Exocytosis Of Secretory Granules Research Articles

Sustained hyperglycemia specifically targets translation of mRNAs for insulin secretion.

Pancreatic β cells are specialized for coupling glucose metabolism to insulin peptide production and secretion. Acute glucose exposure robustly and coordinately increases translation of proinsulin and proteins required for secretion of mature insulin peptide. By contrast, chronically elevated glucose levels that occur during diabetes impair β cell insulin secretion and have been shown experimentally to suppress insulin translation. Whether translation of other genes critical for insulin secretion is similarly downregulated by chronic high glucose is unknown. Here, we used high-throughput ribosome profiling and nascent proteomics in MIN6 insulinoma cells to elucidate the genome-wide impact of sustained high glucose on β cell mRNA translation. Before induction of ER stress or suppression of global translation, sustained high glucose suppressed glucose-stimulated insulin secretion and downregulated translation of not only insulin, but also mRNAs related to insulin secretory granule formation, exocytosis, and metabolism-coupled insulin secretion. Translation of these mRNAs was also downregulated in primary rat and human islets following ex vivo incubation with sustained high glucose and in an in vivo model of chronic mild hyperglycemia. Furthermore, translational downregulation decreased cellular abundance of these proteins. Our study uncovered a translational regulatory circuit during β cell glucose toxicity that impairs expression of proteins with critical roles in β cell function.

An In Vitro Technique to Visualize Exocytosis of Secretory Granules in Mast Cells

An In Vitro Technique to Visualize Exocytosis of Secretory Granules in Mast Cells

An In Vitro Technique to Visualize Exocytosis of Secretory Granules in Mast Cells

An In Vitro Technique to Visualize Exocytosis of Secretory Granules in Mast Cells

A convenient fluorimetry-based degranulation assay using RBL-2H3 cells.

Type I hypersensitivity is triggered by mast cell degranulation, a stimulus-induced exocytosis of preformed secretory granules (SGs) containing various inflammatory mediators. The degree of degranulation is generally expressed as a percentage of secretory granule markers (such as β-hexosaminidase and histamine) released into the external solution, and considerable time and labor are required for the quantification of markers in both the supernatants and cell lysates. In this study, we developed a simple fluorimetry-based degranulation assay using rat basophilic leukemia (RBL-2H3) mast cells. During degranulation, the styryl dye FM1-43 in the external solution fluorescently labeled the newly exocytosed SGs, whose increase in intensity was successively measured using a fluorescence microplate reader. In addition to the rate of β-hexosaminidase secretion, the cellular FM1-43 intensity successfully represented the degree and kinetics of degranulation under various conditions, suggesting that this method facilitates multi-sample and/or multi-time-point analyses required for screening substances regulating mast cell degranulation.

Lipid-driven interleaflet coupling of plasma membrane order regulates FcεRI signaling in mast cells

Interleaflet coupling—the influence of one leaflet on the properties of the opposing leaflet—is a fundamental plasma membrane organizational principle. This coupling is proposed to participate in maintaining steady-state biophysical properties of the plasma membrane, which in turn regulates some transmembrane signaling processes. A prominent example is antigen (Ag) stimulation of signaling by clustering transmembrane receptors for immunoglobulin E (IgE), FcεRI. This transmembrane signaling depends on the stabilization of ordered regions in the inner leaflet for sorting of intracellular signaling components. The resting inner leaflet has a lipid composition that is generally less ordered than the outer leaflet and that does not spontaneously phase separate in model membranes. We propose that interleaflet coupling can mediate ordering and disordering of the inner leaflet, which is poised in resting cells to reorganize upon stimulation. To test this in live cells, we first established a straightforward approach to evaluate induced changes in membrane order by measuring inner leaflet diffusion of lipid probes by imaging fluorescence correlation spectroscopy, by imaging fluorescence correlation spectroscopy (ImFCS), before and after methyl-α-cyclodexrin (mαCD)-catalyzed exchange of outer leaflet lipids (LEX) with exogenous order- or disorder-promoting phospholipids. We examined the functional impact of LEX by monitoring two Ag-stimulated responses: recruitment of cytoplasmic Syk kinase to the inner leaflet and exocytosis of secretory granules (degranulation). Based on the ImFCS data in resting cells, we observed global increase or decrease of inner leaflet order when outer leaflet is exchanged with order- or disorder-promoting lipids, respectively. We find that the degree of both stimulated Syk recruitment and degranulation correlates positively with LEX-mediated changes of inner leaflet order in resting cells. Overall, our results show that resting-state lipid ordering of the outer leaflet influences the ordering of the inner leaflet, likely via interleaflet coupling. This imposed lipid reorganization modulates transmembrane signaling stimulated by Ag clustering of IgE-FcεRI.

