Regulated Restructuring of Mucins During Secretory Granule Maturation In Vivo

Significance

Mucins are large transmembrane and secreted proteins that line and protect epithelial surfaces throughout the body. Defects in mucin production and secretion are associated with diseases of the respiratory and digestive tracts. Here we investigate the factors that regulate the production and packaging of these large proteins. We show that distinct mucins form unique compacted structures within the same membranous secretory granule in cells prior to secretion. Moreover, we show that genes regulating pH, calcium, chloride ions and glycosylation influence the compacted mucin structures. Understanding how mucins are efficiently packaged and secreted may provide insight into diseases resulting from defects in mucin secretion.

Abstract

Mucins are large, highly glycosylated transmembrane and secreted proteins that line and protect epithelial surfaces. However, the details of mucin biosynthesis and packaging in vivo are largely unknown. Here, we demonstrate that multiple distinct mucins undergo intragranular restructuring during secretory granule maturation in vivo, forming unique structures that are spatially segregated within the same granule. We further identify temporally-regulated genes that influence mucin restructuring, including those controlling pH (Vha16-1), Ca²⁺ ions (fwe) and Cl⁻ ions (Clic and ClC-c). Finally, we show that altered mucin glycosylation influences the dimensions of these structures, thereby affecting secretory granule morphology. This study elucidates key steps and factors involved in intragranular, rather than intergranular segregation of mucins through regulated restructuring events during secretory granule maturation. Understanding how multiple distinct mucins are efficiently packaged into and secreted from secretory granules may provide insight into diseases resulting from defects in mucin secretion.

Regulated secretion is an essential process where proteins are packaged into membranous secretory granules that then await a signal to deliver their contents to the extracellular space. While diverse cells and tissues undergo regulated secretion, the details of how specific proteins are transported through the secretory apparatus, appropriately modified and subsequently targeted to and packaged within secretory granules remain largely unknown. The details of protein biosynthesis, compaction and secretion are of particular importance for large extracellular matrix proteins (1, 2) such as mucins, which undergo regulated hydration and expansion as they are secreted from specialized cells (3–5).

Mucins are a family of large, highly O-glycosylated transmembrane and extracellular matrix proteins that line and protect epithelial surfaces (6–8). Within the mammalian digestive tract, the intestinal mucin MUC2 is thought to undergo a regulated unfolding process during secretion that results in the formation of an expanded, hydrated layer that lines and protects the epithelia, serving as the first line of defense against pathogens (4, 7, 9, 10). Indeed, defects in the formation of this secreted layer are associated with diseases of the digestive and respiratory tracts (6, 7). In vitro studies using recombinant, truncated mucins have provided models for how these large mucins may be compacted in a calcium- and pH-dependent manner prior to secretion (into disc-like structures or bundles of parallel rods) (5, 11–15). However, we lack fundamental information on how mucins are packaged/compacted in secretory granules in vivo. Do distinct mucins form unique compacted structures in vivo within secretory granules? Are multiple distinct mucins segregated into separate secretory granules or do they coexist within the same granule in vivo? What genes influence mucin packaging in vivo? And finally, how does O-glycosylation, which is a crucial post-translational modification, impact mucin restructuring in vivo?

Drosophila salivary glands (SGs) are the major secretory organ in the fly and synthesize multiple highly O-glycosylated mucins that are packaged into secretory granules and secreted in response to developmentally regulated hormone pulses (16–20). The size of the glands and their secretory structures, in addition to an array of the fluorescently-labeled proteins, including a fluorescently-labeled mucin (Sgs3-GFP (21)), have allowed visualization of early stages of mucin secretory granule formation through final secretion (21–23) (SI Appendix, Fig. 1 A–E). From prior studies, it is known that mucin-containing secretory granules bud from the trans-Golgi network (24–26), grow through homotypic (granule-granule) fusion (24, 26–28) and undergo a poorly understood maturation process to form large, mature granules that are between 3 to 8 μM in diameter (26–28).

