Arp2/3-mediated F-actin formation controls regulated exocytosis in vivo

The actin cytoskeleton plays crucial roles in many cellular processes, including regulated secretion. However, the mechanisms controlling F-actin dynamics in this process are largely unknown. Through 3D time-lapse imaging in a secreting organ, we show that F-actin is actively disassembled along the apical plasma membrane at the site of secretory vesicle fusion and re-assembled directionally on vesicle membranes. Moreover, we show that fusion pore formation and PIP2 redistribution precedes actin and myosin recruitment to secretory vesicle membranes. Finally, we show essential roles for the branched actin nucleators Arp2/3- and WASp in the process of secretory cargo expulsion and integration of vesicular membranes with the apical plasma membrane. Our results highlight previously unknown roles for branched actin in exocytosis and provide a genetically tractable system to image the temporal and spatial dynamics of polarized secretion in vivo.

R egulated exocytosis is a fundamental cellular process that involves the release of stored secretory cargo from membranous vesicles into the extracellular space. This type of secretion involves the formation of secretory vesicles and their subsequent docking and fusion with the plasma membrane (PM) in response to an external stimulus. Various tissues have different modes of vesicle formation and release, likely due to the different physical and functional properties of the secreted cargo as well as the size/geometry of the secretory vesicles. Regulated secretion is used by specialized tissues to produce and secrete bioactive molecules that mediate diverse functions including neurotransmission, immunity, reproduction and digestion (reviewed in refs 1-3).
Recent studies have highlighted essential, yet often disparate roles for the actin cytoskeleton in regulated exocytosis and in compensatory endocytosis. For example, actin along the cortex of cells acts as a physical barrier to prevent premature fusion of granules with the PM 4-6 . In Xenopus eggs, where secretory vesicles never integrate into the PM but are maintained as shells, actin and myosin recruitment enable vesicles to be retrieved via compensatory endocytosis [7][8][9][10] . Other studies have demonstrated that F-actin is required for proper vesicle compression and expulsion of cargo [11][12][13][14] and to regulate the expansion of the fusion pore 15 . In addition, intravital imaging in rodent salivary glands (SGs) has shown that F-actin is required to regulate both the stabilization and collapse of secretory vesicles after fusion with the PM in vivo 16 . However, the molecular machinery controlling the formation of specific F-actin structures and the spatial kinetics of actin assembly in the process of cargo expulsion and membrane integration has not been fully defined 3 .
Actin exists as monomeric units (G-actin) and in a variety of filaments that can be linear, branched or bundled (F-actin). Linear actin filaments are initiated by the formin-family of actin nucleators/elongation factors that are activated by the concerted action of small Rho-GTPases and phosphoinositides [17][18][19] . Rho and formin influence actin coat formation on lamellar bodies during surfactant secretion 12,13 . Branched actin filaments, which are essential for cell polarity and migration 20,21 , are formed through the coordinated action of the Actin-related protein 2/Actin-related protein 3 (Arp2/3) complex and a variety of nucleation-promoting factors (NPFs), such as WASp, Wash, Whamy and suppressor of cAR (SCAR) 20,[22][23][24] . Arp2/3 present on linear actin filaments is subsequently activated by NPFs, which bind Arp2/3 and induce a structural change. Branched actin is then formed at the site of Arp2/3 attachment at a 70°angle from the existing actin filament 25,26 . Actin nucleators influence glucose-induced biphasic insulin secretion, but their specific effects on actin remodelling in this process are unclear 27 . The role of branched actin assembly and structure is less well understood in other forms of secretion that involve large secretory vesicles containing bulky cargo 3 .
The factors that govern regulated secretion have been challenging to decipher, as many mammalian cells lose polarity and secretory capacity once excised and cultured ex vivo 28 . Additionally, in many systems, the small size of vesicular structures and the lack of markers for cargo impede the ability to image actin dynamics, secretory vesicles and secretion with sufficient resolution. Furthermore, limited genetic tools in many systems have restricted the ability to interrogate the function of specific genes involved in actin formation in vivo.
