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 localized in salivary glands of wandering larvae (A’) and outside 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, fkh-Gal4/ UAS-myr-Tomato) is shown in cyan. At 96 h AEL, Sgs3 synthesis could be detected in the distal cells of the gland. Thereafter, Sgs3 expression gradually expanded proximally, and at 116 h AEL all salivary gland cells expressed Sgs3, with the exception of ductal cells. Exocytosis of SGs began by this time point, and Sgs3 could 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 was 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) did not affect expression of Sgs3-GFP in larvae (B), but blocked Sgs3-GFP release outside the puparium, so the protein was 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 fkh-Gal4 and larvae were cultured at 29°C. All RNAis tested against exocyst complex subunits displayed 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). cherryRNAi n= 7; exo70RNAiV n= 11; exo70RNAiBL n= 8; sec3RNAi n= 6; sec5RNAiBL n= 17; sec5RNAiV n= 6; sec10RNAi n= 9; sec6RNAi n= 11; sec8RNAin= 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 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) were filled with mature SGs (insets). In cells expressing exo84RNAiV (UAS-exo84RNAiV; sgs3-GFP, fkh-Gal4) (B), three different phenotypes could be visualized in a single salivary gland: cells with mature SGs (MSG), cells with immature SGs (ISG) and cells with no SG, in which Sgs3 was retained in a mesh (MLS). Scale bar 30 μm in main panels, and 5 μm in insets. For didactic purposes, MSG, ISG and MLS were drawn on the right, next to the corresponding inset (C) 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 (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); exo84RNAiVn = 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). Raw data use to generate this graph is shown in Supplementary Table 3. (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) displayed the retention phenotype. Downregulation of exocyst subunits provoked significant retention of Sgs3 inside the salivary glands irrespective to 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 fkh-Gal4. “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 was packed in mature SGs. Salivary glands expressing RNAis against each of the exocyst subunits, where Sgs3-GFP exhibited a reticular distribution are shown. SGs were not formed in these cells. 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) distributed in between SGs in control salivary glands (B and F); in sec15RNAi salivary glands (fkh-Gal4/ UAS-sec15RNAiBL) SGs did not form and the Sgs3-dsRed signal overlapped 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 localizes at the Golgi complex before Sgs3 synthesis.

Confocal images of unfixed salivary glands expressing GFP-Sec15 and (A) the ER marker KDEL-RFP or (C) the trans-Golgi marker RFP-Golgi. Sec15 foci did not localize at or associate with the ER (A-B). (C) Sec15 foci were found in close association with trans-Golgi complex cisternae. Examples of association events are shown in insets 1 and 2, including different angles of three-dimensional reconstruction stacks. (D) Sec15 foci and trans-Golgi complex association was lost after Sgs3 synthesis has begun. (E) Quantification of Sec15 foci-Golgi complex association events before and after the onset of Sgs3 synthesis. (Wald Test, p-value < 0.05). “n” represents the number of salivary glands analyzed, n=4.

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) were in mature SGs. In sec15RNAi salivary glands (fkh-Gal4/ UAS-sec15RNAiBL) Sgs3 was retained in a mesh-like structure (B and D), also shown in Figure 4. (A, B) The cis-Golgi complex was labelled with Grasp65-GFP (UAS-Grasp65-GFP), (and the trans-Golgi with RFP-Golgi (UAS-RFP-Golgi), (C, D). The morphology of the cis- and trans-Golgi complexes changed dramatically in sec15-knockdown cells (B”, D”), in comparison to controls (A”, C”). Transgenes were expressed with fkh-Gal4. Scale bar 10 μm. (E) Model of Sgs3 transit from the ER through the cis- and trans-Golgi complex to sprouting SGs from the trans-Golgi complex. (F) The exocyst is needed for tethering the Golgi complex cisternae and to support Golgi complex structure. In the absence of the exocyst Golgi cisternae disconnect, cis- and trans-Golgi become dysfunctional, resulting in Sgs3 retention at the ER.

