A role for serglycin proteoglycan in granular retention and processing of mast cell secretory granule components

In the absence of serglycin proteoglycans, connective tissue-type mast cells fail to assemble mature metachromatic secretory granules, and this is accompanied by a markedly reduced ability to store neutral proteases. However, the mechanisms behind these phenomena are not known. In this study, we addressed these issues by studying the functionality and morphology of secretory granules as well as the fate of the secretory granule proteases in bone marrow-derived mast cells from serglycin(+/+) and serglycin(-/-) mice. We show that functional secretory vesicles are formed in both the presence and absence of serglycin, but that dense core formation is defective in serglycin(-/-) mast cell granules. The low levels of mast cell proteases present in serglycin(-/-) cells had a granular location, as judged by immunohistochemistry, and were released following exposure to calcium ionophore, indicating that they were correctly targeted into secretory granules even in the absence of serglycin. In the absence of serglycin, the fates of the serglycin-dependent proteases differed, including preferential degradation, exocytosis or defective intracellular processing. In contrast, beta-hexosaminidase storage and release was not dependent on serglycin. Together, these findings indicate that the reduced amounts of neutral proteases in the absence of serglycin is not caused by missorting into the constitutive pathway of secretion, but rather that serglycin may be involved in the retention of the proteases after their entry into secretory vesicles.     

Chromogranins A and B are key proteins in amine accumulation, but the catecholamine secretory pathway is conserved without them

Chromogranins are the main soluble proteins in the large dense core secretory vesicles (LDCVs) found in aminergic neurons and chromaffin cells. We recently demonstrated that chromogranins A and B each regulate the concentration of adrenaline in chromaffin granules and its exocytosis. Here we have further studied the role played by these proteins by generating mice lacking both chromogranins. Surprisingly, these animals are both viable and fertile. Although chromogranins are thought to be essential for their biogenesis, LDCVs were evident in these mice. These vesicles do have a somewhat atypical appearance and larger size. Despite their increased size, single-cell amperometry recordings from chromaffin cells showed that the amine content in these vesicles is reduced by half. These data demonstrate that although chromogranins regulate the amine concentration in LDCVs, they are not completely essential, and other proteins unrelated to neurosecretion, such as fibrinogen, might compensate for their loss to ensure that vesicles are generated and the secretory pathway conserved.     

cis-Unsaturated fatty acids induce the fusion of chromaffin granules aggregated by synexin

When isolated chromaffin granules were aggregated by synexin (a Ca2+-binding protein present in chromaffin and other secretory tissues) and then exposed to cis-unsaturated fatty acids at 37 degrees C, they fused together to form large vesicles. The fusion was monitored by phase and electron microscopy and by turbidity measurements on the granule suspension. Arachidonic acid was the most effective fusogen, whereas trans-unsaturated fatty acids, saturated fatty acids, detergents or lysolecithin were inactive. During fusion some of the epinephrine of the granules was released but the soluble core proteins remained trapped in the resulting vesicles. These vesicles swelled to enclose the maximum volume. Although this swelling could be inhibited by increasing the osmotic strength of the medium, it did not appear to depend on the chemiosmotic properties of the granule membranes as it was not influenced by ATP, a proton ionophore, or an anion transport inhibitor. The regulators of this in vitro fusion--Ca2+, synexin, and free, cis-unsaturated fatty acids--may be present in the cytoplasm of the chromaffin cell when it is stimulated to release epinephrine and granule proteins by exocytosis. Therefore, this fusion event may be the same that occurs between chromaffin granules undergoing compound exocytosis.     

Insight in the exocytotic process in chromaffin cells: regulation by trimeric and monomeric G proteins

Catecholamine secretion from chromaffin cells has been used for a long time as a general model to study exocytosis of large dense core secretory granules. Permeabilization and microinjection techniques have brought the possibility to dissect at the molecular level the multi-protein machinery involved in this complex physiological process. Regulated exocytosis comprises distinct and sequential steps including the priming of secretory granules, the formation of a docking complex between granules and the plasma membrane and the subsequent fusion of the granule with the plasma membrane. Key proteins involved in the exocytotic machinery have been identified. For instance, SNAREs which participate in the docking events in most intracellular transport steps along the secretory pathway, play a role in exocytosis in both neuronal and endocrine cells. However, in contrast to intracellular transport processes for which the highest fusion efficiency is required after correct targeting of the vesicles, the number of exocytotic events in activated secretory cells needs to be tightly controlled. We describe here the multistep control exerted by heterotrimeric and monomeric G proteins on the progression of secretory granules from docking to fusion and the molecular nature of some of their downstream effectors in neuroendocrine chromaffin cells.     

