Intragranular vesicles: new organelles in the secretory granules of adrenal chromaffin cells

Summary Chromaffin granules from bovine adrenal medullary chromaffin cells have been found to contain small vesicular structures bounded by unit membranes. Detection of these intragranular vesicles within intact cells requires the use of quick-freezing methods. The intragranular vesicles are labile to fixation by aldehydes which explains why they have not been described in intact cells until now. They are found in approximately 60% of the dense-core chromaffin granules in cells and 85% of isolated granules. They are usually clustered in groups of one to as many as five between the core and the inner surface of the granule membrane. The intragranular vesicles are independent vesicles in that they do not appear as simple invaginations of the granule membrane in either serial thin-section or freeze-etch views. Furthermore, they are released from the cell along with granule contents during nicotine-induced secretion of catecholamines. The structural heterogeneity provided by the intragranular vesicles may be related to the functional heterogeneity of granule contents observed in many recent biochemical studies.     

Calcium dynamics in catecholamine-containing secretory vesicles

We have used an aequorin chimera targeted to the membrane of the secretory granules to monitor the free [Ca(2+)] inside them in neurosecretory PC12 cells. More than 95% of the probe was located in a compartment with an homogeneous [Ca(2+)] around 40 microM. Cell stimulation with either ATP, caffeine or high-K(+) depolarization increased cytosolic [Ca(2+)] and decreased secretory granule [Ca(2+)] ([Ca(2+)](SG)). Inositol-(1,4,5)-trisphosphate, cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate were all ineffective to release Ca(2+) from the granules. Changes in cytosolic [Na(+)] (0-140 mM) or [Ca(2+)] (0-10 microM) did not modify either ([Ca(2+)](SG)). Instead, [Ca(2+)](SG) was highly sensitive to changes in the pH gradient between the cytosol and the granules. Both carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and nigericin, as well as cytosolic acidification, reversibly decreased [Ca(2+)](SG), while cytosolic alcalinization reversibly increased [Ca(2+)](SG). These results are consistent with the operation of a H(+)/Ca(2+) antiporter in the vesicular membrane. This antiporter could also mediate the effects of ATP, caffeine and high-K(+) on [Ca(2+)](SG), because all of them induced a transient cytosolic acidification. The FCCP-induced decrease in [Ca(2+)](SG) was reversible in 10-15 min even in the absence of cytosolic Ca(2+) or ATP, suggesting that most of the calcium content of the vesicles is bound to a slowly exchanging Ca(2+) buffer. This large store buffers [Ca(2+)](SG) changes in the long-term but allows highly dynamic free [Ca(2+)](SG) changes to occur in seconds or minutes.     

Distribution and orientation of microtubules in milk secreting epithelial cells of rat mammary gland

Summary Quantitative ultrastructural analysis of mid-lactation rat mammary gland demonstrated that cytoplasmic microtubules were present in nearly all secretory epithelial cells examined. Most microtubules were oriented perpendicular to the apical membrane and were found in the apical and medial portions of the cell cytoplasm. There was no statistical difference between the number of microtubules associated with vesicles and the number that were not. Most vesicles which were in contact with microtubules were small (50 to 150 nm), appeared electron lucent and were located in a supra-Golgi complex position. Many of these vesicles were seen to be aligned along the axis of longitudinally sectioned microtubules oriented perpendicular to the apical plasma membrane. As measured by a colchicine binding assay, the total tubulin content of mammary tissue from mid-lactation rats was about 107 g/100 mg wet weight. Approximately 19% of the total tubulin was in polymerized form. This study provides evidence that microtubules may be involved in guiding transport of small secretory vesicles to the apical regions of cells for exocytosis.     

