAi) and in salivary glands expressing sec5RNAiBL (fkh-Gal4/ UAS-sec5RNAiBL). Fluorescent intensity around SGs of each of the analyzed maturation factors was quantified using the ImageJ software and plotted (C, F, I and L). Comparison of fluorescent intensity among genotypes and statistical analysis was performed using one-way analysis of variance (ANOVA). “n” represents the number of salivary glands: (C) control RNAi n= 4, sec5RNAiBL n= 5; (F) control RNAi n= 5, sec5RNAiBL n=4; (I) control RNAi n= 6, sec5RNAiBL n= 9; (L) control RNAi n= 11, sec5RNAiBL n= 8. Transgenes were expressed using fkh-Gal4. Scale bar 5 μm. (M) Proposed model of exocyst-dependent SG homotypic fusion and maturation. The exocyst complex is required for homotypic fusion between immature SGs. Immature SGs incorporate Syt-1, CD63 and Rab1 in an exocyst-dependent manner. Syt-1 continues to be recruited to SGs after homotypic fusion. The exocyst complex also inhibits the incorporation of an excess of Rab11 around SGs.

The exocyst is required for secretory granule fusion with the plasma membrane during regulated exocytosis.

Confocal images of unfixed salivary glands of the indicated genotypes. Larvae were grown at the indicated temperatures to attain levels of RNAi mediated silencing that bring about maximal proportion of cells with mature, exocytosis incompetent SGs. (A) Sgs3-GFP localizes within SGs. Control SGs (sgs3-GFP, fkh-Gal4/ UAS-cherryRNAi) are indistinguishable from SGs of salivary cells in which a subunit of the exocyst has been knocked-down. Scale bar 5 μm. (B) In control salivary glands (fkh-Gal4/ UAS-whiteRNAi), the PI(4,5)P2 reporter UAS-PLCη-PH-GFP labels the plasma membrane (dotted line) and also the SGs that have already fused with the plasma membrane (asterisks). (C) SGs of cells expressing exo70RNAiV (UAS-exo70RNAiV; fkh-Gal4) are not labeled with the reporter, indicating that these SGs failed to fuse with the plasma membrane. Scale bar 5 μm. (D) The number of mature SGs positive for PLCη-PH-EGFP per 100 μm of linear plasma membrane was quantified in the indicated genotypes; Exo70 knock-down reduces SG-plasma membrane fusion (Wald test (p value<0.05); 7 salivary glands per genotype were analyzed. (E) During SG exocytosis the exocyst complex, labelled with Sec15-GFP (cyan) localizes as dots in contact sites between SGs and the apical plasma membrane (dotted line) (sgs3-dsRed/ UAS-sec15-GFP; fkh-Gal4). (F) Still panels of Supplementary movie 2 showing a fusion event between a mature SG and the plasma membrane (arrow); a dot of Sec15-GFP indicating the position of the exocyst (arrow) is positioned just at the site where fusion takes place. Scale bar 5 μm. (G) Confocal image of a fixed salivary gland. A mature SG that has fused with the plasma membrane (dotted line), and thus became labelled with phalloidin, displays dots of Sec15-GFP on its side (arrow), just next to the fusion point (asterisk). Transgenes were expressed with a fkh-Gal4 driver. The salivary gland lumen is indicated with “L”. Scale bar 5 μm. (H) Model of the role of the exocyst complex during SG-plasma membrane fusion during regulated exocytosis. The exocyst sits on the membrane of the SG, and tethers the granule to the plasma membrane, favoring the action of fusion molecules. (I) Upon loss of the exocyst complex, mature SGs cannot contact the plasma membrane and fusion does not occur.

Quantification of SG diameter at the indicated time intervals after egg laying.

SGs from salivary gland distal-most cells were analyzed. Columns display: Hours AEL: hours after egg laying; n: number of salivary glands analyzed; number of cells analyzed; number of SGs measured; mean diameter; median diameter; minimum diameter and maximum diameter.