Exocytotic exposure and recycling of membrane antigens of chromaffin granules: ultrastructural evaluation after immunolabeling

The exocytotic exposure and retrieval of an antigen of chromaffin granule membranes were studied with chromaffin cells isolated from bovine adrenal medulla. Cells were incubated with an antiserum against glycoprotein III followed by fluorescein- or gold-labeled anti-IgG. Immunofluorescence on the cell surface was present in a patchy distribution irrespective of whether bivalent antibodies or Fab fragments were used. During subsequent incubation these fluorescent membrane patches were internalized within 45 min. At the ultrastructural level immunogold-labeled patches were present on the surface of stimulated cells. During incubation (5 min to 6 h) these immunolabeled membrane patches became coated, giving rise to coated vesicles and finally to smooth vesicles. These latter vesicles were found spread throughout the cytoplasm including the Golgi region, but Golgi stacks did not become labeled. Part of the immunolabel was transferred to multivesicular bodies, which probably represent a lysosomal pathway. 30 min after incubation immunolabel was also found in electron-dense vesicles apparently representing newly formed chromaffin granules. After 6 h of incubation immunolabel was found in vesicles indistinguishable from mature chromaffin granules. These results provide direct evidence that after exocytosis membranes of chromaffin granules are selectively retrieved from the plasma membrane and are partly recycled to newly formed chromaffin granules, providing a shuttle service from the Golgi region to the plasma membrane.     

Analysis of SCAMP1 function in secretory vesicle exocytosis by means of gene targeting in mice.

Secretory carrier membrane proteins (SCAMPs) comprise a family of ubiquitous membrane proteins of transport vesicles with no known function. Their universal presence in all cells suggests a fundamental role in membrane traffic. SCAMPs are particularly highly expressed in organelles that undergo regulated exocytosis, such as synaptic vesicles and mast cell granules. Of the three currently known SCAMPs, SCAMP1 is the most abundant. To investigate the possible functions of SCAMP1, we generated mice that lack SCAMP1. SCAMP1-deficient mice are viable and fertile. They exhibit no changes in the overall architecture or the protein composition of the brain or alterations in peripheral organs. Capacitance measurements in mast cells demonstrated that exocytosis could be triggered reliably by GTPgammaS in SCAMP1-deficient cells. The initial overall capacitance of mast cells was similar between wild type and mutant mice, but the final cell capacitance after completion of exocytosis, was significantly smaller in SCAMP1-deficient cells than in wild type cells. Furthermore, there was an increased proportion of reversible fusion events, which may have caused the decrease in the overall capacitance change observed after exocytosis. Our data show that SCAMP1 is not essential for exocytosis, as such, and does not determine the stability or size of secretory vesicles, but is required for the full execution of stable exocytosis in mast cells. This phenotype could be the result of a function of SCAMP1 in the formation of stable fusion pores during exocytosis or of a role of SCAMP1 in the regulation of endocytosis after formation of fusion pores.     

Exocytosis of single chromaffin granules in cell-free inside-out membrane patches

In chromaffin cells, exocytosis of single granules and properties of the fusion pore--the first connection between vesicular lumen and extracellular space --can be studied by cell-attached patch amperometry, which couples patch-clamp capacitance measurements with simultaneous amperometric recordings of transmitter release. Here we have studied exocytosis of single chromaffin granules and endocytosis of single vesicles in cell-free inside-out membrane patches by patch capacitance measurements and patch amperometry. We excised patches from chromaffin cells by using methods developed for studying properties of single ion channels. With low calcium concentrations in the pipette and bath, the patches showed no spontaneous exocytosis, but exocytosis could be induced in some patches by applying calcium to the cytoplasmic side of the patch. Exocytosis was also stimulated by calcium entry through the patch membrane. Initial conductances of the fusion pore were undistinguishable in cell-attached and excised patch recordings, but the subsequent pore expansion was slower in excised patches. The properties of exocytotic fusion pores in chromaffin cells are very similar to those observed in mast cells and granulocytes. Excised patches provide a tool with which to study the mechanisms of fusion pore formation and endocytosis in vitro.     