Metabolic Role of GABA in the Secretory Function of Pancreatic β-Cells: Its Hypothetical Implication in β-Cell Degradation in Type 2 Diabetes.

The stimulus-secretion coupling of a glucose-induced release is generally attributed to the metabolism of the hexose in the β-cells in the glycolytic pathway and the citric acid cycle. Glucose metabolism generates an increased cytosolic concentration of ATP and of the ATP/ADP ratio that closes the ATP-dependent K+-channel at the plasma membrane. The resultant depolarization of the β-cells opens voltage-dependent Ca2+-channels at the plasma membrane that triggers the exocytosis of insulin secretory granules. The secretory response is biphasic with a first and transient peak followed by a sustained phase. The first phase is reproduced by a depolarization of the β-cells with high extracellular KCl maintaining the KATP-channels open with diazoxide (triggering phase); the sustained phase (amplifying phase) depends on the participation of metabolic signals that remain to be determined. Our group has been investigating for several years the participation of the β-cell GABA metabolism in the stimulation of insulin secretion by three different secretagogues (glucose, a mixture of L-leucine plus L-glutamine, and some branched chain alpha-ketoacids, BCKAs). They stimulate a biphasic secretion of insulin accompanied by a strong suppression of the intracellular islet content of gamma-aminobutyric acid (GABA). As the islet GABA release simultaneously decreased, it was concluded that this resulted from an increased GABA shunt metabolism. The entrance of GABA into the shunt is catalyzed by GABA transaminase (GABAT) that transfers an amino group between GABA and alpha-ketoglutarate, resulting in succinic acid semialdehyde (SSA) and L-glutamate. SSA is oxidized to succinic acid that is further oxidized in the citric acid cycle. Inhibitors of GABAT (gamma-vinyl GABA, gabaculine) or glutamic acid decarboxylating activity (GAD), allylglycine, partially suppress the secretory response as well as GABA metabolism and islet ATP content and the ATP/ADP ratio. It is concluded that the GABA shunt metabolism contributes together with the own metabolism of metabolic secretagogues to increase islet mitochondrial oxidative phosphorylation. These experimental findings emphasize that the GABA shunt metabolism is a previously unrecognized anaplerotic mitochondrial pathway feeding the citric acid cycle with a β-cell endogenous substrate. It is therefore a postulated alternative to the proposed mitochondrial cataplerotic pathway(s) responsible for the amplification phase of insulin secretion. It is concluded the new postulated alternative suggests a possible new mechanism of β-cell degradation in type 2 (perhaps also in type 1) diabetes.

Multiple pathways and independent functional pools in insulin granule exocytosis.

In contrast to synaptic vesicle exocytosis, secretory granule exocytosis follows a much longer time course, and thus allows for different prefusion states prior to stimulation. Indeed, total internal reflection fluorescence microscopy in living pancreatic β cells reveals that, prior to stimulation, either visible or invisible granules fuse in parallel during both early (first) and late (second) phases after glucose stimulation. Therefore, fusion occurs not only from granules predocked to the plasma membrane but also from those translocated from the cell interior during ongoing stimulation. Recent findings suggest that such heterogeneous exocytosis is conducted by a specific set of multiple Rab27 effectors that appear to operate on the same granule; namely, exophilin-8, granuphilin, and melanophilin play differential roles in distinct secretory pathways to final fusion. Furthermore, the exocyst, which is known to tether secretory vesicles to the plasma membrane in constitutive exocytosis, cooperatively functions with these Rab27 effectors in regulated exocytosis. In this review, the basic nature of insulin granule exocytosis will be described as a representative example of secretory granule exocytosis, followed by a discussion of the means by which different Rab27 effectors and the exocyst coordinate to regulate the entire exocytic processes in β cells.