Here, we use the SG system to address how mucins are packaged in vivo. Through genetics and multimodal imaging, we find that each secretory granule contains multiple distinct mucins that undergo organized folding/restructuring events during granule maturation to generate distinct structures that become spatially segregated. Moreover, we identify the temporally regulated genes controlling pH and ion concentrations that influence the restructuring events. Our data elucidate key steps and factors involved in intragranular, rather than intergranular segregation of mucins through regulated restructuring in vivo.

Results

Unique Spatial Association of Distinct Structures in Individual Secretory Granules.

We used transmission electron microscopy (TEM) and focused ion beam scanning electron microscopy (FIB-SEM) to characterize the paracrystalline-like structures (29) present in mature secretory granules of Drosophila stage 2 SGs (Fig. 1). The majority of the granule is occupied by bundles of electron-dense filaments that are evenly spaced apart by ∼28 nm (Fig. 1 A and B and SI Appendix, Fig. 1F). Bundles of electron-dense dots that have the same spacing seen for the filaments are also present (Fig. 1 A and C and SI Appendix, Fig. 1F), suggesting that they may represent cross-sections of the filament bundles. Additionally, electron-lucent discs that are ∼78 nm in diameter (Fig. 1 A and D and SI Appendix, Fig. 1G) and contain an electron-dense node (black arrowhead in Fig. 1D ), and an electron-dense matrix (Fig. 1D, white arrowhead) are also present. Comparison of aldehyde and cryofixed (high-pressure frozen, HPF) secretory granules demonstrated that the structures seen are independent of fixation procedures (SI Appendix, Fig. 1 H and I). Additionally, TEM performed on wild-type (w¹¹¹⁸) SGs that do not express the Sgs3-GFP transgene revealed similar structures, indicating that these structures are present independent of the transgene (SI Appendix, Fig. 1J). To better understand the spatial relationship between the electron-lucent discs (hereafter referred to as discs) and the electron-dense matrix, we performed FIB-SEM on SGs. We acquired a three-dimensional (3D) volume of a single granule with an isotropic resolution of 6 nm (Fig. 1E) and used a cropped region (160 × 334 × 200, xyz) within the granule in different orientations for segmentation and 3D reconstruction (Fig. 1 F–J). Semiautomated segmentation was performed and the discs were pseudocolored in red and the electron-dense matrix in green (Fig. 1F’). Analysis of the 3D reconstruction reveals that the electron-dense matrix has indentations (arrows in Fig. 1K) into which the discs are embedded (Fig. 1 K’–K’’). The discs were found to be at the peripheral surface of the electron-dense matrix when viewed at different rotational angles (Fig. 1 L–N) and not within the electron-dense matrix as revealed by orthogonal sections through the 3D volume (Fig. 1O). Taken together, our data indicate each mature secretory granule contains multiple distinct structures with defined dimensions. Moreover, two of these structures (discs and electron-dense matrix) are associated with one another spatially to become partially segregated from the third structure (filament bundles).

Fig. 1.

Mature secretory granules have distinct intragranular structures. TEM of a mature secretory granule from a stage 2 SG (A). Magnified views of the colored boxed regions show that the mature granule has at least three distinguishable structures: electron-dense filaments arranged in parallel bundles (B and C), electron-lucent discs (D; black arrowhead) and an electron-dense matrix (D; white arrowhead). Scale bars, 500 nm for A and 100 nm for B–D. Focused ion beam scanning electron microscopy (FIB-SEM) of stage 2 secretory granules (E–J) shows slices through the region encompassing the discs and electron-dense matrix. Pseudocolored discs (red) and electron-dense matrix (green) (F’) were reconstructed in 3 dimensions in K–N. White arrows in K highlight indented regions of the electron-dense matrix in which the discs are embedded. Images are rotated 90° (L), 180° (M) and 270° (N). Orthogonal slice showing only the red pseudocolored discs is shown in O. Scale bars, 600 nm for E and 100 nm for F–O. Representative images from three independent experiments are shown.

Distinct Mucins Form Unique Intragranular Structures In Vivo.

