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 study the molecular machinery regulating the formation of the granules from the TGN and their fusion with the APM. We set up an experimental system aimed at imaging and tracking the secretory granules in the SGs of live animals and based on high-resolution intra-vital microscopy performed on a series of transgenic mouse models expressing selected fluorescently labeled molecules. Among them, a mouse expressing the soluble green fluorescent protein (GFP) in both the sub-mandibular and the parotid glands, enables a clear visualization of both the secretory granules and the APM. Our analysis on the effect of various agonists of GPCRs has revealed two major differences between in vivo and ex-vivo models: 1) the stimulation of the beta-adrenergic but not the muscarinic receptors, enhances the mobility of the secretory granules promoting their docking and subsequent fusion at the APM and 2) muscarinic receptors do not play any synergistic role with the adrenergic receptor during exocytosis. Furthermore, by using another mouse model, which expresses the Tomato fluorescent protein fused with a di-palmitoylated peptide, a well-established marker for the plasma membrane, we discovered that the secretory granules after fusing with the plasma membrane completely collapse within 30-40 seconds. This result underscores another major differences between in vivo 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. Notably, we also showed that after fusion with the APM, the granules recruit filamentous actin (F-actin) and two isoforms of the actin motor myosin II (myosin IIa and IIb). The impairment of either the dynamics of F-actin or the motor activity of myosin II did not affect the fusion of the secretory granules with the APM, but affected substantially their collapse leading to the accumulation of fused granules, which often expanded in size. This expansion is due to the hydrostatic pressure generated by fluid secretion and the stimulation of compound exocytosis. These results suggest that the acto-myosin complex provides a contractile scaffold around the secretory granules that performs multiple tasks, such as i) preventing the hydrostatic pressure to disrupt the plasma membrane, ii) preventing compound exocytosis, and iii) facilitating the completion of the fusion at the APM. We have proposed that this machinery operates in secretory systems in which the collapse of the granules is not energetically favorable due to geometrical constrains. Indeed, we found that the actomyosin complex is recruited in other exocrine glands such as pancreas, lacrimal glands, and mammary glands, but not in endocrine organs. 2) Molecular machinery regulating endocytosis in salivary glands of live animals The role of endocytosis in the physiology of the SGs has never been elucidated. Uptake of proteins from either the apical or the basolateral domain of the ductal system have been described but never thoroughly investigated due to the lack of an appropriate experimental model. The presence under physiological conditions of salivary proteins in the bloodstream and of serum proteins in the saliva, argues strongly in favor of a constant and bi-directional transcytotic movement of proteins across the salivary gland epithelium.
Our aim i s to first define the endocytic pathways operating in SGs in vivo, and then to investigate the contribution of the endocytic events in the patho-physiology of the glands and especially during secretion. We set up various experimental systems in the SGs of live rodents aimed at imaging and studying endocytic events, which occur at either the apical or the basolateral membrane of the SGs epithelium, and in stromal cells. Interestingly, endocytosis in the SGs epithelium of a live animal is significantly reduced when compared with cell cultures, whereas endocytosis in stromal cells appears to occur at a faster rate than in vitro systems. Furthermore, we found that stimulation of the secretory activity of the SGs enhances endocytosis from the apical pole whereas does not have any effect on basolateral endocytosis or on uptake from stromal cells. Indeed, using both fluid-phase markers, such as fluorescently-labeled dextrans or smaller molecules, and probes that selectively label the PM, we found that in resting conditions the endocytic activity at the APM is extremely low, whereas stimulation of protein but not water secretion enhances internalization via compensatory endocytosis. Since the SGs are target organs for gene therapy we sought to investigate the endocytosis of plasmid DNA. By using a combination of pharmacological inhibitors, immunocytochemistry and IVM, we found that DNA is internalized in all the components of the SGs epithelium (intercalated ducts, large ducts and acini) by an unconventional endocytic pathway that is sensitive to amiloride, an inhibitor of macropinocytosis. Moreover, only a small proportion of internalized DNA is localized in the early endosomal compartments suggesting that lysosomal degradation is bypassed via endosomal escape. Finally, we observed that stimulation of protein secretion enhances the uptake of the DNA in acinar cells by the same compensatory endocytic pathway observed before. These results revealed unconventional endocytic pathways in live animals that may be exploited to better design non viral-based gene therapy. Endocytosis from the basolateral membrane of the SG epithelium was assessed by three main strategies: i) by injecting fluorescently-labeled probes either systemically or directly into the SGs (fluid-phase endocytosis and non-selective membrane internalization);ii) by expressing fluorescently labeled receptors that are selectively targeted to the basolateral membrane and are known to be internalized either upon agonist stimulation (beta-adrenergic receptor and EGFR) or constitutively (TfnR, GFP-GPI, GFP-Farnesyl);and iii) by using transgenic mice expressing fluorescently tagged plasma membrane markers (m-Tomato, m-GFP, GPI-GFP). Surprisingly, we found that in live animals, basolateral endocytosis is extremely slow under both basal and stimulated conditions. On the other hands, in ex-vivo preparations or primary cell cultures derived from the SGs, endocytosis was comparable to what reported for other cell culture models. By using intravital fluorescence recovery after photo-bleaching (iFRAP) we were able to show that these discrepancies are due to differences in the lateral mobility of plasma membrane lipids and proteins. Indeed, in vivo membrane mobility is reduced 3-5 folds with respect to in vitro model system preventing either the lateral segregation of membranes into endocytic vesicles or vesicle biogenesis.
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