Ca2+ signals in pancreatic acinar cells in response to physiological stimulation in vivo.

The exocrine pancreas secretes fluid and digestive enzymes in response to parasympathetic release of acetylcholine (ACh) via the vagus nerve and the gut hormone cholecystokinin (CCK). Both secretion of fluid and exocytosis of secretory granules containing enzymes and zymogens are dependent on an increase in the cytosolic [Ca2+ ] in acinar cells. It is thought that the specific spatiotemporal characteristics of the Ca2+ signals are fundamental for appropriate secretion and that these properties are disrupted in disease states in the pancreas. While extensive research has been performed to characterize Ca2+ signalling in acinar cells, this has exclusively been achieved in ex vivo preparations of exocrine cells, where it is difficult to mimic physiological conditions. Here we have developed a method to optically observe pancreatic acinar Ca2+ signals in vivo using a genetically expressed Ca2+ indicator and imaged with multi-photon microscopy in live animals. In vivo, acinar cells exhibited baseline activity in fasted animals, which was dependent on CCK1 receptors (CCK1Rs). Both stimulation of intrinsic nervous input and administration of systemic CCK induced oscillatory activity in a proportion of the cells, but the maximum frequencies were vastly different. Upon feeding, oscillatory activity was also observed, which was dependent on CCK1Rs. No evidence of a vago-vagal reflex mediating the effects of CCK was observed. Our in vivo method revealed the spatial and temporal profile of physiologically evoked Ca2+ signals, which will provide new insights into future studies of the mechanisms underlying exocrine physiology and that are disrupted in pathological conditions. KEY POINTS: In the exocrine pancreas, the spatiotemporal properties of Ca2+ signals are fundamentally important for the appropriate stimulation of secretion by the neurotransmitter acetylcholine and gut hormone cholecystokinin. These characteristics were previously defined in ex vivo studies. Here we report the spatiotemporal characteristics of Ca2+ signals in vivo in response to physiological stimulation in a mouse engineered to express a Ca2+ indicator in acinar cells. Specific Ca2+ 'signatures' probably important for stimulating secretion are evoked in vivo in fasted animals, by feeding, neural stimulation and cholecystokinin administration. The Ca2+ signals are probably the result of the direct action of ACh and CCK on acinar cells and not indirectly through a vago-vagal reflex.

Functional hierarchy among different Rab27 effectors involved in secretory granule exocytosis.

The Rab27 effectors are known to play versatile roles in regulated exocytosis. In pancreatic beta cells, exophilin-8 anchors granules in the peripheral actin cortex, whereas granuphilin and melanophilin mediate granule fusion with and without stable docking to the plasma membrane, respectively. However, it is unknown whether these coexisting effectors function in parallel or in sequence to support the whole insulin secretory process. Here, we investigate their functional relationships by comparing the exocytic phenotypes in mouse beta cells simultaneously lacking two effectors with those lacking just one of them. Analyses of prefusion profiles by total internal reflection fluorescence microscopy suggest that melanophilin exclusively functions downstream of exophilin-8 to mobilize granules for fusion from the actin network to the plasma membrane after stimulation. The two effectors are physically linked via the exocyst complex. Downregulation of the exocyst component affects granule exocytosis only in the presence of exophilin-8. The exocyst and exophilin-8 also promote fusion of granules residing beneath the plasma membrane prior to stimulation, although they differentially act on freely diffusible granules and those stably docked to the plasma membrane by granuphilin, respectively. This is the first study to diagram the multiple intracellular pathways of granule exocytosis and the functional hierarchy among different Rab27 effectors within the same cell.