To determine how these unique intragranular structures relate to the mucins expressed in vivo, we next performed RNA interference (RNAi)-mediated knockdown of the genes encoding the major secretory mucins known to be expressed in the SGs, Sgs1, and Sgs3 (16–18, 30–32). RNAi-mediated knockdown of Sgs1 (using Sgs1^{TRIP.HMC02393} crossed with the driver c135-Gal4, Sgs3-GFP, which expresses in the larval SG) resulted in a complete loss of the disc structures typically seen in mature control granules (Fig. 2 A vs. B). However, no discernable changes in the organization/arrangement of the bundled filaments or the electron-dense matrix were observed (Fig. 2 A vs. B). qPCR of all Sgs genes verified that Sgs1 was specifically knocked down (>95% reduction) in this experiment (Fig. 2D). Western blots of protein extracts from Sgs1^{TRIP.HMC02393} SGs probed with the lectin peanut agglutinin (PNA), which is known to detect the highly O-glycosylated Sgs1 and Sgs3 proteins, showed a specific loss of the Sgs1 band (33) (Fig. 2F). Additionally, an antibody raised to Sgs1 (SI Appendix, Fig. 2 A and B) also verified the specific loss of the Sgs1 band in Sgs1^{TRIP.HMC02393} SG extracts (Fig. 2F). Taken together, these results suggest that Sgs1 is a component of the disc structures present in mature secretory granules.

Fig. 2.

Distinct mucins form unique structures within secretory granules. TEMs on control (A) secretory granules were compared to those in which RNAi was performed to Sgs1 (Sgs1^{TRiP.HMC02393}) (B) and Sgs3 (Sgs3^{TRiP.HMJ30021}) (C). qPCR confirmed the specific knockdown of Sgs1 (D) and Sgs3 (E) expression. Western blots probed with the lectin PNA, which detects the glycosylated Sgs1, shows the specific loss of the Sgs1 band (F; red arrow). Westerns probed with an antibody to Sgs1 showed the loss of the specific Sgs1 band (F; red arrow). Loss of the Sgs3 protein upon Sgs3^{TRiP.HMJ30021} RNAi was confirmed by Western blots probed with the lectin PNA, which showed the specific loss of the glycosylated Sgs3 bands (G; red arrows). Westerns probed with the Sgs3 antibody (33) also showed the specific loss of Sgs3 upon RNAi to Sgs3 (G; red arrows). (F) Quantitation of the diameter of secretory granules from control and Sgs3^{TRiP.HMJ30021} SGs is shown. ****P < 0.0001. (Scale bars, 600 nm for A–C). Error bars show SD. Representative images from three independent experiments are shown.

We next performed RNAi-mediated knockdown of Sgs3 (Sgs3^{TRIP.HMJ30021} crossed with c135-Gal4, Sgs3-GFP), the most abundant mucin present in the SGs. Upon performing TEM on Sgs3^{TRIP.HMJ30021} SGs, we found complete loss of the filament bundles and the electron-dense dots (that likely represented cross-sections of the filament bundles) (Fig. 2 A vs. C). While the filament bundles were absent, the discs and the electron-dense matrix were still present in the Sgs3^{TRIP.HMJ30021} secretory granules. Additionally, we also observed that the Sgs3^{TRIP.HMJ30021} secretory granules were significantly smaller in diameter than the controls (quantitated in Fig. 2H), likely due to the loss of Sgs3, which is one of the most abundantly expressed proteins in the SG. qPCR demonstrated that RNAi to Sgs3 resulted in a specific reduction in Sgs3 expression by >95% (Fig. 2E). Western blots probed with an antibody to Sgs3 and the PNA lectin verified the specific loss of Sgs3 and the highly O-glycosylated Sgs3 bands (33) (Fig. 2G). Therefore, these data suggest that the Sgs3 mucin is a component of the bundled filament structures present within mature secretory granules. Attempts to immunolocalize Sgs1 and Sgs3 to structures in granules did not result in specific staining, likely because antibody binding sites are no longer accessible once these proteins have undergone restructuring and compaction within secretory granules. Taken together, our results suggest that two distinct mucins adopt unique intragranular structures within the same secretory granule. Additionally, given that mature wt secretory granules always contained both filaments and discs, our results support a model where distinct mucins are not segregated into unique secretory granules, but rather are packaged into the same secretory granule and segregated through specific restructuring events.

Mucins Undergo Regulated Restructuring During Secretory Granule Maturation.