To investigate the role of actin dynamics and branched actin in secretion, we performed high-resolution, real-time imaging at single-granule resolution in Drosophila SGs, which are amenable to genetic manipulation and contain large secretory granules (3-8 mM in diameter) filled with high-molecular-weight cargo. Using Drosophila lines expressing fluorescently-labelled molecules, we are able to directly image secretory vesicle cargo, apical membrane dynamics and actin and myosin recruitment in real time to define the temporal and spatial sequence of events occurring during regulated exocytosis. By performing 4-dimensional (4D) imaging of actin dynamics during secretion, we show rearrangement of cortical actin at the point of vesicle fusion followed by coordinated recruitment of actin to the fused vesicle from the PM. We further demonstrate that fusion pore formation and redistribution of the phosphoinositide, PIP 2 , occurs before actin recruitment. By imaging cargo directly, we show that fusion pore formation and cargo secretion are temporally distinct events, with the latter being dependent on actin for completion. Finally, using in vivo RNA interference (RNAi), we identify essential roles for the branched actin nucleators Arp2, Arp3 and their activator, WASp, in mediating cargo expulsion and secretory granule membrane integration with the PM. This study demonstrates a previously unknown role for branched actin nucleators in exocytosis and offers a high-resolution, genetically tractable platform for interrogating the role of any factor involved in actin dynamics and regulated secretion in a living organ in real time.

Results
Spatial and temporal dynamics of regulated secretion. The Drosophila larval salivary system consists of two glands comprised of secretory epithelial cells that connect to a common duct (Fig. 1a). Secretory cells are filled with large secretory granules (3-8 mM in diameter) containing high molecular weight, highly glycosylated mucins (salivary gland secretion proteins or sgs proteins) 29,30 . Regulated secretion in Drosophila SGs begins with a small pulse of the steroid hormone 20-hydroxyecdysone (20E) during early third instar larval stage. This stimulus induces the expression of the sgs genes that encode the cargo that will be packaged into secretory vesicles; then, a second pulse of 20E during late third instar instructs these newly formed secretory granules to fuse with the apical PM to begin secretion [31][32][33] . To image secretion events in real time, we took advantage of Drosophila lines expressing fluorescent markers for secretory cargo (Sgs3-GFP) 32 , F-actin (Lifeact-Ruby, Lifeact-RFP or Lifeact-GFP) 34,35 and the apical membrane (Myr-tdTomato) 36 . SGs from Sgs3-GFP-expressing larvae were harvested during late third instar and cultured ex vivo. Cultured SGs maintain their polarity and respond to the exogenous addition of 20E, which triggers fusion of the secretory vesicles with the apical PM and release of their contents into the lumen (Fig. 1b). As secretion proceeds, the diameter of the lumen expands as it fills with the Sgs3-GFP cargo ( Fig. 1b and Supplementary Movie 1), similar to what is seen in vivo ( Supplementary Fig. 1a). (Real-time videos of all still images shown throughout this paper are in the Supplementary Movies section.) Imaging the dynamics of individual secretory granules using Drosophila lines expressing Sgs3-GFP cargo and the apical PM marker Myr-tdTomato revealed apical membrane deformations at the point of vesicle fusion (Fig. 1c). In addition, individual secretory vesicles undergo a slight expansion after fusion with the apical PM, likely due to hydration-related expansion of the mucin cargo. Real-time imaging of secretory cargo revealed that secretion proceeds with complete expulsion of cargo followed by integration of the vesicle membranes with the PM (Fig. 1c and Supplementary Movie 2).