Identification of secretory vesicles in homogenates of pea stem segments

In homogenates of stem sections from etiolated pea (Pisum sativum L.) seedlings, secretory vesicles can be separated from Golgi-apparatus cisternae by rate-zonal centrifugation in renografin gradients. Optically, two bands of turbidity are observed, the uppermost containing the secretory vesicles and the lower one the Golgi-apparatus cisternae. The absence of glutaraldehyde in the homogenizing medium has allowed the effective characterization of marker-enzyme activities. Golgi-apparatus cisternae have been recognized by the presence of inosine-diphosphatase and glucan-synthase I activities as well as by electron microscopy. In contrast, although secretory vesicles also bear inosine diphosphatase they do not appear to possess glucan-synthase activity. Three plasma-membrane markers, NPA-binding, glucan synthase II, and KCl,Mg²⁺-adenosine triphosphatase (pH 6.5), were not detected in secretory vesicles. Pulse-chase experiments with [³H]glucose support our designation of secretory vesicles and Golgi-cisternal fractions.Abbreviations ER endoplasmic reticulum - GSI, GSII glucan, synthase I, II, respectively - IDPase inosine diphosphatase - PM plasma membrane - SV(s) secretory vesicle(s)     

Micro-vesicles,pyriform vesicles and macro-vesicles associated with the plasma membrane in the root hairs ofVicia hirsuta after freeze-substitution

After freeze-substitution, micro-vesicles were found only in close proximity to the plasma membrane. Macro and pyriform vesicles were found throughout the cytosol, but also ‘packaged’ close to the plasma membrane, the package delineated by electron transparent outlines similar to the endoplasmic reticulum. These outlines appeared to be continuous with nearby endoplasmic reticulum and were always associated with Golgi bodies and microtubules. Micro-vesicles were found only in grazing sections of the plasma membrane made between the apical dome and the region of the nucleus, where the cell is the most cytoplasmic, and only in close proximity to the plasma membrane. Micro-vesicles were also found in close proximity to microtubules as well as other vesicle types. From the results it is suggested that pyriform and micro-vesicles may have specialised roles in root hair tip growth.     

Lithium enhances secretion from large dense-core vesicles in nerve growth factor-differentiated PC12 cells

Considerable attention has been focused on the therapeutic role of lithium (Li) in bipolar disorders. Although no consensus has emerged, Li presumably influences the behavior of neurons that regulate mood and behavior. Using PC12 cells to study cellular and molecular actions of Li, we previously reported that Li modulates the expression of proteins associated with large dense-core vesicles (LDCVs; organelles typically containing monoamines, neuropeptides and other cargo proteins). The current investigation indicates that this enhanced expression of LDCV proteins correlates with an altered secretory phenotype in Li-treated cells. Immunoblotting detects significant increases in the cellular content and secretion of the LDCV cargo proteins chromogranin B and secretogranin II. Amperometry reveals an increase of spike number elicited by K+-depolarization of Li-treated cells but no change of spike amplitude or kinetics. Electron microscopy reveals no significant change in LDCV number per unit area in Li-treated cells. However, there is a significant increase (about 15%) in the diameter of LDCVs after Li. Thus, Li induces changes in the properties of LDCVs that culminate in augmented regulated secretion in nerve growth factor-differentiated PC12 cells. These results extend our understanding of Li-dependent changes of cellular function that may be germane to the therapeutic action of Li.     

Imaging exocytosis of ATP-containing vesicles with TIRF microscopy in lung epithelial A549 cells

Nucleotide release constitutes the first step of the purinergic signaling cascade, but its underlying mechanisms remain incompletely understood. In alveolar A549 cells much of the experimental data is consistent with Ca²⁺-regulated vesicular exocytosis, but definitive evidence for such a release mechanism is missing, and alternative pathways have been proposed. In this study, we examined ATP secretion from A549 cells by total internal reflection fluorescence microscopy to directly visualize ATP-loaded vesicles and their fusion with the plasma membrane. A549 cells were labeled with quinacrine or Bodipy-ATP, fluorescent markers of intracellular ATP storage sites, and time-lapse imaging of vesicles present in the evanescent field was undertaken. Under basal conditions, individual vesicles showed occasional quasi-instantaneous loss of fluorescence, as expected from spontaneous vesicle fusion with the plasma membrane and dispersal of its fluorescent cargo. Hypo-osmotic stress stimulation (osmolality reduction from 316 to 160 mOsm) resulted in a transient, several-fold increment of exocytotic event frequency. Lowering the temperature from 37°C to 20°C dramatically diminished the fraction of vesicles that underwent exocytosis during the 2-min stimulation, from ~40% to ≤1%, respectively. Parallel ATP efflux experiments with luciferase bioluminescence assay revealed that pharmacological interference with vesicular transport (brefeldin, monensin), or disruption of the cytoskeleton (nocodazole, cytochalasin), significantly suppressed ATP release (by up to ~80%), whereas it was completely blocked by N-ethylmaleimide. Collectively, our data demonstrate that regulated exocytosis of ATP-loaded vesicles likely constitutes a major pathway of hypotonic stress-induced ATP secretion from A549 cells.     

