Endoplasmic reticulum stress impedes regulated secretion by governing key exocytotic and granulogenic molecular switches.

Dense core vesicles (DCVs) and synaptic vesicles are specialised secretory vesicles in neurons and neuroendocrine cells, and abnormal release of their cargo is associated with various pathophysiologies. Endoplasmic reticulum (ER) stress and inter-organellar communication are also associated with disease biology. To investigate the functional status of regulated exocytosis arising from the crosstalk of a stressed ER and DCVs, ER stress was modelled in PC12 neuroendocrine cells using thapsigargin. DCV exocytosis was severely compromised in ER-stressed PC12 cells and was reversed to varying magnitudes by ER stress attenuators. Experiments with tunicamycin, an independent ER stressor, yielded similar results. Concurrently, ER stress also caused impaired DCV exocytosis in insulin-secreting INS-1 cells. Molecular analysis revealed blunted SNAP25 expression, potentially attributed to augmented levels of ATF4, an inhibitor of CREB that binds to the CREB-binding site. The effects of loss of function of ATF4 in ER-stressed cells substantiated this attribution. Our studies revealed severe defects in DCV exocytosis in ER-stressed cells for the first time, mediated by reduced levels of key exocytotic and granulogenic switches regulated via the eIF2α (EIF2A)-ATF4 axis.

Getting Nervous: An Evolutionary Overhaul for Communication.

The evolution of a nervous system as a control system of the body's functions is a key innovation of animals. Its fundamental units are neurons, highly specialized cells dedicated to fast cell-cell communication. Neurons pass signals to other neurons, muscle cells, or gland cells at specialized junctions, the synapses, where transmitters are released from vesicles in a Ca2+-dependent fashion to activate receptors in the membrane of the target cell. Reconstructing the origins of neuronal communication out of a more simple process remains a central challenge in biology. Recent genomic comparisons have revealed that all animals, including the nerveless poriferans and placozoans, share a basic set of genes for neuronal communication. This suggests that the first animal, the Urmetazoan, was already endowed with neurosecretory cells that probably started to connect into neuronal networks soon afterward. Here, we discuss scenarios for this pivotal transition in animal evolution.

Advanced Imaging Approaches to Reveal Molecular Mechanisms Governing Neuroendocrine Secretion.

Identification of the molecular mechanisms governing neuroendocrine secretion and resulting intercellular communication is one of the great challenges of cell biology to better understand organism physiology and neurosecretion disruption-related pathologies such as hypertension, neurodegenerative, or metabolic diseases. To visualize molecule distribution and dynamics at the nanoscale, many imaging approaches have been developed and are still emerging. In this review, we provide an overview of the pioneering studies using transmission electron microscopy, atomic force microscopy, total internal reflection microscopy, and super-resolution microscopy in neuroendocrine cells to visualize molecular mechanisms driving neurosecretion processes, including exocytosis and associated fusion pores, endocytosis and associated recycling vesicles, and protein-protein or protein-lipid interactions. Furthermore, the potential and the challenges of these different advanced imaging approaches for application in the study of neuroendocrine cell biology are discussed, aiming to guide researchers to select the best approach for their specific purpose around the crucial but not yet fully understood neurosecretion process.

The Regulated Secretion and Models of Intracellular Transport: The Goblet Cell as an Example.

Transport models are extremely important to map thousands of proteins and their interactions inside a cell. The transport pathways of luminal and at least initially soluble secretory proteins synthesized in the endoplasmic reticulum can be divided into two groups: the so-called constitutive secretory pathway and regulated secretion (RS) pathway, in which the RS proteins pass through the Golgi complex and are accumulated into storage/secretion granules (SGs). Their contents are released when stimuli trigger the fusion of SGs with the plasma membrane (PM). In specialized exocrine, endocrine, and nerve cells, the RS proteins pass through the baso-lateral plasmalemma. In polarized cells, the RS proteins secrete through the apical PM. This exocytosis of the RS proteins increases in response to external stimuli. Here, we analyze RS in goblet cells to try to understand the transport model that can be used for the explanation of the literature data related to the intracellular transport of their mucins.

