1) Molecular machinery regulating protein secretion in the acinar cells of salivary glands. In the SGs, the major secretory units are the acini that are formed by pyramidal polarized cells, which form small canaliculi at the apical plasma membrane (APM) where salivary proteins and water are secreted. Proteins destined to secretion are packed in secretory granules (SCGs) that are released into the cytoplasm, and transported to the cell periphery. Here, upon stimulation of the appropriate G protein-coupled receptor (GPCR), the granules fuse with the APM, releasing their content into the lumen of the canaliculi.
Our aim i s to elucidate the molecular machinery regulating their fusion with the APM and how the homeostasis of the APM is maintained. We developed an experimental system in the SGs of live rodents aimed at imaging and tracking individual secretory granules, and visualizing the dynamics of the APM. We used high-resolution intravital microscopy performed on a series of transgenic mouse models, which ubiquitously express: i) the soluble green fluorescent protein (GFP-mouse), or ii) the tandem-Tomato fluorescent protein fused with a membrane-targeted peptide (m-Tomato mouse), iii) or both molecules (GFP/m-Tomato mouse). The GFP-mouse provided clear visualization of both the secretory granules and the APM, and enabled an accurate quantitative analysis of the exocytic events. On the other hand, the m-Tomato mouse enabled imaging the APM and the secretory granules after fusion has occurred. Although the morphology of the acinar cells has been analyzed by transmission and scanning electron microscopy, several key aspects of the structure of these cells and their three dimensional arrangement were not fully elucidated. By performing z-scans and generating volume renderings of individual acini, we determined that each acinar cell contains an average of 250-300 secretory granules with diameters between 1.0-1.5 m. Moreover, we found that each acinar cell contributes to the formation of at least two canaliculi that are 10-15 m in length, and have diameters of 0.3-0.4 m. We have shown that the stimulation of the β-adrenergic, but not the muscarinic receptors, enhances the mobility of the secretory granules and promotes their docking and subsequent fusion with the APM. Moreover, we determined that after the opening of the fusion pore the secretory granules completely collapse within 40-60 seconds and their membranes are integrated into the canaliculi. Overall, these results reveal two major differences between in vivo observations and ex-vivo models in which, i) muscarinic receptors have been shown to elicit exocytosis, and ii) compound exocytosis (i.e. the sequential fusion of strings of secretory granules) has been described as the primary modality of fusion. By using a mouse expressing both the m-Tomato and the small peptide lifeact fused with GFP (a novel tool to label dynamically F-actin) we determined that upon the opening of the fusion pore, the APM diffuses into the limiting membrane of the granules triggering the recruitment of F-actin onto their surface. The assembly of F-actin initiates and facilitates the gradual collapse of the secretory granules, as shown by impairing the dynamics of the actin cytoskeleton through pharmacological agents, such as cytochalasin D (cyto D) or latrunculin A (lat A). Indeed, under these conditions the fused granules were arrested at the APM after the opening of the fusion pore, and expanded in size 2-3 fold with respect to their original diameters. This expansion is the result of two processes: i) the increase of the hydrostatic pressure in the acinar canaliculi due to the stimulation of fluid secretion, and ii) the sequential fusion of adjacent secretory granules. These observations strongly suggest that actin plays three major roles during regulated exocytosis: i) facilitates the completion of the gradual collapse of the secretory granules at the APM, ii) protects the granules from the hydrostatic pressure, and iii) prevents the unregulated fusion of the adjacent granules. We postulated that in order to produce the contractile activity necessary to drive the gradual collapse of the secretory granules, a myosin motor would be required as well. Indeed, we found that two of the three isoforms of the myosin II actin motor (IIa and IIb) were recruited onto the secretory granules upon stimulation of regulated exocytosis. Interestingly, blocking the motor activity of myosin II with blebbistatin resulted in a delay in the gradual collapse of the secretory granules. We sought to determine how both isoforms of myosin II are recruited onto the membrane of the secretory granules. We first asked whether myosin II was recruited through F-actin. To this end, we crossed knock-in mice expressing either GFP-myosin IIa or GFP-myosin IIb with mice expressing RFP-lifeact. We found that both myosin IIa and IIb are recruited after the assembly of F-actin onto the granules. However, we observed that i) both myosins continue to be actively recruited onto the membranes even after F-actin disassembles, and ii) the recruitment of both myosins is only slightly affected by actin depolymerizing agents. These results suggested that other molecules may be required for myosin II recruitment and retention and in order to identify them we performed the proteomic analysis of secretory granules purified from rat submandibular glands. Among the proteins associated with the granules we identified Septin2, a small GTPase previously suggested to work as a scaffold protein facilitating the phosphorylation of myosin II via the recruitment of the myosin light chain kinase (MLCK). Septin2 was indeed expressed in the SGs, and recruited onto the secretory granules after their fusion with the plasma membrane. Moreover, inhibition of the septin2 activity with the specific inhibitor forchlorfenuron (FCF) resulted in the impairment of the assembly of the actin cytoskeleton during regulated exocytosis. Interestingly, we found that in the SGs of Drosophila larvae, a system that shares similarities with the rodent salivary glands, siRNA-mediated downregulation of septin2 resulted in a severe impairment of regulated exocytosis of mucin-containing granules and highlighted an additional role for septin2 in pre-fusion events. Finally, we determined whether the requirement for a functional actomyosin complex is unique to the rodent salivary glands or is a general mechanism shared by other secretory systems. We found that this machinery is conserved in the mouse exocrine pancreas, as shown by imaging regulated exocytosis in the acini of live mice expressing Lifeact and mTomato or Lifeact and either Myosin IIa or IIb. In animals that were starved for 24 hours, no exocytosis was detected. On the other hand, administration of food elicited exocytosis of secretory granules, which underwent a gradual collapse and, upon fusion with the APM, recruited sequentially actin and myosin II. Based on our findings we proposed that the assembly of the actomyosin complex is required in exocrine glands and in those secretory systems that possess a unique geometry in which the secretory vesicles have a lower membrane tension (diameters >1 m) than the target membrane (0.3-0.4 m) making the gradual collapse of the secretory granules energetically unfavorable. The contractile activity of the actomyosin complex may work to overcome this energy barrier by i) contributing to the expansion of the fusion pore, and ii) facilitating the movement of membranes from the granules toward the APM.

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National Institute of Dental & Craniofacial Research
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