To examine the temporal formation of these mucin structures, we performed TEM on SGs at different stages of secretory granule development. SGs display a gradient of secretory granules during stage 1, with granule biogenesis beginning at the distal tip and proceeding proximally (Fig. 3 A’’–D’’) (21). We therefore sliced stage 1 SGs from proximal to distal (at 25-μm intervals) to capture secretory granules at various stages of development (Fig. 3 A’’–D’’). In the initial proximal sections captured, we observed several small granules scattered throughout the cytoplasm ranging between 200 to 400 nm in diameter (Fig. 3 A-A’ and SI Appendix, Fig. 1K). These small granules often had no discernable internal structures or the beginnings of what appear to be irregularly spaced filaments (Fig. 3A’). In subsequent sections moving more distally, we observed granules ranging from 600 to 900 nm in diameter that contained more irregularly spaced filaments and a few small regions with higher-electron density (Fig. 3 B-B’ and SI Appendix, Fig. 1L). Further distal sections revealed larger diameter granules (900-1200 nm) with more distinct filaments in addition to larger electron-dense regions (Fig. 3 C-C’ and SI Appendix, Fig. 1M). In sections with secretory granules ranging from 1,200 to 1,800 nm in diameter, filaments were very distinct and arranged in parallel stacks, along with scattered electron-dense and electron-lucent zones (Fig. 3 D-D' and SI Appendix, Fig. 1N). Finally, in stage 2 SGs, where most cells have secretory granules >1,800 nm in diameter, all three structures were present (Fig. 3E-E’ and SI Appendix, Fig. 1O). Taken together, these results suggest that mucins undergo regulated restructuring during secretory granule maturation, with disordered filaments forming first, followed by electron-dense aggregates and electron-lucent regions. Sgs3 filaments gradually arrange in parallel while Sgs1 electron-lucent discs associate with the periphery of the electron-dense regions. Our results support a model where multiple distinct mucins exist in each granule and adopt distinct structures as secretory granules mature.

Fig. 3.

Mucins undergo regulated restructuring during secretory granule maturation. TEMs show the formation of distinct structures over time during secretory granule maturation. Stage 1 SGs were sectioned and imaged from proximal to distal (A’’–D’’). Secretory granules moving proximal to distal have diameters ranging from 200 to 400 nm (A), 600 to 900 nm (B), 900 to 1,200 nm (C), and 1,200 to 1,800 nm (D). In stage 2 SGs (E’’), secretory granules are greater than 1,800 nm in diameter (E) and display ordered filament bundles and distinct electron-lucent discs in close association with the electron-dense matrix. Boxed areas are shown magnified in A’–E’. (Scale bars, 200 nm for A, 600 nm for B–E, and 100 nm for A’–E’). Representative images from three independent experiments are shown.

Mucin Restructuring Is Dependent on Genes Controlling Calcium and pH In Vivo.

We next set out to identify genes involved in secretory granule maturation and the restructuring of mucins. To begin, we performed qPCR on SGs at various stages (stage 0, 1, 2, and 3; SI Appendix, Fig. 1 A–D) of secretory granule development to identify genes that are up-regulated as development proceeds (SI Appendix, Fig. 2 C–E). Previous in vitro studies have suggested that an acidic pH is required for conformational changes necessary to package large proteins, such as mucins, into more compact structures within secretory granules (5, 34). We therefore examined the expression of genes that encode subunits of the vacuolar-type ATPase (v-ATPase) proton pump, which regulates the pH within intracellular compartments (35) (SI Appendix, Fig. 2C). Vha16-1, which encodes the 16-kD subunit-1 of the v-ATPase proton pump, was highly expressed during all stages of secretory granule development and peaked in expression during stage 2 (SI Appendix, Fig. 2C). RNAi to Vha16-1 (using two independent lines, Vha16-1^VDRC49291 or Vha16-1^VDRC10449 , which were crossed with the driver c135-Gal4, Sgs3-GFP) resulted in secretory granules that were circular and swollen in appearance when compared to control granules (Fig. 4 A vs. B and SI Appendix, Fig. 3 A–C). Quantitation of circularity showed statistically significant differences between control and Vha16-1^VDRC49291 or Vha16-1 ^VDRC104490 secretory granules (Fig. 4E). To further test the specific role of the proton pump and pH in secretory granule morphology, we treated SGs with the v-ATPase inhibitor Bafilomycin A1 (36). Bafilomycin A1 treatment likewise resulted in swollen and circular secretory granules (Fig. 4 C vs. D), mimicking the phenotype seen upon knockdown of Vha16-1. To further understand the ultrastructural changes resulting from the loss of the v-ATPase, we next performed TEM on Vha16-1^VDRC49291 SGs. RNAi to Vha16-1 resulted in the loss of the Sgs3 filament bundles, suggesting that an acidic pH is required for the formation of these structures (Fig. 4 J vs. K). The Sgs1 discs and the electron-dense matrix were still present but were no longer aggregated within the granule, but dispersed along the periphery of the granule. These results suggest that Vha16-1 is required for the appropriate folding/restructuring of Sgs3 in vivo.