We next imaged the dynamics of actin recruitment to individual secretory granules using Drosophila lines expressing both Sgs3-GFP and Lifeact-Ruby. Secretory vesicles in proximity to the apical membrane are rapidly coated with actin, which precedes vesicle compression and membrane integration ( Fig. 1d and Supplementary Movie 3). To image actin recruitment around the entirety of the vesicle, we performed three-dimensional imaging in real time (4D imaging) in Drosophila lines expressing Lifeact-GFP. Interestingly, 4D reconstruction of actin dynamics showed that cortical actin present along the apical membrane is cleared or disrupted at the site of vesicle fusion, forming a small hole that continues to expand in size ( Fig. 1e and Supplementary  mucinous cargo at single granule resolution that involves dynamic rearrangement of both the cortical actin and the apical membrane at the site of secretory vesicle fusion. This process culminates in complete expulsion of cargo and integration of the granular membrane with the PM. We next addressed the temporal order of membrane fusion (fusion pore formation) and actin recruitment to the secretory vesicles. By infusing a small molecular weight (MW) fluorescent dextran (A647-10 kDa dextran, stoke radius B2 nm (ref. 15)) into the lumens of SGs expressing Lifeact-GFP, we demonstrate that the fusion pore between the vesicle and the PMs forms before F-actin recruitment to the secretory vesicle (Fig. 2a, d and Supplementary Fig. 2a). Indeed, dextran diffusion from the SG lumen into the vesicle was detected before Lifeact-GFP (F-actin) on vesicle membranes (Supplementary Movie 6). The average time difference in the detection of dextran relative to actin was 57 ± 18 s (n ¼ 11; ± refers to standard deviation). In addition, we did not see any evidence for compound exocytosis (for example, the sequential fusion of vesicles with one another at the PM), consistent with what was previously reported in mammalian SGs 16 .
Membrane mixing and the redistribution of phosphoinositides are known to regulate actin polymerization and membrane fusion events in other systems 37 . In particular, the membrane phosphoinositide PI(4, 5)P 2 (PIP 2 ) is known to stimulate actin polymerization by regulating the activity of actin nucleators 38,39 . Therefore, we next examined whether redistribution of PIP 2 occurs during SG secretion by using Drosophila lines expressing both the PIP 2 reporter PLCd-PH-EGFP 40 and Lifeact-RFP. Indeed, evidence for membrane mixing via PIP 2 movement from the apical PM to secretory vesicle membranes is seen (Fig. 2b,e and Supplementary Movie 7). Interestingly, PIP 2 recruitment precedes F-actin recruitment (by 51 ± 19 s (n ¼ 12)), consistent with a role for PIP 2 in F-actin formation along secretory vesicle membranes ( In rodent exocrine glands, it was shown that the actin motor non-muscle myosin II is recruited onto the secretory granules, but the temporal sequence of events is not known 16 . To determine the kinetics of myosin recruitment relative to actin recruitment, we performed time-lapse imaging in flies co-expressing the myosin marker non-muscle myosin II (Zip-GFP) 41,42 and Lifeact-Ruby. As shown in Fig. 2c,f and Supplementary Fig. 3a and Supplementary Movie 8, myosin is detected on fused vesicles after F-actin. The average time difference in the detection of actin relative to myosin was 25±13 s (n ¼ 11). Altogether, our data show that secretion in Drosophila SGs begins with fusion pore formation and redistribution of PIP 2 , followed by directional actin recruitment to secretory vesicle membranes and, finally, myosin recruitment. Moreover, secretion of the cargo is followed by complete membrane integration of secretory vesicles with the apical PM. Therefore, our real-time imaging system provides spatial and temporal resolution to image dynamic events occurring on the membranes of individual secretory granules in a living organ.