Vesicular Ca(2+) mediates granule motion and exocytosis

Secretory vesicles of chromaffin cells are acidic organelles that maintain an increasing pH gradient towards the cytosol (5.5 vs. 7.3) that is mediated by V-ATPase activity. This gradient is primarily responsible for the accumulation of large concentrations of amines and Ca(2+), although the mechanisms mediating Ca(2+) uptake and release from granules, and the physiological relevance of these processes, remain unclear. The presence of a vesicular matrix appears to create a bi-compartmentalised medium in which the major fractions of solutes, including catecholamines, nucleotides and Ca(2+), are strongly associated with vesicle proteins, particularly chromogranins. This association appears to be favoured at acidic pH values. It has been demonstrated that disrupting the pH gradient of secretory vesicles reduces their rate of exocytosis and promotes the leakage of vesicular amines and Ca(2+), dramatically increasing the movement of secretory vesicles and triggering exocytosis. In this short review, we will discuss the data available that highlights the importance of pH in regulating the association between chromogranins, vesicular amines and Ca(2+). We will also address the potential role of vesicular Ca(2+) in two major processes in secretory cells, vesicle movement and exocytosis.     

Bovine chromaffin granule membranes undergo Ca(2+)-regulated exocytosis in frog oocytes

We have devised a new method that permits the investigation of exogenous secretory vesicle function using frog oocytes and bovine chromaffin granules, the secretory vesicles from adrenal chromaffin cells. Highly purified chromaffin granule membranes were injected into Xenopus laevis oocytes. Exocytosis was detected by the appearance of dopamine-beta-hydroxylase of the chromaffin granule membrane in the oocyte plasma membrane. The appearance of dopamine-beta-hydroxylase on the oocyte surface was strongly Ca(2+)-dependent and was stimulated by coinjection of the chromaffin granule membranes with InsP3 or Ca2+/EGTA buffer (18 microM free Ca2+) or by incubation of the injected oocytes in medium containing the Ca2+ ionophore ionomycin. Similar experiments were performed with a subcellular fraction from cultured chromaffin cells enriched with [3H]norepinephrine-containing chromaffin granules. Because the release of [3H]norepinephrine was strongly correlated with the appearance of dopamine-beta-hydroxylase on the oocyte surface, it is likely that intact chromaffin granules and chromaffin granule membranes undergo exocytosis in the oocyte. Thus, the secretory vesicle membrane without normal vesicle contents is competent to undergo the sequence of events leading to exocytosis. Furthermore, the interchangeability of mammalian and amphibian components suggests substantial biochemical conservation of the regulated exocytotic pathway during the evolutionary progression from amphibians to mammals.     

Adrenal chromaffin granules and secretory granules from thyroid parafollicular cells have several common antigens

The presence of various antigens in two types of isolated endocrine vesicles (chromaffin granules and secretory vesicles of thyroid parafollicular cells) was investigated by immunoblotting. The two types of vesicles have three common secretory proteins: chromogranin A, chromogranin B and secretogranin II. Furthermore, six common membrane antigens were found: cytochrome b-561, carboxypeptidase H, glycoprotein II, glycoprotein III, synaptin/synaptophysin and SV 2. These results demonstrate that vesicles obtained from neural crest-derived endocrine cells not only share several common secretory peptides and proteins, but also have common properties as far as their membrane antigens are concerned.     

Presence of syntaxin 1A in secretory granules of chromaffin cells and interaction with chromogranins A and B

Syntaxin 1A and synaptotagmin I are key participants of fusion complex formation during exocytotic processes, and syntaxin 1A is known to be present in the plasma membrane. Here, we show the presence of not only synaptotagmin I but also syntaxin 1A in secretory granules of bovine adrenal chromaffin cells by immunogold electron microscopy, and further demonstrate the interaction of these proteins with chromogranins A and B (CGA and CGB), two major proteins of secretory granules. Interaction between chromogranins and the components of fusion complex also suggests active participation of CGA and CGB in fusion complex formation and subsequent exocytosis.     

Exocytotic release from individual granules exhibits similar properties at mast and chromaffin cells.

