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 synthesized in the endoplasmic reticulum and transported through the Golgi apparatus to the Trans-Golgi Network (TGN), where they are packed in secretory granules that are released into the cytoplasm and transported to the cell periphery. There, 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 the fusion of the secretory granules with the APM in live animals, with a specific focus on the actin cytoskeleton, whose role in exocytosis has not been elucidated yet. To this aim, we set up an experimental system to image and track the secretory granules in the SGs of live animals, which is based on the use of selected transgenic mouse models expressing various fluorescently labeled molecules. Among them, a mouse expressing the soluble green fluorescent protein (GFP) and a mouse expressing a membrane targeted peptide fused with the fluorescent protein Tomato, enable clear visualization of both the secretory granules and the APM. We have found that: 1) the stimulation of the beta-adrenergic, but not the muscarinic receptors, enhances the mobility of the secretory granules and promotes their docking and subsequent fusion at the APM, and 2) that after fusing with the PM the secretory granules completely collapse within 30-40 seconds. This result underscores a major difference between in vivo observations and ex-vivo models in which compound exocytosis (i.e. the sequential fusion of strings of secretory granules), has been described as the primary modality of fusion. Next, we investigated when and where the actin cytoskeleton is required for the exocytosis of the secretory granules. By using immunocytochemistry we observed that during the beta-adrenergic stimulation actin is recruited onto a subpopulation of secretory granules that is localized in close proximity of the APM. To study the dynamics of the actin recruitment we have transduced the salivary glands of live rats with the small peptide Lifeact fused with GFP, a novel tool to label dynamically F-actin. We determined that actin is recruited onto the surface of the granules only after fusion has occurred, and it is released into the cytoplasm only after their complete collapse. Impairment of the dynamics of the actin cytoskeleton, using pharmacological agents such as cytochalasin D (cyto D) or latrunculin A (lat A), did not affect the fusion of the secretory granules with the APM, but it blocked substantially their collapse leading to the accumulation of fused granules which often expanded in size reaching a diameter of 3-5 m. Based on this finding, we have hypothesized that one of the roles of actin is to provide a scaffold around the fusing granule, which facilitates the completion of the fusion process and prevents the diffusion of the APM within the membrane of the granule. 2) Molecular machinery regulating endocytosis in salivary glands The role of endocytosis in the physiology of the SGs has not been examined. Uptake of proteins from either the apical or the basolateral domain of the SGs epithelium has been described but never thoroughly investigated. Notably, in other secretory systems, endocytosis from the APM has been shown to be upregulated during exocytosis. This process, called compensatory endocytosis, is thought to mediate the retrieval of both membranes and key molecules that are needed for the subsequent rounds of secretion. Furthermore, the endocytosis of the GPCRs at the basolateral membrane is thought to be a key step in the regulation of the signaling controlling exocytosis.
Our aim i s to understand the contribution of the endocytic pathways in the SGs during secretion, with a particular focus in elucidating their nature and molecular machinery. To image the endocytic events in the SGs of live rodents we first determined whether systemically injected fluorescent molecules would reach the target cells by diffusing out from the vasculature. We showed that the fenestrated capillaries in the submandibular glands permit the diffusion of molecules up to 70-80 kDa, which is in the range of physiologically relevant molecules such as transferrin (Tfn) or albumin. As probes to study endocytosis we primarily used fluorescently labeled dextrans, Tfn and bovine serum albumin (BSA), which are known to be internalized through different endocytic routes. Notably, all these molecules were internalized within the first few minutes after their injection, primarily by the stromal cells in the SGs, as determined by immunocytochemistry. Their trafficking from the early to late endosomal/lysosomal compartments was followed dynamically and we were able for the first time to image fusion events at a resolution comparable to that achieved in cell culture by confocal microscopy. This enabled us to determine that after the early endosomal fusion step, molecules are delivered to the late endosomes and lysosomes through a mixed process of maturation and formation of transient tubular interconnections. Furthermore, we determined that the internalization step is dependent on the actin cytoskeleton, since administration of drugs such as cyto D and lat A, which disrupt actin dynamics, results in a significant reduction in endocytosis of the injected probes. Both under resting or stimulated conditions, none of the probes injected was internalized by any cell in the SGs epithelium, suggesting that the access of molecules to the basal membrane of the SGs epithelium is tightly regulated, most likely by the basement membrane. Finally, we analyzed the internalization of the same molecules from the apical domain of the SG epithelium. Probes were administered by either injection or slow gravity-mediated infusion though the Whartons duct. Under resting conditions most of the probes underwent a low but detectable level of endocytosis in both the ducts and the acinar cells. However, upon stimulation of protein but not water secretion, the probes were primarily internalized by the acini, most likely by the stimulation of compensatory endocytosis. 3) Molecular machinery regulating the sorting of proteins from the trans-Golgi network to the apical or the basolateral domain of the plasma membrane In addition to the secretory granules, proteins are sorted constitutively from the TGN to the apical or the basolateral domain of the PM. In the SGs, these pathways are thought to contribute to the protein composition of the saliva either directly, by transporting some of the salivary proteins, or indirectly, by targeting some component of the secretory granule fusion machinery to the exocytic sites, and this aspect has not been fully elucidated yet.
Our aim i s to understand how constitutive apical sorting is regulated by 1) elucidating whether proteins follow a direct route to the PM or they are first diverted towards other intracellular compartments, and 2) dissecting the molecular machineries regulating the sorting events. To this aim we have chosen to study the sorting of two members of the Aquaporin family of water channels that are expressed in acinar cells in the SG: aquaporin 3 (AQP3) and aquaporin 5 (AQP5). These molecules are localized to the basolateral and apical domains, respectively, and their sorting signals have been proposed to reside in the cytoplasmic domains. As first step, we have determined whether the ectopically expressed aquaporins were correctly targeted to the proper domain in the SGs of live animals
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