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 was packed in mature SGs. RNAis targeting any of the subunits of the exocyst were expressed at the indicated temperatures, giving rise to salivary cells with immature granules. Scale bar 5 μm. (B) GFP-Sec15 (cyan) and Sgs3-dsRed (red) were expressed in salivary glands (sgs3-dsRed/ UAS-GFP-sec15; fkh-Gal4) at 18 °C. GFP-Sec15 foci were mostly found in between SGs (yellow arrows) in cells bearing immature SGs (upper panel) and not in cells with mature SGs (lower panel), in which most foci did not localize in between SGs (green arrowheads). Scale bar 1 μm. (C) Quantification of Sec15 foci in between SGs relative to the total number of Sec15 foci (%) (Wald Test, p-value < 0.05). Eight salivary glands with ISGs and 5 salivary glands with MSGs were analyzed. (D) Still panels of two different frames of Supplementary movie 6 showing that during a homotypic fusion event, GFP-Sec15 accumulated precisely at the contact site between two neighboring SGs (dotted circle); scale bar 5 μm. (E-G) Expression of EGFP (sgs3-dsRed; UAS-EGFP, fkh-Gal4) (E) or Sec8 (sgs3-dsRed/ UAS-Sec8; fkh-Gal4) (F) did not affect SG size (red). Expression of GFP-Sec15 (G) (sgs3-dsRed/ UAS -GFP-sec15; fkh-Gal4) generated giant SGs (asterisks). Scale bar 10 μm. (H) Quantification of the percentage of salivary gland cells with at least one SG larger than 8 μm diameter; (Likelihood ratio test followed by Tuckeýs test, p-value < 0.05). “n” represents the number of salivary glands analyzed. UAS-EGFP n=7; UAS-Sec8 n=5; UAS-GFP-Sec15 n=5. Transgenes were expressed using fkh-Gal4. “ns”= not significant.

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 to 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, sec5RNAiBLn=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 localized within SGs. Control SGs (sgs3-GFP, fkh-Gal4/ UAS-cherryRNAi) were 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-PLCγ-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) were 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 PLCγ-PH-EGFP per 100 μm of linear plasma membrane was quantified in the indicated genotypes; Exo70 knock-down reduced 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 GFP-Sec15 (cyan) localized as dots in contact sites between SGs and the apical plasma membrane (dotted line) (sgs3-dsRed/ UAS-GFP-sec15; 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 GFP-Sec15 indicating the position of the exocyst (arrow) was positioned just at the site where fusion was taking 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, displayed dots of GFP-Sec15 on its side (arrow), just next to the fusion point (asterisk). Transgenes were expressed with fkh-Gal4. 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 a=er egg laying.

SGs from salivary gland distal-most cells were analyzed. Columns display: Hours AEL: hours aAer 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.

Raw data for experiments of Figure 3C and Supplementary Figure 1A.

Salivary gland developmental staging.

Salivary glands analyzed in experiments of the indicated figures. Columns display: Experimental temperature, larval hours of development after egg laying (AEL), equivalent hours of development at 25°C (based on SG phenotype and salivary gland general appearance), presence or absence of Sgs3 in salivary glands, at the developmental time studied, and expected stage of SGs (Mature or Immature).

Penetrance of phenotypes observed after silencing subunits of the exocyst.

Additional RNA 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 RNAi expression of exocyst subunits. “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 fkh-Gal4. 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. “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.

Remaining mRNA levels after RNAi-mediated knock-down of exocyst subunits correlate with the observed phenotypes.

mRNA levels of the indicated genes were measured by qRT-PCR in homogenates from salivary glands. sec3 (A) or exo70 (B) mRNA levels from larva grown at the indicated temperatures were determined in salivary glands expressing sec3RNAi (sgs3-GFP, fkh-Gal4/ UAS-sec3RNAi (A) or exo70RNAiBL(sgs3-GFP, fkh-Gal4/ UAS-exo70RNAiBL) (B) relative to salivary glands expressing cherryRNAi (sgs3-GFP, fkh-Gal4/ UAS-cherryRNAi) from larvae grown at the same temperatures. (C) exo70 mRNA levels from larva grown at 29 °C were determined in salivary glands expressing exo70RNAiV (sgs3-GFP, fkh-Gal4/ UAS-exo70RNAiV) or exo70RNAiBL (sgs3-GFP, fkh-Gal4/ UAS-exo70RNAiBL) relative to salivary glands expressing cherryRNAi (sgs3-GFP, fkh-Gal4/ UAS-cherryRNAi). n = 4 for all genotypes and conditions. Statistical analysis was performed by one-way ANOVA followed by Tukey’s test with a confidence interval higher than 95% (p < 0.05). “ns”= not significant.

Chronic or Acute knock-down of exocyst subunits generate comparable phenotypes.