Mast cell–mediated inflammation relies on insulin-regulated aminopeptidase controlling cytokine export from the Golgi

On activation, mast cells rapidly release preformed inflammatory mediators from large cytoplasmic granules via regulated exocytosis. This acute degranulation is followed by a late activation phase involving synthesis and secretion of cytokines, growth factors, and other inflammatory molecules via the constitutive pathway that remains ill defined. We investigated the role for an insulin-responsive vesicle-like endosomal compartment, marked by insulin-regulated aminopeptidase (IRAP), in the secretion of TNF-α and IL-6 in mast cells and macrophages. Murine knockout (KO) mouse models (IRAP-KO and kit-Wsh/sh) were used to study inflammatory disease models and to measure and mechanistically investigate cytokine secretion and degranulation in bone marrow-derived mast cells invitro. IRAP-KO mice are protected from TNF-α-dependent kidney injury and inflammatory arthritis. In the absence of IRAP, TNF-α and IL-6 but not IL-10 fail to be efficiently secreted. Moreover, chemical targeting of IRAP endosomes reduced proinflammatory cytokine secretion. Mechanistically, impaired TNF-α export from the Golgi and reduced colocalization of vesicle-associated membrane protein (VAMP) 3-positive TNF-α transport vesicles with syntaxin 4 (aka Stx4) was observed in IRAP-KO mast cells, while VAMP8-dependent exocytosis of secretory granules was facilitated. IRAP plays a novel role in mast cell-mediated inflammation through the regulation of exocytic trafficking of cytokines.

Quantification of Secretory Granule Exocytosis by TIRF Imaging and Capacitance Measurements.

Hormones and neurotransmitters are released from (neuro)endocrine cells by regulated exocytosis of secretory granules. During exocytosis, the granule membrane fuses with the plasma membrane, which allows release of the stored content into the bloodstream or the surrounding tissue. Here, we give a detailed description of two complementary methods to observe and quantify exocytosis in single cells: high-resolution TIRF microscopy and patch-clamp capacitance recordings. Precise stimulation of exocytosis is achieved by local pressure application or voltage-clamp depolarizations. While the chapter is focused on insulin-secreting cells as an accessible and disease-relevant model system, the methodology is applicable to a wide variety of secretory cells including chromaffin and PC12 cells.

Electrophysiological Characterization of Stem-cell Derived β-cells to Help Produce Clinically Relevant Cells for Transplant

Although significant progress has been made in treating type 1 diabetes with islet transplantation, new cell sources are needed since organ donors remain limited. Stem cell-derived β cells (SCβ-cells) offer a promising alternative for cell replacement therapy; however, key characteristics displayed by mature primary human β cells, such as glucose-regulated excitability and insulin secretion via the exocytosis of secretory granules, remain blunted in SCβ-cells, reflecting immaturity after cellular differentiation.

Secretory granule exocytosis and its amplification by cAMP in pancreatic β-cells.

The sequence of events for secreting insulin in response to glucose in pancreatic β-cells is termed "stimulus-secretion coupling". The core of stimulus-secretion coupling is a process which generates electrical activity in response to glucose uptake and causes Ca2+ oscillation for triggering exocytosis of insulin-containing secretory granules. Prior to exocytosis, the secretory granules are mobilized and docked to the plasma membrane and primed for fusion with the plasma membrane. Together with the final fusion with the plasma membrane, these steps are named the exocytosis process of insulin secretion. The steps involved in the exocytosis process are crucial for insulin release from β-cells and considered indispensable for glucose homeostasis. We recently confirmed a signature of defective exocytosis process in human islets and β-cells of obese donors with type 2 diabetes (T2D). Furthermore, cyclic AMP (cAMP) potentiates glucose-stimulated insulin secretion through mechanisms including accelerating the exocytosis process. In this mini-review, we aimed to organize essential knowledge of the secretory granule exocytosis and its amplification by cAMP. Then, we suggest the fatty acid translocase CD36 as a predisposition in β-cells for causing defective exocytosis, which is considered a pathogenesis of T2D in relation to obesity. Finally, we propose potential therapeutics of the defective exocytosis based on a CD36-neutralizing antibody and on Apolipoprotein A-I (ApoA-I), for improving β-cell function in T2D.