List of the Drosophila lines utilized in this work.

Stock number and repository center from where each line was obtained are indicated.

Additional RNAi lines targeting exocyst subunits utilized to assess reduction-of-function phenotypes in the pathway of regulated exocytosis. (A) Quantification of the penetrance of each of the three phenotypes observed upon downregulation of each of the exocyst subunits. Larvae were grown at four different temperatures to achieve different levels of exocyst subunits RNAi expression. “n” = Number of salivary glands analyzed. controlRNAi (cherryRNAi), n = 4 (29°C), n = 5 (25, 21 and 19°C); exo70RNAiV, n = 5 (29°C), n = 6 (25°C), n = 6 (21°C), n = 4 (19°C); sec15RNAiBL, n = 5 (29°C), n = 6 (25°C), n = 5 (21°C), n = 5 (19°C); exo84RNAiBL, n = 7 (29°C), n = 5 (25°C), n = 6 (21°C), n = 5 (19°C); sec5RNAiV, n = 5 (29°C), n = 5 (25°C), n = 6 (21°C), n = 6 (19°C); (B) Quantification of the penetrance of the Sgs3-GFP retention phenotype in salivary glands of prepupae with the indicated RNAis at different temperatures. “n” = Number of vials containing 20-30 larvae each. controlRNAi (cherryRNAi), n = 7 (25°C), n = 9 (21°C), n = 19 (19°C); exo70RNAiV, n = 6 (25°C), n = 8 (21°C), n = 6 (19°C); sec5RNAiV, n = 4 (25°C), n = 6 (21°C), n = 5 (19°C); sec15RNAiBL, n = 7 (25°C), n = 8 (21°C), n = 7 (19°C); exo84RNAiBL, n = 5 (25°C), n = 4 (21°C), n = 8 (19°C). RNAis were expressed using a fkh-Gal4 driver. Comparisons of the retention phenotype were carried out between genotypes at each different temperature, and statistical analysis was performed using a Likelihood ratio test followed by Tuckeýs test (“*”=p-value< 0.05). For those RNAis with 100% penetrance no statistical analysis was performed due to the lack of standard error.. “ns”= not significant. This figure represents an extension of the experiments of Figure 3C-D; all RNAis were expressed with controls at the same time, and therefore the controls are identical to those of Figure 3.

The exocyst is required for Sgs3-GFP exit from ER and SG biogenesis.

Confocal images of unfixed salivary glands of the indicated genotypes. Larvae were grown at 29°C to achieve maximal activation of the Gal4-UAS system, and maximal downregulation of exocyst complex subunits. The ER was labeled with GFP-KDEL (UAS-GFP-KDEL), (A and C) or with Bip-sfGFP-HDEL (UAS-Bip-sfGFP-HDEL) (E and G). In control salivary glands (fkh-Gal4/ UAS-whiteRNAi) (A and E) the ER has a network-like appearance and lays in between mature SGs that contain Sgs3-dsRed, and thus no colocalization between the ER marker and Sgs3-dsRed is detected (B and F). Upon expression of sec15RNAi (fkh-Gal4/ UAS-sec15RNAiBL) (C) or sec3RNAi (UAS-sec3RNAi; fkh-Gal4) SGs do not form, and Sgs3-dsRed is retained in the ER as demonstrated by colocalization of both markers in the two-dimensional line scan analysis (D and H). RNAis were expressed using a fkh-Gal4 driver. Scale bar 10 μm.

The exocyst is required to maintain normal Golgi complex morphology.

Confocal images of unfixed salivary glands of the indicated genotypes. Larvae were grown at 29°C to achieve maximal activation of the Gal4-UAS system, and maximal downregulation of exocyst complex subunits. In control salivary glands (fkh-Gal4/ UAS-whiteRNAi) SGs, filled with Sgs3-dsRed (A) or with Sgs3-GFP (C), reach a mature size, and the cis-Golgi (UAS-Grasp65-GFP) (A) or trans-Golgi (UAS-RFP-Golgi) (C) markers distribute as cisternae in the cytoplasm. Upon expression of sec3RNAi (UAS-sec3RNAi; fkh-Gal4) SGs do not form, and both the cis- and trans-Golgi complexes appear disorganized: the cis-Golgi appearing more fragmented (B) and the trans-Golgi swollen and enlarged (D). RNAis were expressed using a fkh-Gal4 driver. Scale bar 10 μm.

