Regulated Restructuring of Mucins During Secretory Granule Maturation In Vivo

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.

Keywords: O-glycosylation; mucin; salivary gland; secretion; secretory granules.

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.

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.

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.

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.

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.