CLC Anion/Proton Exchangers Regulate Secretory Vesicle Filling and Granule Exocytosis in Chromaffin Cells.

ClC-3, ClC-4, and ClC-5 are electrogenic chloride/proton exchangers that can be found in endosomal compartments of mammalian cells. Although the association with genetic diseases and the severe phenotype of knock-out animals illustrate their physiological importance, the cellular functions of these proteins have remained insufficiently understood. We here study the role of two Clcn3 splice variants, ClC-3b and ClC-3c, in granular exocytosis and catecholamine accumulation of adrenal chromaffin cells using a combination of high-resolution capacitance measurements, amperometry, protein expression/gene knock out/down, rescue experiments, and confocal microscopy. We demonstrate that ClC-3c resides in immature as well as in mature secretory granules, where it regulates catecholamine accumulation and contributes to the establishment of the readily releasable pool of secretory vesicles. The lysosomal splice variant ClC-3b contributes to vesicle priming only with low efficiency and leaves the vesicular catecholamine content unaltered. The related Cl-/H+ antiporter ClC-5 undergoes age-dependent downregulation in wild-type conditions. Its upregulation in Clcn3-/- cells partially rescues the exocytotic mutant defect. Our study demonstrates how different CLC transporters with similar transport functions, but distinct localizations can contribute to vesicle functions in the regulated secretory pathway of granule secretion in chromaffin cells.SIGNIFICANCE STATEMENT Cl-/H+ exchangers are expressed along the endosomal/lysosomal system of mammalian cells; however, their exact subcellular functions have remained insufficiently understood. We used chromaffin cells, a system extensively used to understand presynaptic mechanisms of synaptic transmission, to define the role of CLC exchangers in neurosecretion. Disruption of ClC-3 impairs catecholamine accumulation and secretory vesicle priming. There are multiple ClC-3 splice variants, and only expression of one, ClC-3c, in double Cl-/H+ exchanger-deficient cells fully rescues the WT phenotype. Another splice variant, ClC-3b, is present in lysosomes and is not necessary for catecholamine secretion. The distinct functions of ClC-3c and ClC-3b illustrate the impact of expressing multiple CLC transporters with similar transport functions and separate localizations in different endosomal compartments.

Intravital subcellular and single molecule imaging reveals multiple actin filament populations collaborate in the remodelling of the secretory granule membrane

The actin cytoskeleton contributes to many cell functions that involve remodelling of membranes. One of these processes is exocytosis of large secretory granules in the salivary gland. We used intravital subcellular microscopy (ISMic) to identify the types of actin filaments that are required for granule integration into the apical membrane and intravital single molecule microscopy (iSiMM) to study the molecular behaviour and nanostructures at the basolateral membrane. We find at least two types of actin filaments are associated with exocytosis at the apical membrane.

Secretome and Tunneling Nanotubes: A Multilevel Network for Long Range Intercellular Communication between Endothelial Cells and Distant Cells.

As a cellular interface between the blood and tissues, the endothelial cell (EC) monolayer is involved in the control of key functions including vascular tone, permeability and homeostasis, leucocyte trafficking and hemostasis. EC regulatory functions require long-distance communications between ECs, circulating hematopoietic cells and other vascular cells for efficient adjusting thrombosis, angiogenesis, inflammation, infection and immunity. This intercellular crosstalk operates through the extracellular space and is orchestrated in part by the secretory pathway and the exocytosis of Weibel Palade Bodies (WPBs), secretory granules and extracellular vesicles (EVs). WPBs and secretory granules allow both immediate release and regulated exocytosis of messengers such as cytokines, chemokines, extracellular membrane proteins, coagulation or growth factors. The ectodomain shedding of transmembrane protein further provide the release of both receptor and ligands with key regulatory activities on target cells. Thin tubular membranous channels termed tunneling nanotubes (TNTs) may also connect EC with distant cells. EVs, in particular exosomes, and TNTs may contain and transfer different biomolecules (e.g., signaling mediators, proteins, lipids, and microRNAs) or pathogens and have emerged as a major triggers of horizontal intercellular transfer of information.