The mechanisms of chromogranin B-regulated Cl- homeostasis.

Chloride is the most abundant inorganic anions in almost all cells and in human circulation systems. Its homeostasis is therefore important for systems physiology and normal cellular activities. This topic has been extensively studied with chloride loaders and extruders expressed in both cell surfaces and intracellular membranes. With the newly discovered, large-conductance, highly selective Cl- channel formed by membrane-bound chromogranin B (CHGB), which differs from all other known anion channels of conventional transmembrane topology, and is distributed in plasma membranes, endomembrane systems, endosomal, and endolysosomal compartments in cells expressing it, we will discuss the potential physiological importance of the CHGB channels to Cl- homeostasis, cellular excitability and volume control, and cation uptake or release at the cellular and subcellular levels. These considerations and CHGB's association with human diseases make the CHGB channel a possible druggable target for future molecular therapeutics.

Rab27a Contributes to Cathepsin S Secretion in Lacrimal Gland Acinar Cells.

Altered lacrimal gland (LG) secretion is a feature of autoimmune dacryoadenitis in Sjögren's syndrome (SS). Cathepsin S (CTSS) is increased in tears of SS patients, which may contribute to disease. Rab3D and Rab27a/b isoforms are effectors of exocytosis in LG, but Rab27a is poorly studied. To investigate whether Rab27a mediates CTSS secretion, we utilized quantitative confocal fluorescence microscopy of LG from SS-model male NOD and control male BALB/c mice, showing that Rab27a-enriched vesicles containing CTSS were increased in NOD mouse LG. Live-cell imaging of cultured lacrimal gland acinar cells (LGAC) transduced with adenovirus encoding wild-type (WT) mCFP-Rab27a revealed carbachol-stimulated fusion and depletion of mCFP-Rab27a-enriched vesicles. LGAC transduced with dominant-negative (DN) mCFP-Rab27a exhibited significantly reduced carbachol-stimulated CTSS secretion by 0.5-fold and ß-hexosaminidase by 0.3-fold, relative to stimulated LGAC transduced with WT mCFP-Rab27a. Colocalization of Rab27a and endolysosomal markers (Rab7, Lamp2) with the apical membrane was increased in both stimulated BALB/c and NOD mouse LG, but the extent of colocalization was much greater in NOD mouse LG. Following stimulation, Rab27a colocalization with endolysosomal membranes was decreased. In conclusion, Rab27a participates in CTSS secretion in LGAC though the major regulated pathway, and through a novel endolysosomal pathway that is increased in SS.

Proteomic Analysis of Lipid Rafts from RBL-2H3 Mast Cells.

Lipid rafts are highly ordered membrane microdomains enriched in cholesterol, glycosphingolipids, and certain proteins. They are involved in the regulation of cellular processes in diverse cell types, including mast cells (MCs). The MC lipid raft protein composition was assessed using qualitative mass spectrometric characterization of the proteome from detergent-resistant membrane fractions from RBL-2H3 MCs. Using two different post-isolation treatment methods, a total of 949 lipid raft associated proteins were identified. The majority of these MC lipid raft proteins had already been described in the RaftProtV2 database and are among highest cited/experimentally validated lipid raft proteins. Additionally, more than half of the identified proteins had lipid modifications and/or transmembrane domains. Classification of identified proteins into functional categories showed that the proteins were associated with cellular membrane compartments, and with some biological and molecular functions, such as regulation, localization, binding, catalytic activity, and response to stimulus. Furthermore, functional enrichment analysis demonstrated an intimate involvement of identified proteins with various aspects of MC biological processes, especially those related to regulated secretion, organization/stabilization of macromolecules complexes, and signal transduction. This study represents the first comprehensive proteomic profile of MC lipid rafts and provides additional information to elucidate immunoregulatory functions coordinated by raft proteins in MCs.