Fig. 4.

Intragranular restructuring of mucins is dependent on genes controlling pH, calcium, and chloride. Confocal images of control (A) and RNAi-mediated knockdown of Vha16-1 (Vha16-1^VDRC49291) (B) are shown. SGs untreated (C) or incubated with the v-ATPase inhibitor Bafilomycin A1 (D) recapitulate the round secretory granule phenotype seen upon knockdown of Vha16-1. Circularity measurements for two independent RNAi lines, Vha16-1^VDRC49291 and Vha16-1^VDRC104490, are shown in (E). (F) The protein trap fwe-YFP shows that fwe localizes to secretory granule membranes. Confocal images of the RNAi lines fwe^VDRC39596 (G), Clic^VDRC105975 (H) and ClC-c^VDRC6466 (I) demonstrate irregular granule morphologies. TEMs of control (J), Vha16-1^VDRC49291 (K), fwe^VDRC39596 (L), Clic^VDRC105975 (M) and ClC-c^VDRC6466 (N) and pgant9^Δ/Df(2R) (O) are shown. Higher magnification TEMs for control (P) and pgant9^Δ/Df(2R) (Q) are shown. (R) Quantitation of the distance between Sgs3 filaments in control and pgant9Δ/Df(2R) granules is shown. (Scale bars, 10 μm for A–I, 600 nm for J–O, 200 nm for P and Q). ****P < 0.0001. Representative images from three independent experiments are shown.

In vitro reconstitution experiments have indicated that calcium is required for proper mucin folding (5, 37, 38). To determine how genes controlling calcium may influence mucin restructuring in vivo, we analyzed the gene flower (fwe), which encodes a transmembrane calcium ion channel that regulates synaptic vesicle exo- and endocytosis (39) and is up-regulated in the larval SG (SI Appendix, Fig. 2D). Confocal imaging of stage 2 SGs expressing a fluorescently tagged fwe genomic rescue construct (fwe-YFP) (39) shows that fwe localizes to secretory granule membranes (Fig. 4F). RNAi knockdown of fwe (using two independent RNAi lines, fwe^VDRC#39596 and fwe^TRiP.GL01498) (SI Appendix, Fig. 3 D–F) resulted in SGs containing very large granules with low-GFP intensity (Fig. 4G and SI Appendix, Fig. 3F). TEM analysis of SGs from fwe^VDRC39596 animals showed large granules that lacked the Sgs1 discs and the electron-dense matrix (Fig. 4 J vs. L). Additionally, the organized bundled filaments typical of Sgs3 were no longer present. Instead, secretory granules were filled with very long winding filaments that spanned the entire lumen of the granule, making it look like a ball of yarn (Fig. 4 J vs. L). Therefore, loss of fwe disrupts the restructuring of both Sgs1 and Sgs3, suggesting that the formation of these structures is calcium-dependent in vivo.

Loss of Clic or ClC-c Affects Secretory Granule Morphology.