Arp2/3 F-actin formation is required for secretion. The structural complexities associated with large, highly glycosylated cargo such as mucins 43,44 , may require actomyosin contractile forces for cargo expulsion and membrane integration. We therefore examined the role of F-actin in SG secretion. We had previously shown that pharmacological disruption of the actin cytoskeleton by latrunculin A (LA) or cytochalasin D (CD) treatment impairs the gradual collapse of the secretory granules in rodent SGs in vivo 16 . Likewise, treatment of Drosophila SGs with LA or CD also resulted in disrupted secretion ( Fig. 3a,b, Supplementary Fig. 5a,c and Supplementary Movies 9-12). Treatment with either drug severely inhibited vesicle collapse as fused vesicles expanded dramatically in size, underwent compound fusion and failed to undergo membrane integration (Fig. 3a,b, Supplementary Fig. 5a,c and Supplementary Movies 9-12). The efficacy of each treatment on the actin cytoskeleton was assessed by examining both Lifeact-Ruby in real time and by staining fixed glands with phalloidin (Fig. 3c, Supplementary  Figs 4a,b and 5b and Supplementary . Taken together, these results demonstrate a requirement for F-actin in regulated secretion in this system and highlight its role in vesicle collapse and the prevention of compound fusion. Conventional light microscopy cannot resolve the organization of actin filaments on the surface of the membranes of the secretory granules in Drosophila SGs. Nonetheless, based on the fact that F-actin on the granules prevents compound exocytosis, we speculated that filaments may be tightly organized around the membranes, possibly by employing extensive branching. Branched actin is nucleated from linear filaments via the Arp2/3 complex and NPFs. To determine which of these proteins may be involved in regulated secretion within Drosophila SGs, we performed immunofluorescent staining on actively secreting glands. Arp3, WASp, Whamy and SCAR (but not Wash) each co-localized with phalloidin on granules that had fused with the apical PM, suggesting a role for these factors in secretion (Fig. 4a). We next examined the temporal order of recruitment of the Arp3 complex onto the secreting granules by creating a Drosophila line expressing both GFP-labelled Arp3 (Arp3-GFP) and Lifeact-Ruby. Real-time imaging of Lifeact-Ruby and Arp3-GFP during secretion revealed that F-actin is seen on granules before Arp3 recruitment (Fig. 4b,c, Supplementary Fig. 3b and Supplementary Movie 17). The average time difference in the detection of F-actin relative to Arp3 was 29±14 s (n ¼ 13). As the Arp2/3 complex is known to bind linear actin and is required for branched actin formation, our results suggest a model where fused vesicles are initially coated with linear actin, which is detected by Lifeact, and then subsequently bound by Arp2/3 to form a branched actin network.
To investigate whether branched actin is required for regulated exocytosis, we next performed in vivo knockdown of components of the Arp2/3 complex in flies expressing Sgs3-GFP or Lifeact-Ruby. RNAi to Arp3 substantially decreased Arp3 expression and resulted in granules that fused with the apical membrane, but failed to secrete cargo and failed to undergo membrane integration (Fig. 5a, Supplementary Figs 6a,b and 7a and Supplementary Movies 18 and 19). Upon loss of Arp3, fused granules continued to enlarge, similar to what was observed in SGs treated with LA. However, unlike LA treatment, no evidence of compound exocytosis was seen, suggesting that linear actin still present on granules may function to block compound fusion. Interestingly, some F-actin (likely linear actin) was still detected on fused granules in the Arp3 RNAi background, although no longer in a homogenous distribution ( Supplementary Fig. 6c . We next examined the role of the Arp2/3 activator, WASp. RNAi to WASp, an NPF that can activate Arp2/3, also resulted in granules that grew larger in size, failed to expel cargo and failed to undergo membrane integration; these granules also maintained limited and unevenly distributed staining for F-actin (likely detecting linear actin) and did not show evidence of compound fusion (Fig. 5c and Supplementary Figs 6a,b,c,e and 7c and Supplementary . Finally, large, expanded granules could also be seen in the SGs in the intact pupae in Arp3 RNAi, Arp2 RNAi or WASp RNAi backgrounds ( Supplementary Fig. 8), again indicating that our ex vivo SG system faithfully recapitulates what is occurring in vivo. Taken together, our studies demonstrate an essential role for the branched actin nucleators Arp2, Arp3 and WASp in secretory cargo expulsion and membrane integration during regulated exocytosis in vivo (Fig. 5d).

