Homeostasis of the apical plasma membrane during regulated exocytosis in the salivary glands of live rodents

Abstract

In exocrine organs such as the salivary glands, fluids and proteins are secreted into ductal structures by distinct mechanisms that are tightly coupled. In the acinar cells, the major secretory units of the salivary glands, fluids are secreted into the acinar canaliculi through paracellular and intracellular transport, whereas proteins are stored in large granules that undergo exocytosis and fuse with the apical plasma membranes releasing their content into the canaliculi. Both secretory processes elicit a remodeling of the apical plasma membrane that has not been fully addressed in in vitro or ex vivo models. Recently, we have studied regulated exocytosis in the salivary glands of live rodents, focusing on the role that actin and myosin plays in this process. We observed that during exocytosis both secretory granules and canaliculi are subjected to the hydrostatic pressure generated by fluid secretion. Furthermore, the absorption of the membranes of the secretory granules contributes to the expansion and deformation of the canaliculi. Here we suggest that the homeostasis of the apical plasma membranes during exocytosis is maintained by various strategies that include: (1) membrane retrieval via compensatory endocytosis, (2) increase of the surface area via membrane folds and (3) recruitment of a functional actomyosin complex. Our observations underscore the important relationship between tissue architecture and cellular response, and highlight the potential of investigating biological processes in vivo by using intravital microscopy.

Figure 1. Expansion of the acinar canaliculi during regulated exocytosis. (A)The APM of the SG acini were imaged upon subcutaneous (SC) injection of iso by intravital confocal microscopy, as previously described. A canaliculus (arrows) expanded over time as shown in the micrographs. Arrowheads point to a SCGs in the cytoplasm. Scale bar, 2µm. Lower graph shows the quantification of the diameter of the canaliculi. (B) Fluid expulsion during regulated exocytosis. A fluorescently labeled dextran (red) was retro-infused by gravity into the ducts of the SGs of a live rat. Iso (upper panels) or carb (lower panels) were injected SC and the SGs were imaged by intravital two-photon microscopy. The parenchyma of the glands is revealed by the excitation of endogenous fluorescence (cyan). After 40 sec from the injection of the agonists, the dextran was completely cleared from the ductal system (arrows). Scale bar, 20 µm. (C) Compensatory endocytosis. A fluorescently labeled dextran (red) was retro-infused in the ducts of the SGs of a live rat. The acinar canaliculi (arrowheads) are rapidly filled with the dye. The dotted line delimits an acinus (Ac). Iso was injected SC and the glands were imaged by intravital two-photon microscopy. As shown in (B), the dextran is cleared from the canaliculi. However, a fraction of it is internalized in small vesicles (arrows) generated by compensatory endocytosis from the APM. Scale bar, 10 µm. (D) Membrane folds in the acinar canaliculi. GFP-mice were injected SC with either iso (upper panels) or carb (lower panel). Acinar canaliculi were imaged by intravital confocal microscopy after 30 min from the injection. When SCGs exocytosis is elicited with iso, membrane folds appear in the canaliculi (upper panel and enlargement). Scale bar, 2 µm. (E) Model for the expansion of the canaliculi. After injection of iso, the SCG (blue) fuse with the APM (red). The hydrostatic pressure generated by fluids secretion (light blue arrows) contributes to the initial expansion of the canaliculi. SCGs are absorbed at the APM and compensatory endocytosis is stimulated (light blue vesicles). The excess of membranes that are not retrieved by compensatory endocytosis is accommodated by expanding the diameter of the canaliculi and by generating membrane folds. Actin (green) and myosin II (blue) are localized to the SCGs to counteract the hydrostatic pressure.

Figure 2. The actomyosin complex provides a scaffold to counteract the effect of the hydrostatic pressure on the secretory granules. (A)Mice expressing the m-Tomato probe were pre-treated with cytochalasin D, blebbistatin or the vehicle (DMSO, ctrl) and injected with iso to stimulate SCGs exocytosis. When the actin cytoskeleton was disrupted with cytochalasin D or the motor activity of myosin II was inhibited with blebbistatin the SCGs expanded in size due to the hydrostatic pressure generated by fluids secretion (light blue arrows). Scale bar, 2 µm. Actin (green) and myosin II (blue) are recruited to the SCGs to counteract the hydrostatic pressure and to facilitate the gradual collapse of the SCGs.^,(B) SCGs were imaged after their fusion with the APM by using time-lapse intravital confocal microscopy. The diameter of the SCGs was measured in animals treated with cytochalasin D (red circles), blebbistatin (blue circles) or DMSO (black circles). (C) Fluorescently labeled dextran (red) was retro-infused in the ducts of the SGs of a live rat by using a cannula introduced in the major excretory duct as described in legend to Figure 1. The same acinus was imaged before (upper panel) and after the SC injection of iso and the artificial increase of the hydrostatic pressure achieved with a syringe connected to the cannula (lower panel). Acinar canaliculi (arrows) were filled with the dye (upper panel). Enlarged SCGs (arrowheads) appeared from the APM (lower panel). Scale bar, 10 µm.