Along with acidic pH, in vitro studies have suggested a role for various ions in charge shielding to promote packaging of mucin polymers (5, 37, 38). qPCR data from SGs at various stages revealed that genes encoding two chloride channels (Clic and ClC-c) are highly expressed and up-regulated during stages 1 and 2 (SI Appendix, Fig. 2E). Clic encodes an intracellular chloride transporter (40) and ClC-c is a homolog of human CLCN3(CLC-3), which encodes a voltage-gated chloride transporter that is involved in endosomal acidification (41, 42). RNAi to Clic (using two independent lines, Clic^VDRC#105975 and Clic^{VDRC #28303}), resulted in granules that were circular, swollen and much larger in diameter than control granules (Fig. 4 A vs. H and SI Appendix, Fig. 3 G–I). TEM examination revealed a notable loss of the electron-dense matrix relative to control and dispersal of the Sgs1 discs throughout the granule (Fig. 4 J vs. M). Sgs3 filament bundles could still be seen but appeared to be shorter and less distinct relative to control (Fig. 4 J vs. M). RNAi to ClC-c (using two independent lines, ClC-c^VDRC#6466 and ClC-c^{VDRC #106844}) (SI Appendix, Fig. 3 J–L) and resulted in granules that were grossly misshapen (Fig. 4 A vs. I and SI Appendix, Fig. 3 K vs. L). TEM analysis of ClC-c^VDRC6466 revealed that the electron-dense matrix was fragmented into smaller islands and scattered throughout the granule lumen (Fig. 4 J vs. N). The Sgs1 discs were still present around the periphery of the electron-dense matrix but were much less distinct in structure. The Sgs3 filaments maintained their parallel stacked arrangement. These results suggest that the loss of ClC-c is specifically affecting the restructuring of the electron-dense matrix and the organization of Sgs1.

Mucin Glycosylation Influences Packaging and Secretory Granule Morphology.

Previous studies from our group have shown that loss of an O-glycosyltransferase (PGANT9B), which glycosylates Sgs3, results in secretory granules that are grossly abnormal in morphology (33). To further examine how the loss of PGANT9B affects secretory granule morphology, we examined pgant9 mutants (pgant9^Δ/Df(2R)) by TEM (Fig. 4O). pgant9^Δ/Df(2R) granules displayed significantly reduced spacing between the Sgs3 filaments relative to control (Fig. 4 P–R), suggesting that O-glycosylation of Sgs3 may regulate the distance between filaments. Additionally, the Sgs1 discs were less pronounced than the control (Fig. 4 P vs. Q). We hypothesize that the irregular granule shape may be due to the less mobile nature of the more tightly packed Sgs3 filaments. Indeed, examination of several TEM micrographs of pgant9^Δ/Df(2R) granules suggests that the mixing of granule contents may be constrained upon homotypic fusion of granules (Fig. 4O). Taken together, we demonstrate that distinct mucins undergo ordered restructuring during secretory granule maturation in vivo, which is influenced by specific genes controlling pH, ion changes and mucin glycosylation. We propose a model where multiple distinct mucins are packaged into the same secretory granule, where they undergo temporally regulated restructuring events that allow for intragranular spatial segregation (Fig. 5).

Fig. 5.

Secretory granule maturation and intragranular mucin restructuring/segregation. Secretory granule maturation involves the growth of granules by homotypic (granule-granule) fusion along with the regulated restructuring of mucins, which is dependent on genes that control pH, calcium and chloride ions and O-glycosylation. Restructuring of the mucins Sgs1 and Sgs3 during secretory granule maturation results in the intragranular segregation of these distinct cargo proteins.

Discussion

Here we address fundamental questions regarding mucin packaging/compaction within secretory granules in vivo using a combination of genetics and multimodal imaging (confocal, TEM and FIB-SEM) within Drosophila salivary glands. We demonstrate that individual secretory granules contain multiple distinct mucins that undergo temporally ordered restructuring during secretory granule maturation to adopt distinct intragranular structures. Our results support a model where multiple mucins are packaged into the same secretory granule and segregated via restructuring events during secretory granule maturation (Fig. 5). Indeed, evidence for distinct mucins being present within the same secretory granule within cells of human and mouse respiratory systems was recently reported (43), suggesting this is not unique to Drosophila. The packaging of multiple proteins into a single immature granule obviates the need for specific sorting receptors for individual proteins, and instead relies on segregation via intragranular compartmentalization. Our results highlight how secretory cells under a high secretory burden may rely on intragranular rather than intergranular segregation of distinct secretory cargo through the formation of biological condensates.