Discussion
Here, we show temporal and spatial dynamics of F-actin formation during regulated exocytosis and define essential roles for the branched actin nucleators Arp2, Arp3 and WASp in cargo secretion and membrane integration (Fig. 5d). By imaging actin dynamics in four dimensions at single granule resolution in a secreting organ, we demonstrate that actin along the PM is dynamically cleared at the point of vesicle fusion. This observation supports a role for cortical actin acting as a barrier to premature vesicle fusion, as has been proposed for other modes of secretion [4][5][6]45 . Shortly after this cortical rearrangement, actin begins to expand over the secretory vesicle from the site of PM contact. Previous studies imaging actin (as it is involved in exocytosis) in two dimensions suggest that recruitment occurs simultaneously over all regions of the vesicle 46 . However, our 4D renderings provide spatial and temporal resolution that has not been seen previously and show that actin recruitment to the secretory vesicle begins at the apical PM. These dynamic changes in actin organization suggest a highly ordered process whereby factors on the PM and vesicle act in concert to remodel actin to mediate secretory vesicle docking/fusion as well as to reform the actomyosin machinery around the fused vesicle. This mode of actin recruitment may provide the continuity with cortical actin required to generate the appropriate forces needed for cargo expulsion/release, especially in cases of large, highly glycosylated cargo, such as mucins.
Actin recruitment to secretory vesicles was preceded by the formation of the fusion pore between the apical PM and the secretory vesicle membrane. In addition, membrane mixing and redistribution of the membrane lipid PIP 2 also occurred before F-actin recruitment. As PIP 2 is known to mediate actin formation in other systems 38,39 , our results suggest that this membrane mixing may trigger actin polymerization on secretory granule membranes. Interestingly, actin polymerization appears to occur in two distinct steps, as the recruitment of Lifeact to secretory granules is detected before Arp3 recruitment. As the Arp2/3 complex is known to bind linear actin, this suggests that actin formation on fused secretory granules begins with linear actin formation (detected by Lifeact), which originates from the apical PM. Linear actin is then bound by the Arp2/3 complex, which catalyses the formation of branched actin. Myosin recruitment occurs after actin is present, as has been seen in cell-based systems 12 . Future work will employ this system to temporally dissect the recruitment of other factors involved in secretion.
The ordered recruitment of linear and branched forms of actin suggests essential roles for each in regulated secretion. However, specific roles for branched actin in regulated secretion were previously unknown. Taking advantage of genetic tools, we directly interrogated the role of branched actin in regulated  secretion by performing RNAi to the genes that regulate its formation. In vivo knockdown of Arp2 or Arp3, both of which are involved in the synthesis of branched actin, resulted in similar phenotypes, where secretory vesicles fused with the PM but were unable to secrete their cargo and unable to undergo membrane integration. These vesicles grew very large and in some instances, separated from the PM and floated back into the cytoplasm. In addition, in vivo knockdown of the NPF WASp resulted in similar phenotypes, identifying it as an additional regulator of exocytosis in this system. Taken together, our results suggest that branched actin is required to generate the force necessary to complete expulsion of the secretory cargo and integration of vesicular membranes with the PM. In the absence of branched actin, the fused secretory vesicles continue to expand in size, likely due to the hydration and expansion of the highly glycosylated mucinous cargo present within. These results have implications for the importance of branched actin formation in other systems such as the digestive tract, where secretion of large, highly glycosylated/ cross-linked cargo is essential for extracellular matrix formation, microbial interactions and protection of epithelial cell surfaces 47,48 .
Our results also suggest unique roles for linear actin in regulated exocytosis. LA and CD, which disrupt the formation of both linear and branched actin, resulted in compound fusion of secretory vesicles with one another, which was not seen upon knockdown of Arp2, Arp3 or WASp. This suggests that the linear actin first recruited to the fused secretory vesicle may act as a barrier to prevent the fusion of other secretory vesicles. However, we cannot rule out the possibility that residual branched actin present in our knockdown animals may be able to prevent compound fusion events. Future studies combining fluorescent imaging and electron microscopy of genetically manipulated SGs will aim to identify specific actin structures present on fused secretory granules and how they influence vesicle interactions.