The effects of temperature on granular secretion were studied in individual bovine adrenal chromaffin and rat peritoneal mast cells. It was found that more molecules are released from individual granules at physiological temperature than at room temperature, where such experiments are normally performed. In mast cells, there is also a dramatic decrease in the time required for exocytosis to be complete at 37 degrees C compared to room temperature. In the presence of some cations, the amount released from individual granules at room temperature from both types of cells could be altered. The amount of secretion decreased with the divalent cation zinc but increased with the monovalent cation cesium. These experiments used two electrochemical techniques: cyclic voltammetry and amperometry. With amperometry, the concentration gradient created by the electrode near the cell further increased the amount of release. Similar responses to changes in the extracellular environment in chromaffin and mast cells suggest that the mechanism of extrusion of the granule contents is similar in both cell types.     

Secretory granules: and the last shall be first..

By tagging secretory granules with the fluorescent protein dsRed-E5, which changes its emission from green to red over time, Duncan et al. analysed the age-dependent distribution of secretory vesicles within chromaffin cells. This elegant study illustrates as never before how age is a critical factor that segregates granules with respect to their localization and mobility and the probability of them undergoing exocytosis in response to different stimuli.     

An unexpected role for autophagy in degranulation of mast cells

Mast cells play a crucial role in allergic inflammatory reactions through releasing cytosolic granules upon antigen stimulation. However, the mechanisms underlying maturation and release of secretory granules are not fully understood. We found that autophagy is constitutively induced in mast cells under full nutrition conditions, and type II LC3 (LC3-II), a marker for autophagosomes, localizes on secretory granules. While deletion of Atg7 does not impair the development of bone marrow-derived mast cells (BMMCs), Atg7-deficient BMMCs show severe impairment of degranulation, but not cytokine production, upon antigen stimulation. Moreover we found that LC3-II, but not LC3-I, colocalizes with CD63, a marker for secretory lysosomes and is released extracellularly along with degranulation in wild-type BMMCs, but not Atg7-deficient BMMCs. Finally, passive cutaneous anaphylaxis reactions are almost completely abolished in mast celldeficient mice reconstituted with Atg7-deficient BMMCs. Collectively, these results suggest that autophagy is not essential for the development, but plays a crucial role in degranulation, of mast cells.     

Changes in mobility of chromaffin granules in actin network with its assembly and Ca(2+)-dependent disassembly by gelsolin.

As a final stage of cell signal transduction, secretory cells release hormones by exocytosis. Before secretory granules contact with the cell membrane for fusion, an actin-network barrier must dissociate as a prelude. To elucidate dynamical behaviors of secretory granules in actin networks, in vitro assembly and disassembly processes of actin networks were examined by means of dynamic light-scattering spectroscopy. We studied actin polymerization in the presence of chromaffin granules isolated from bovine adrenal medullas and found that the entanglement of actin filaments rapidly formed cages that confined granules in them. We also studied the effect of gelsolin, one of actin-severing proteins, on the network of actin filaments preformed in the presence of chromaffin granules. It turned out that the cages that confined granules rapidly disappeared when gelsolin was added in the presence of free Ca2+ ions. A semiquantitative analysis of dynamic light-scattering spectra permitted us to estimate the changes in the mobility (or the translational diffusion coefficient) of chromaffin granules in the actin network with its assembly and Ca(2+)-dependent disassembly by gelsolin. Based on the present results and some pieces of evidence in the literature, a model is proposed for biophysical situations before, during, and after an exocytotic event.     

Mechanisms underlying phasic and sustained secretion in chromaffin cells from mouse adrenal slices.

Many neurosecretory preparations display two components of depolarization-induced exocytosis: a phasic component synchronized with Ca2+ channel opening, followed by a slower sustained component. We evaluated possible mechanisms underlying this biphasic behavior by stimulating mouse chromaffin cells in situ with both depolarizations and flash photolysis of caged Ca2+. From a direct comparison of the secretory responses to both stimuli, we conclude that phasic and sustained release components originate from a readily releasable pool (RRP) of equally fusion-competent vesicles, suggesting that differences in the vesicles' proximity to Ca2+ channels underlie the biphasic secretory behavior. An intermediate pool in dynamic equilibrium with the RRP ensures rapid recruitment of release-ready vesicles after RRP depletion. Our results are discussed in terms of a refined model for secretion in chromaffin cells.     