The exocyst mediates the acquisition of secretory granule maturation factors.

Incorporation of the SG maturation markers Syt1 (sgs3-dsRed/ UAS-syt1-GFP; fkh-Gal4), (A-D) and CD63 (sgs3-dsRed/ UAS-CD63-GFP; fkh-Gal4), (E-H) to developing granules. Prior to SG biogenesis (<96 Hours AEL) Syt1 localizes at the basolateral plasma membrane of salivary cells (A), while CD63 localizes at the apical plasma membrane (E). Scale bar 100 μm. Both markers localize at the membrane of immature SGs and mature SGs (B-D and F-H). Scale bar 5 μm. Note that Syt1 signal intensity increases around SGs as they mature (compare B”, C” and D’’), whereas CD63 signal remains fairly stable during SG maturation (compare F”, G” and H”).

The exocyst is required for secretory granule maturation.

Confocal images of unfixed salivary glands of the indicated genotypes. Larvae were grown at 25 or 21°C to modulate the activity of the Gal4-UAS system and generate a maximal proportion of immature (A, B, G, H, M and N) or mature SGs (D, E, J, K, P and Q) respectively. (A, B, D and E) Recruitment of Syt1-GFP (UAS-Syt1-GFP); of (G, H, J and K) CD63-GFP (UAS-CD63-GFP); or of (M, N, P and Q) YFP-Rab11 was analyzed in control salivary glands expressing whiteRNAi (fkh-Gal4/ UAS-whiteRNAi) and in salivary glands expressing sec3RNAi (UAS-sec3RNAi; fkh-Gal4). SGs were labelled with Sgs3-dsRed. Fluorescent intensity around SGs of each of the analyzed maturation factors was quantified using the ImageJ software and plotted (C, F, I, L, O and R). Comparison of fluorescent intensity among genotypes and statistical analysis was performed using one-way analysis of variance (ANOVA). “n” represents the number of salivary glands: (C) control RNAi n= 7, sec3RNAi n=6; (F) control RNAi n=7, sec3RNAi n=13; (I) control RNAi n= 12, sec3RNAi n= 9; (L) control RNAi n= 8, sec3RNAi n= 8; (O) control RNAi n= 7, sec3RNAi n= 10; (R) control RNAi n= 7, sec3RNAi n= 4. Transgenes were expressed using fkh-Gal4. “ns” = not significant. Scale bar 5 μm.

The exocyst negatively regulates incorporation of Rab11 to SGs.

Salivary gland cells expressing Sgs3-dsRed and endogenously tagged YFP-Rab11 (A), or overexpressing a constitutively active form of Rab11 at 29°C (fkh-Gal4/ UAS-YFP-Rab11ca) (B). YFP-Rab11 localizes around mature SGs (A). Overexpression of Rab11CA arrests SG maturation (B), as shown in the quantification of SG diameter (C). YFP-Rab11, n = 6; UAS-YFP-RAB11CA, n = 5. “n” = Number of salivary glands. (D) In control salivary glands, Sec15-GFP localizes as foci on the periphery of mature SGs (sgs3-dsRed/ UAS-sec15-GFP; fkh-Gal4). (E) Knock-down of Rab11 (sgs3-dsRed; UAS-rab11RNAi/ fkh-Gal4) generates immature SGs and prevents Sec15-GFP from localizing on SGs. (F) Proposed mechanism of genetic interactions between Rab11 and Sec15. A single-negative feedback loop regulates the levels of these proteins on the SGs, and recruitment of the maturation factors Syt-1, CD63 and Rab1. Scale bar 10 μm.