Our study is unique in that we image secretory cargo as a direct indicator of secretion. Previous studies have used visualization of the fusion pore as evidence of cargo secretion 8 . However, in our system, fusion pore formation and cargo expulsion are temporally distinct events, indicating that fusion pore formation alone is not sufficient for secretion of vesicle contents. In addition to the forces mediated by branched actin, other factors may be required for the secretion of large, glycosylated cargos. This system can be used to interrogate other genes that regulate the processing, packaging and release of complex cargos.
In summary, our study highlights coordinated actin clearance and directional recruitment from the PM as well as essential roles for branched actin nucleators in regulated exocytosis in a living, secreting organ. The powerful combination of Drosophila genetics with in vivo and ex vivo imaging allows one to rapidly interrogate the role of factors in many aspects of secretion, including vesicle biogenesis, vesicle movement, fusion with the PM, release of granule cargo and expansion of cargo once in the lumen. Given that many genes and mechanisms involved in secretion are conserved across species, this system can provide insight into the factors that regulate mammalian secretion.  50 and were the following stocks: VDRC #29944 (P{UAS-Arp2IR), VDRC #108951 (P{UAS-Arp3IR), VDRC #108220 (P{UAS-WaspIR), VDRC #60008 (w 1118 ; P{UAS-dicer2 w þ }) and VDRC 60009 (w 1118 ; P{UAS-dicer2 w þ }). The wild-type stocks used were either Oregon R or w 1118 (VDRC #60000). The Gal4 driver line used in these studies was Bloomington stock #6978 (w 1118 ; P{GawB}c135), which drives expression in the Drosophila SG beginning during embryonic stage 15 and continuing through the third instar larval stage. The c135-Gal4 driver stock was recombined with Sgs3-GFP to generate c135-Gal44Sgs3-GFP. VDRC #60008, which expresses dicer2 under the control of the UAS promoter, was crossed to VDRC #108951 and VDRC #108220 to enhance RNAi-mediated knockdown. VDRC #60009, which also expresses dicer2, was crossed to VDRC #29944 to enhance gene knockdown. Sgs3-GFP was recombined with Bloomington #3554 to generate Sgs3-GFP, UAS-Lifeact-Ruby. Sgs3-GFP was recombined with Bloomington #3222 1to generate Sgs3-GFP, UAS-Myr-tdTomato. Bloomington #3554 was recombined with Flytrap line #CC01626 to generate Zip-GFP, UAS-Lifeact-Ruby. The c135-Gal4 driver was recombined with Zip-GFP, UAS-Lifeact-Ruby to generate c135-Gal44Zip-GFP, UAS-Lifeact-Ruby. Bloomington #3554 was recombined with Bloomington #39723 to generate UAS-Arp3-GFP, UAS-Lifeact-Ruby. The c135-Gal4 driver was recombined with Bloomington #58362 to generate c135-Gal44 UAS-Lifeact-RFP. All Drosophila crosses were kept on MM media (KD Medical, Inc.) at 25°C unless specified otherwise. Crosses to generate expression of dsRNA were performed using flies from a UAS-IR transgenic line and the c135-Gal44Sgs3-GFP or c135-Gal44Zip-GFP, UAS-Lifeact-Ruby stocks.