Phenotypes of control (whiteRNAi) (A-C), sec3RNAi (D-F) or sec15RNAiBL (G-I) salivary glands. The thermo-sensitive Gal80 system (Gal80ts) was utilized to compare salivary glands after silencing the indicated genes throughout larval development (chronic silencing: A, D, G) or at the 3rd larval instar only (acute silencing: B, E, H). Chronic RNAi expression was achieved by growing larvae at the restrictive temperature (29 °C) from larval eclosion onwards (A, D and G). Acute RNAi expression was achieved by growing larvae at the permissive temperature (18 °C) from larval eclosion until early L3 (larval 3rd instar), (B, E and H) and then shifting larvae to the restrictive temperature (29 °C) until analyzed. E: embryogenesis; L1: larval 1st instar; L2: 2nd instar; and L3: 3rd instar. The penetrance of each phenotype observed (Mature Secretory Granules, Immature Secretory Granules or Mesh-Like Structure) was quantified for each genotype and experimental condition (C, F and I). No substantial differences were detected between chronic and acute silencing of exocyst subunits. “n” represents the total number of salivary glands analyzed. controlRNAi (whiteRNAi) (A) n=5, (B) n= 3; sec3RNAi (D) n=6, (E)n=7; sec15RNAiBL(G, H) n= 4.

Expression of GFP-Sec15 can rescue SG maturation after sec15 knock-down.

Expression of sec15RNAiBLat 18 °C (sgs3-dsRed; UAS-PLCγPH-GFP, fkh-Gal4/UAS-sec15RNAiBL) (A) generates salivary glands that do not form mature SGs (MSG) in any of the cells analyzed. Simultaneous expression of GFP-Sec15 (UAS-GFP-sec15/ Sgs3-dsRed; UAS-sec15RNAiBL/ fkh-Gal4) (B) generates salivary glands with MSGs in 67% of the cells. (C) Quantification of each of the three possible phenotypes Mature SGs (MSG), Immature SGs (ISG) or Mesh-like structure (MLS) in control larvae (sgs3-dsRed; UAS-PLCγPH-GFP, fkh-Gal4/UAS-sec15RNAiBL) or after expression of the rescue construct (UAS-GFP-sec15/ sgs3-dsRed; UAS-sec15RNAiBL/ fkh-Gal4). “n” represents the total number of salivary glands analyzed. Control genotype (UAS-PLCγPH-GFP) n=7; Rescue genotype (UAS-GFP-sec15) n=5.

Exocyst down-regulation does not affect general cellular health or homeostasis.

Mitochondrial distribution and morphology (A-B), cis- and trans-Golgi complex distribution and morphology (C-D), nuclear size (E), and autophagy (F) were analyzed in salivary glands of control (UAS-whiteRNAi) and exocyst knock-down salivary glands. Mitochondria were labelled with mito-GFP (UAS-mito-GFP), cis-Golgi with GRASP65 (UAS-GRASP65-GFP), trans-Golgi with RFP-Golgi (UAS-RFP-Golgi), nuclei with EGFP (UAS-EGFP), and autophagy with mChAtg8 (UAS-GFP-mCh-atg8). Larvae were grown at 25 °C to achieve a condition at which knock-down of the exocyst subunits mostly generate cells with immature SGs. RNAis were expressed using fkh-Gal4. (E) Quantification of nuclear area in the indicated genotypes was performed using image J. (F) Quantification of the area coved by mCh-Atg8 foci over the total area of salivary gland analyzed (%) was performed using image J. Statistical analysis was carried-out using one-way analysis of variance (ANOVA) “n” represents the total number of salivary glands analyzed. (E) controlRNAi (whiteRNAi) n= 8; exo70RNAiBL n=5; sec3RNAi n=4. (F) starved controlRNAi(whiteRNAi) n= 5; fed controlRNAi (whiteRNAi) n= 8; fed exo84RNAiV n=5; fed sec3RNAi n=5.

Exocyst down-regulation does not affect apical polarity markers.

(A) CD63 (UAS-CD63-GFP), (B) CD8 (UAS-CD8-mCherry), (C) Rab11 (YFP-Rab11), (D) PI(4,5)P2 (UAS-PLCγ-PH-GFP) and (E) filamentous actin (phalloidine-647) distribution were analyzed in salivary glands of control (UAS-whiteRNAi) or exocyst knock-down salivary glands (UAS-exo70RNAiBL, UAS-sec3RNAi or UAS-exo84RNAiV). Larvae were grown at 25 °C (UAS-exo70RNAiBL or UAS-sec3RNAi) or at 29°C (UAS-exo84RNAiV) to achieve a condition at which exocyst downregulation generate mostly cells with immature SGs. Note that in (A) and (E), for accurate comparisons, the control genotype (UAS-whiteRNAi) was also assayed at different temperatures. Transgenes were expressed with fkh-Gal4. Statistical analysis was performed using (A-B) one-way analysis of variance (ANOVA), (D) Likelihood ratio test followed by Tuckeýs test or (E) Wald Test (p value < 0.05, “ns”=not significant). “n” represents the total number of salivary glands analyzed. (A) controlRNAi(whiteRNAi) n= 6; exo70RNAiBL n=5; controlRNAi(whiteRNAi) n=3; exo84RNAiV n=3. (B) controlRNAi(whiteRNAi) n= 8; exo70RNAiBL n=5; sec3RNAi n=6. (D) n=4. (E) controlRNAi (whiteRNAi) n= 4; exo70RNAiBL n=10; controlRNAi(whiteRNAi) n=6; exo84RNAiV n=4.