Interestingly, each mucin adopted a structure reminiscent of structures seen in in vitro reconstitution experiments performed with segments of mammalian mucins. Sgs1 adopted a disc-like structure, similar to that seen upon reconstitution of a portion of MUC5B in vitro, where head-to-head N-terminal stacking is proposed to result in this compaction (13–15). Likewise, Sgs3 was a component of the parallel filament bundles, similar to those seen upon in vitro reconstitution of portions of MUC2 (5, 12). Biophysical and single-particle EM analysis suggest that the MUC2 mucin is packaged as concatenated polygonal rings, with the mucin-domains projecting perpendicular from the base of the rings as parallel rods (5). Our results suggest that the highly O-glycosylated mucin domains are components of the parallel rods, as disruption of glycosylation results in tighter spacing of these rods. The packaging of MUC2 is proposed to be biologically significant, as it allows the mucin to undergo regulated expansion into a net-like structure that will line the intestinal environment, conferring protection from microbial and mechanical damage (7). Like mammalian mucins, Drosophila SG mucins also form a lining (that mediates attachment to a substrate to enable metamorphosis), suggesting that their packaging and expansion upon secretion may be similarly regulated.

We have further identified the factors that influence the ordered restructuring of the Drosophila mucins during secretory granule maturation in vivo. The intermediate steps of secretory granule formation/granule maturation after export from the trans-Golgi are poorly understood for mammalian mucins, although roles for calcium ions and pH have been shown to be important for this process in vitro (5, 13–15). Indeed, we find that genes controlling pH and calcium (Vha16-1 and fwe, respectively) are differentially regulated during secretory granule maturation and are essential for mucin restructuring in vivo. While RNAi to Vha16-1 preferentially affected Sgs3 structures, RNAi to fwe affected all structures, resulting in granules filled with long, winding fibers, suggestive of extensive mucin polymerization without further compaction. Previous in vitro studies have suggested that calcium-dependent charge shielding is necessary for the compaction of MUC2 into bundled rod-like structures (5). Similarly, in vitro studies of MUC5B identified calcium-dependent stacking and condensation of this mucin into round disc-like structures (13–15). Our results further suggest that calcium may be necessary for the proper folding/compaction of both Sgs1 and Sgs3 polymers in vivo. Taken together, our in vivo studies support a role for pH and calcium in intragranular packaging of endogenous mucins and are well-aligned with previous in vitro reconstitution/folding studies that have highlighted roles for calcium and pH in the restructuring of mucins (5, 13–15).

Finally, we show a potential role for genes involved in chloride transport and demonstrate that O-glycosylation, a protein modification abundant on mucins (44, 45), influences the structures of the packaged mucins, thereby influencing secretory granule morphology. Ultrastructural analysis demonstrated that the loss of pgant9 results in Sgs3 filaments that are more tightly spaced, likely due to a reduction in the presence of O-linked glycans. We hypothesize that the irregular granule shapes in the absence of pgant9 may be due to the less mobile nature of the more tightly packed Sgs3 filaments. These observations are in line with our previous study showing that abnormal granule morphology became evident as homotypic fusion was occurring (33). In summary, we demonstrate that distinct mucins undergo temporally regulated restructuring during secretory granule maturation to form unique structures and we identify genes involved in this process. We propose a model for regulated intragranular cargo segregation to ensure efficient storage and secretion of large extracellular matrix proteins such as mucins. The formation of these unique biological condensates may allow cargo segregation within secretory granules in the absence of membranes. Future studies will examine additional factors involved in mucin packaging as well as their effects on the biophysical properties of the secreted mucins.

Materials and Methods

Full details on the materials and methods are described in the SI Appendix. The methods include fly strains and genetics; TEM; FIB-SEM imaging and segmentation; antibody preparation and validation; Western blotting; real-time PCR; morphometric analysis; salivary gland imaging; and v-ATPase inhibitor treatment.

Data, Materials, and Software Availability

All study data are included in the article and/or supporting information.