Neurotransmitter release: fusion or 'kiss-and-run'?

The clear synaptic vesicles of neurons release their contents at the presynaptic membrane and are then quickly retrieved. However, it is unclear whether a complete cycle of exocytosis and endocytosis is always involved or whether neurotransmitter can be released by a transient interaction. Recent findings in chromaffin and mast cells suggest that exocytosis is preceded by the formation of a pore that has similar conductance properties to ion channels. The content of the secretory organelle partially escapes at this early step, but the pore can close before the vesicle fuses fully. This article looks at the evidence that quantal release of neurotransmitter from clear synaptic vesicles may occur by a similar 'kiss-and-run' mechanism.     

‘Neurosecretion’ by a classic cholinergic innervation apparatus

Summary Nerve terminals forming typical synapses with adrenal chromaffin tissues have been examined in the goldfish, frog (Rana pipiens), hamster and rat. Presumptive secretory inclusions present in the terminals are of two distinct types. Electron-lucent synaptic vesicles 30–50 nm in diameter are densely clustered adjacent to membrane thickenings and presumably discharge their contents into the synaptic clefts. Secretory granules (i.e. large dense-cored vesicles) 60–100 nm in diameter are more abundant in other parts of the terminals. Sites of granule exocytosis have been observed in each of the animals investigated. They are usually encountered within apparently undifferentiated areas of plasmalemma and only rarely occur within synaptic thickenings. Granule exocytosis from within synaptic terminals and chromaffin gland cells is most readily observed in specimens exposed, prior to fixation, to saline solutions containing both tannic acid, and 4-aminopyridine and/or elevated levels of K⁺. These findings show that the pattern of secretory discharge, involving both synaptic and non-synaptic release, which is widespread in invertebrate central nervous systems, is also characteristic of vertebrate, peripheral cholinergic terminals.     

Membrane recycling after exocytosis: An ultrastructural study of cultured chromaffin cells

When exocytosis of granule contents is induced by nicotine stimulation, glycoprotein III (a chromaffin granule membrane constituent) is exposed on the surface of cultured chromaffin cells, where it may be labeled with an immunocytochemical tracer. The subsequent fate of this glycoprotein after endocytosis was followed at the ultrastructural level using immunogold methods and was analyzed by morphometry. After stimulation exocytosis membranes newly inserted into the plasma membrane labeled with gold particles for glycoprotein III were found to be endocytosed via coated vesicles and finally found in organelles devoid of chromogranin A, the major secretory granule protein. At intervals between 30 min and 24 h after cell stimulation and immunolabeling, most labeled structures were identified by two different morphological approaches as prelysosomes and lysosomes. In contrast with results obtained on freshly isolated chromaffin cells, it is thus concluded that in cultured cells granule membrane recycling into new granules does not occur. It is suggested that the fate of granule membrane endocytosed after cell stimulation may be influenced by the external conditions to which cells are previously exposed.     

Rab3 Proteins Involved in Vesicle Biogenesis and Priming in Embryonic Mouse Chromaffin Cells

The four Rab3 paralogs A–D are involved in exocytosis, but their mechanisms of action are hard to study due to functional redundancy. Here, we used a quadruple Rab3 knockout (KO) (rab3a, rab3b, rab3c, rab3d null, here denoted as ABCD^?/?) mouse line to investigate Rab3 function in embryonic mouse adrenal chromaffin cells by electron microscopy and electrophysiological measurements. We show that in cells from ABCD^?/? animals large dense‐core vesicles (LDCVs) are less abundant, while the number of morphologically docked granules is normal. By capacitance measurements, we show that deletion of Rab3s reduces the size of the releasable vesicle pools but does not alter their fusion kinetics, consistent with an altered function in vesicle priming. The sustained release component has a sigmoid shape in ABCD^?/? cells when normalized to the releasable pool size, indicating that vesicle priming follows at a higher rate after an initial delay. Rescue experiments showed that short‐term (4–6 h) overexpression of Rab3A or Rab3C suffices to rescue vesicle priming and secretion, but it does not restore the number of secretory vesicles. We conclude that Rab3 proteins play two distinct stimulating roles for LDCV fusion in embryonic chromaffin cells, by facilitating vesicle biogenesis and stabilizing the primed vesicle state.