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, C and E) or with Bip-sfGFP-HDEL (UAS-Bip-sfGFP-HDEL) (G and I). In control salivary glands (fkh-Gal4/UAS-whiteRNAi) (A and G), the ER displayed a network-like appearance localized in between mature SGs that contain Sgs3-dsRed, and thus no colocalization between the ER marker and Sgs3-dsRed was detected (B and H). Upon expression of sec15RNAi (fkh-Gal4/UAS-sec15RNAiBL) (C), sec10RNAi (UAS-sec10RNAi/ fkh-Gal4) (E) or sec3RNAi (UAS-sec3RNAi; fkh-Gal4) SGs did not form, and Sgs3-dsRed was retained in the ER, as demonstrated by colocalization of both markers in the two-dimensional line scan analysis (D, F and J). RNAis were expressed using fkh-Gal4. Scale bar 10μm.

Quantification of colocalization between ER markers and Sgs3.

Pearsońs coefficient to evaluate colocalization between the ER markers UAS-GFP-KDEL (A) or UAS-Bip-sfGFP-HDEL (B) and Sgs3-dsRed in control salivary glands (UAS-whiteRNAi) or after exocyst knock-down (UAS-sec15RNAiBL). Larvae were grown at 29 °C. Statistical analysis was performed using one-way analysis of variance (ANOVA). Analysis corresponds to data shown in Figure 4. “n” represents the total number of salivary glands analyzed. (A) n=3, (B) n=2.

The exocyst is required to maintain the typical morphology of the Golgi complex.

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. cis-Golgi (UAS-Grasp65-GFP) (A) and trans-Golgi (UAS-RFP-Golgi) (B) markers were analyzed In control salivary glands (fkh-Gal4/ UAS-whiteRNAi) or in exocyst-down-regulated salivary glands (UAS-sec15RNAiBL, UAS-sec3RNAi or UAS-exo84RNAiV). RNAis were expressed using fkh-Gal4. Scale bar 10 μm. Bar graphs show the quantification of the penetrance of the Golgi complex phenotype. Statistical analysis was performed using a Likelihood ratio test followed by Tuckeýs test (p value < 0.05, “ns”=not significant). “n” represents the total number of salivary glands analyzed. (A) controlRNAi(whiteRNAi) n= 3; sec15RNAiBL n=11; sec3RNAin=3. (B) controlRNAi (whiteRNAi) n= 9; sec15RNAiBL n=4; sec3RNAi n=4; exo84RNAiV n=3.

Incorporation of maturation markers to SGs in wild type salivary glands.

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 localized at the basolateral plasma membrane of salivary cells (A), while CD63 localized at the apical plasma membrane (E). Scale bar 100 μm. Both markers localized 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 mediates the acquisition of secretory granule maturation factors.

Maturation proteins Syt-1 and CD63 were gradually incorporated to the membrane of SG. 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 fluorescence intensity among genotypes, and statistical analysis were performed using one-way analysis of variance (ANOVA). “n” represents the number of salivary glands: (C) controlRNAi n= 7, sec3RNAin=6; (F) controlRNAi n=7, sec3RNAi n=13; (I) controlRNAi n= 12, sec3RNAi n= 9; (L) controlRNAi n= 8, sec3RNAi n= 8; (O) controlRNAi n= 7, sec3RNAi n= 10; (R) controlRNAi 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 localized around mature SGs (A). Overexpression of Rab11CA arrested 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, GFP-Sec15 localized as foci on the periphery of mature SGs (sgs3-dsRed/ UAS-GFP-sec15; fkh-Gal4). (E) Knock-down of Rab11 (sgs3-dsRed; UAS-rab11RNAi/ fkh-Gal4) generated immature SGs and prevented GFP-Sec15 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.