The immunophilin Zonda controls regulated exocytosis in endocrine and exocrine tissues.

Exocytosis is a fundamental process in physiology, that ensures communication between cells, organs and even organisms. Hormones, neuropeptides and antibodies, among other cargoes are packed in exocytic vesicles that need to reach and fuse with the plasma membrane to release their content to the extracellular milieu. Hundreds of proteins participate in this process and several others in its regulation. We report here a novel component of the exocytic machinery, the Drosophila transmembrane immunophilin Zonda (Zda), previously found to participate in autophagy. Zda is highly expressed in secretory tissues, and regulates exocytosis in at least three of them: the ring gland, insulin-producing cells and the salivary gland. Using the salivary gland as a model system, we found that Zda is required at final steps of the exocytic process for fusion of secretory granules to the plasma membrane. In a genetic screen we identified the small GTPase RalA as a crucial regulator of secretory granule exocytosis that is required, similarly to Zda, for fusion between the secretory granule and the plasma membrane.

Normal and defective pathways in biogenesis and maintenance of the insulin storage pool.

Both basal and glucose-stimulated insulin release occur primarily by insulin secretory granule exocytosis from pancreatic β cells, and both are needed to maintain normoglycemia. Loss of insulin-secreting β cells, accompanied by abnormal glucose tolerance, may involve simple exhaustion of insulin reserves (which, by immunostaining, appears as a loss of β cell identity), or β cell dedifferentiation, or β cell death. While various sensing and signaling defects can result in diminished insulin secretion, somewhat less attention has been paid to diabetes risk caused by insufficiency in the biosynthetic generation and maintenance of the total insulin granule storage pool. This Review offers an overview of insulin biosynthesis, beginning with the preproinsulin mRNA (translation and translocation into the ER), proinsulin folding and export from the ER, and delivery via the Golgi complex to secretory granules for conversion to insulin and ultimate hormone storage. All of these steps are needed for generation and maintenance of the total insulin granule pool, and defects in any of these steps may, weakly or strongly, perturb glycemic control. The foregoing considerations have obvious potential relevance to the pathogenesis of type 2 diabetes and some forms of monogenic diabetes; conceivably, several of these concepts might also have implications for β cell failure in type 1 diabetes.

Granular detail of β cell structures for insulin secretion.

Pancreatic β cells secrete insulin in response to increased glucose concentrations. Müller et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202010039) use 3D FIB-SEM to study the architecture of these cells and to elucidate how glucose stimulation remodels microtubules to control insulin secretory granule exocytosis.

Melanophilin Accelerates Insulin Granule Fusion without Predocking to the Plasma Membrane.

Direct observation of fluorescence-labeled secretory granule exocytosis in living pancreatic β-cells has revealed heterogeneous prefusion behaviors: some granules dwell beneath the plasma membrane before fusion, while others fuse immediately once they are recruited to the plasma membrane. Although the former mode seems to follow sequential docking-priming-fusion steps as found in synaptic vesicle exocytosis, the latter mode, which is unique to secretory granule exocytosis, has not been explored well. Here, we show that melanophilin, one of the effectors of the monomeric guanosine-5'-triphosphatase Rab27 on the granule membrane, is involved in such an accelerated mode of exocytosis. Melanophilin-mutated leaden mouse and melanophilin-downregulated human pancreatic β-cells both exhibit impaired glucose-stimulated insulin secretion, with a specific reduction in fusion events that bypass stable docking to the plasma membrane. Upon stimulus-induced [Ca2+]i rise, melanophilin mediates this type of fusion by dissociating granules from myosin-Va and actin in the actin cortex and by associating them with a fusion-competent, open form of syntaxin-4 on the plasma membrane. These findings provide the hitherto unknown mechanism to support sustainable exocytosis by which granules are recruited from the cell interior and fuse promptly without stable predocking to the plasma membrane.