Amyloid-like aggregation of provasopressin in diabetes insipidus and secretory granule sorting.

BACKGROUND: Aggregation of peptide hormone precursors in the trans-Golgi network is an essential process in the biogenesis of secretory granules in endocrine cells. It has recently been proposed that this aggregation corresponds to the formation of functional amyloids. Our previous finding that dominant mutations in provasopressin, which cause cell degeneration and diabetes insipidus, prevent native folding and produce fibrillar aggregates in the endoplasmic reticulum (ER) might thus reflect mislocalized amyloid formation by sequences that evolved to mediate granule sorting. RESULTS: Here we identified two sequences responsible for fibrillar aggregation of mutant precursors in the ER: the N-terminal vasopressin nonapeptide and the C-terminal glycopeptide. To test their role in granule sorting, the glycopeptide was deleted and/or vasopressin mutated to inactivate ER aggregation while still permitting precursor folding and ER exit. These mutations strongly reduced sorting into granules and regulated secretion in endocrine AtT20 cells. CONCLUSION: The same sequences - vasopressin and the glycopeptide - mediate physiological aggregation of the wild-type hormone precursor into secretory granules and the pathological fibrillar aggregation of disease mutants in the ER. These findings support the amyloid hypothesis for secretory granule biogenesis.

Redirecting intracellular trafficking and the secretion pattern of FSH dramatically enhances ovarian function in mice.

FSH and luteinizing hormone (LH) are secreted constitutively or in pulses, respectively, from pituitary gonadotropes in many vertebrates, and regulate ovarian function. The molecular basis for this evolutionarily conserved gonadotropin-specific secretion pattern is not understood. Here, we show that the carboxyterminal heptapeptide in LH is a gonadotropin-sorting determinant in vivo that directs pulsatile secretion. FSH containing this heptapeptide enters the regulated pathway in gonadotropes of transgenic mice, and is released in response to gonadotropin-releasing hormone, similar to LH. FSH released from the LH secretory pathway rescued ovarian defects in Fshb-null mice as efficiently as constitutively secreted FSH. Interestingly, the rerouted FSH enhanced ovarian follicle survival, caused a dramatic increase in number of ovulations, and prolonged female reproductive lifespan. Furthermore, the rerouted FSH vastly improved the in vivo fertilization competency of eggs, their subsequent development in vitro and when transplanted, the ability to produce offspring. Our study demonstrates the feasibility to fine-tune the target tissue responses by modifying the intracellular trafficking and secretory fate of a pituitary trophic hormone. The approach to interconvert the secretory fate of proteins in vivo has pathophysiological significance, and could explain the etiology of several hormone hyperstimulation and resistance syndromes.

A Membrane-Fusion Model That Exploits a ß-to-α Transition in the Hydrophobic Domains of Syntaxin 1A and Synaptobrevin 2.

Parallel zippering of the SNARE domains of syntaxin 1A/B, SNAP-25, and VAMP/synaptobrevin 2 is widely regarded as supplying the driving force for exocytotic events at nerve terminals and elsewhere. However, in spite of intensive research, no consensus has been reached concerning the molecular mechanism by which these SNARE proteins catalyze membrane fusion. As an alternative to SNARE-based models, a scenario was developed in which synaptotagmin 1 (or, 2) can serve as a template to guide lipid movements that underlie fast, synchronous exocytosis at nerve terminals. This "dyad model" advanced a novel proposal concerning the membrane disposition of the palmitoylated, cysteine-rich region of these synaptotagmins. Unexpectedly, it now emerges that a similar principle can be exploited to reveal how the hydrophobic, carboxyl-terminal domains of syntaxin 1A and synaptobrevin 2 can perturb membrane structure at the interface between a docked synaptic vesicle and the plasma membrane. These "ß-to-α transition" models will be compared and contrasted with other proposals for how macromolecules are thought to intervene to drive membrane fusion.