Ex vivo SG culture and real-time imaging. SGs from third instar wandering larvae were dissected in Schneider's Drosophila medium (Gibco) and transferred to glass bottom culture dishes (MatTek) containing 50 ml media. To immediately image glands that were actively secreting, the majority of the media were then removed from the dish and a 13-mm, 0.05 mm, polycarbonate membrane filter (Sterlitech) was gently placed on top of the gland. The media that were removed from the dish were then placed on top of the membrane filter and the sample was subsequently imaged. For imaging sessions longer than 2 h, 0.5% low melt agarose in Schneider's Drosophila medium was layered on top of glands and the sample was subsequently imaged. To induce secretion in non-secreting glands, 20-hydroxyecdysone (Sigma) was added to glands in glass bottom dishes to a final concentration of 700 mM and dishes were placed in a humidified chamber at room temperature for 90 min. The media from these dishes were then removed, a membrane was gently placed on top of the glands and media were added back to the top of the membrane. For experiments involving fluorescent dextran, SGs from non-secreting third instar wandering larvae were dissected and transferred to glass bottom culture dishes containing 50 ml media. Alexa Fluor 647, Alexa Fluor 680 or Rhodamine B Dextran (Invitrogen) was subsequently added to the media to a final concentration of 1 mM and then nutated at room temperature for 45 min. 20-Hydroxyecdysone was then added to a final concentration of 700 mM and placed in a humidified chamber at room temperature for 90 min. The media from these dishes were then removed, a membrane was gently placed on top of the glands and media were added back to the top of the membrane. Quantitative RT-PCR. Quantitative RT-PCR was used to determine efficiency of gene silencing when RNAi was induced. cDNA prepared from SGs from third instar wandering larvae of c135-Gal4,Sgs3-GFP4IR crosses or controls (c135-Gal4,Sgs3-GFP 4VDRC#60000 or OregonR) was used in qPCR reactions. Briefly, RNA was isolated using the RNAqueous-Micro Total RNA Isolation Kit (Ambion). cDNA synthesis was performed using iScript cDNA Synthesis Kit (Bio-Rad). PCR primers ( Supplementary Fig. 7d) were designed using Beacon Designer software (Bio-Rad). QRT-PCR was performed on a CFX96 real-time PCR thermocycler (Bio-Rad) using the SYBR-Green PCR Master Mix (Bio-Rad). RNA levels were normalized to 18S rRNA. Values represent mean value ± s.e.m. Significance values were calculated using two-tailed Student's t-test.
Statistical analyses. Number of replicates used for each analysis is specified in figure legends. To measure percent fluorescent intensity for Lifeact/Dextran, Lifeact/PLCd-PH, Lifeact/Zip or Lifeact/Arp3 experiments, Nikon Elements software was used. A rectangular or oval region of interest (ROI) was drawn around individual secreting granules and the Time Measurement analysis tool was used to measure average fluorescent intensity within the ROI for each channel.
Values were exported to Microsoft Excel and percent fluorescent intensity was calculated. The percent fluorescent intensity of each channel at each time point was calculated as: % fluorescent intensity ¼ (Fluorescent intensity-Fluorescent intensity of T 0 )/(Fluorescent intensity of T max -Fluorescent intensity of T 0 ) Â 100. T 0 is the time frame at which one of the two channels being measured begins to continually increase in fluorescent intensity. T max is the maximum fluorescent intensity value reached for each respective channel being measured. Each graph was plotted from T 0 until at least two time point beyond T max for each channel. Percent fluorescent intensity values that were negative due to fluctuations in intensity values as a result of granule movement were set to 0. Quantification was only performed on granules that did not move within or through the imaging plane during the entire process of secretion. To determine the average time difference in the detection of each component in the pairwise comparisons, we calculated the time between T 0 and when fluorescent intensity of the second component began to continually increase and was at least 1% higher than the previous time point. Measurements were performed on 11 granules for Lifeact/Dextran, 12 granules for Lifeact/PLCd-PH, 11 granules for Lifeact/Zip and 13 granules for Lifeact/Arp3. Averages were calculated for each pair and are expressed as mean values ± s.d. To measure fold change in size of secreting Sgs3-GFP granules, Fiji (http://fiji.sc/Fiji) was used to outline and measure the area of individual secreting granules as a function of time after fusion. Granule fusion with the apical membrane correlates with a change in fluorescent intensity of the fusing granule. Time 0 s represents the frame immediately before a change in fluorescent intensity of a given granule is detected. Values were exported to Microsoft Excel and fold change in granule size was calculated. Graphs represent mean values of all granules measured ± s.d. Quantification was only performed on granules where fusion with the apical membrane could be detected and could be clearly visualized throughout the time frame of analysis. No statistical method was used to predetermine sample size.