1)Molecular basis of the actomyosin-driven membrane remodeling during regulated exocytosis in salivary glands. The major secretory units of the salivary glands (SGs) are the acini that are formed by pyramidal polarized cells in which the apical plasma membrane (APM) forms small canaliculi where proteins and water are released. Proteins destined to secretion are packed in secretory granules at the trans-Golgi network (TGN) and transported to the cell periphery where they fuse with the APM upon stimulation of GPCRs.
Our aim i s to elucidate the molecular machinery regulating the fusion and integration of the secretory granules with the APM and the maintenance of APM homeostasis. To this end, 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 have established that upon stimulation of the β-adrenergic receptor, the granules fuse with the APM, releasing their content into the lumen of the canaliculi. In addition, we found that a complex composed of F-actin and two isoforms of non-muscle myosin II (NMIIA and NMIIB) is recruited onto the secretory granules after the fusion step. We showed that the actomyosin contractile activity is required for the integration of the granular membranes into the APM and the completion of exocytosis. In the last year our work has been focusing on elucidating two aspects of the regulation of the actomyosin complex during regulated exocytosis, and specifically: the machinery regulating F-actin assembly onto the secretory granules, and the mechanisms of recruitment and regulation of NMII. Our work has shown that F-actin is assembled around the secretory granules after their fusion with the APM, and plays three distinct roles during regulated exocytosis: first, stabilizes the granular membranes, second, prevents compound exocytosis, and finally, facilitates their gradual collapse. We have hypothesized that the F-actin scaffold is progressively assembled around the granules in two steps: the first requires the formation of linear filaments and provides the stabilization of the membranes, and the second requires the formation of branched filaments, and regulates the collapse of the granules. Consistent with this hypothesis, we found that Profilin1 and mDia2, two components of the machinery initiating the formation of linear filaments, are recruited onto the secretory granules right after fusion, whereas the actin branching factors Arp2/3, cortactin, WASP, and Wave2 are recruited at a later stage. Moreover, by using a pharmacological approach we found that blocking mDia2 resulted in the expansion of the secretory granules, whereas blocking the activity of Arp2/3 significantly delayed their gradual collapse. In addition, we have shown that β-adrenergic stimulation results in the appearance of specific phosphorylated forms of Arp2 (T237 and T238) and cortactin (Y421 and Y466) onto the secretory granules, thus establishing a link between signaling and cytoskeleton. We have also shown that NMIIA and NMIIB are recruited onto the secretory granules after their fusion with the APM, and by using a pharmacological approach we have determined that their contractile activity drives the gradual integration of the secretory granules into the APM. This contrasts with other cellular processes where actomyosin-based contractions employ only one isoform of NMII. Notably, by using conditional knock-out mice we determined that NMIIA regulates the gradual collapse of the secretory granules, whereas NMIIB is required for stabilizing the granular membranes. Since both NMIIA and NMIIB are recruited after the formation of the F-actin scaffold, we had assumed that this process would be mediated by their well-characterized actin-binding site. Unexpectedly, we found that both NMII isoforms are recruited in an actin-independent fashion, and that the main role of F-actin is to facilitate the proper assembly of the myosin filaments. We determined that this step is regulated by the phosphorylation of the regulatory chain of NMII that is catalyzed by the myosin light chain kinase (MLCK), which is also recruited onto the secretory granules. Moreover, we discovered that the phosphorylation of NMII is controlled by the recruitment of filaments composed of septin2 and septin6, two small GTPase that regulate the actin cytoskeleton during cytokinesis. Interestingly, blocking the ability of septins to form filaments severely impairs exocytosis, by slowing the gradual integration of the granular membranes. 2) Bioenergetics of regulated exocytosis. The integration of the membranes of the secretory granules and the APM is an energetically unfavorable process. A single exocytic event consumes significant energy to bring together and fuse a single secretory granule and the plasma membrane, as estimated for neurotransmission. We found that 150-200 secretory granules undergo exocytosis after β-adrenergic stimulation of an acinar cell and that each granule is retrieved by 50-75 endocytic vesicles. Therefore, a central question is: how is cell metabolism regulated during exocytosis? Our hypothesis is that mitochondrial function is increased during β-adrenergic stimulation and is tightly coupled to the exo-endocytic events. We have developed a method to follow the dynamics of the mitochondrial metabolic activity in the SGs of live rats. Since there are no reliable tools to quantitatively image the levels of cellular ATP in vivo, we used two-photon microscopy to determine NADH levels (the main substrate of the electron transport chain, ETC) and mitochondrial potential. Unexpectedly, we discovered that mitochondrial metabolism undergoes rapid and spontaneous oscillations in SGs under basal conditions (period: 10-15 sec). This finding contrast with what reported in exocrine glands in vitro where transient metabolic oscillations were observed only after agonist stimulation. These oscillations are regulated by reactive oxygen species but are insensitive to increase in the levels of intracellular Ca2+, indicating a novel regulation completely different than what described in ex vivo organs. Most notably, we found that mitochondrial oscillations are highly coordinated throughout the SG epithelium via the activity of gap junctions, which may transport a not yet identified small molecule that regulates the synchronization of the oscillations. Finally, our work has shown that β-adrenergic stimulation transiently halts NADH oscillations and increases mitochondrial potential, the latter indicating an increased production of ATP, as would be required to drive regulated exocytosis. Consistently, blocking the mitochondrial ATP-synthase inhibits the assembly of the actomyosin complex (a major energy requiring step). Interestingly, under β-adrenergic stimulations blocking the activity of complex I does not affect exocytosis, suggesting that a different pathway is used to regulate mitochondrial metabolism during stimulated secretion.
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