Association of glycoproteins and pepsinogen in the secretory granules of fundic epithelial cells isolated from guinea pig stomach

Epithelial cells were isolated from the fundic portion of the guinea pig stomach. Cells were separated by velocity sedimentation at unit gravity in a Ficoll 70 gradient and pooled in three fractions. By morphological and biochemical criteria, each fraction was characterized as a population highly enriched in one of the three main functional types: oxyntic cell; chief cell and mucus-secreting cell. Measure of the pepsinogen content and specific stainings of the secretory granules for light and electron microscopy led to the definition of two types of mucus-secreting cells in nearly equal quantity; mucous cells with smaller secretory granules entirely glycoproteic in nature and muco-peptic cells containing larger heterogeneous secretory granules. These granules were made of a proteic core containing pepsinogen surrounded by a thin membrane and a voluminous cap, both containing carbohydrates. The cap appeared as if built of orderly packed layers of glycoproteins. Secretory granules of chief cells were also surrounded by a membrane containing glycoproteins and occasionally a small glycoproteic cap. Pepsinogen content was estimated to be three times higher in a single chief cell than in a muco-peptic cell.     

Control of secretory granule interaction and exocytosis in pancreatic cells

Application of the laser-based technique of photon correlation spectroscopy to anin vitro study of the ionic stability and interaction kinetics of zymogen granules isolated from rat exocrine pancreas is described here. In addition the separation from pancreatic acinar cell cytosol of a factor which stabilizes isolated zymogen granules and inhibits cation-induced granule aggregation is outlined. The basis of this action and the significance of the cytosolic inhibitory factor in the regulation of granule mobility and exocytosisin vivo is discussed.     

Spatial and structural interrelationships between secretory cells of the subcommissural organ and blood vessels

Summary In 76 specimens (amphibians, reptilians, mammals) belonging to 25 different vertebrate species, the region of the subcommissural organ (SCO) was investigated with the use of a primary antiserum raised against an extract of bovine Reissner's fiber+the immunoperoxidase procedure according to Sternberger et al. (1970).In the SCO of a toad (Bufo arenarum) and several species of reptiles (lacertilians, ophidians, crocodilians), the ependymal cells were the only type of secretory cell displaying vascular contacts, whereas in mammals ependymal and hypendymal cells established intimate spatial contacts with blood vessels. In Bufo arenarum, but especially in the reptilian species examined, the ependymo-vascular relationship was exerted by a population of ependymal cells having a rather constant location within the SCO and projecting to capillaries that showed a remarkably constant pattern of anatomical distribution. In the SCO of mammals the modality and degree of the structural relationships between secretory cells and blood vessels varied greatly from species to species. In the SCO of the armadillo and dog the secretory tissue was organized as a thick, highly vascularized layer with most of the cells oriented toward the capillaries. A rather opposite situation was found in the SCO of New-and Old-World monkeys, where vascular contacts were restricted to a few ependymal cells.Supported by Grant I/38259 from the Stiftung Volkswagenwerk, Federal Republic of Germany, and Grant RS-82-18 from the Dirección de Investigaciones, Universidal Austral de Chile     

Characteristics of rat normal mammary epithelial cells and dimethylbenzanthracene-induced mammary adenocarcinoma cells grown in monolayer culture

Summary The characteristics of normal mammary epithelial and 7,12-dimethylbenz[a]anthracene (DMBA)-induced adenocarcinoma cells derived from rats and grown in monolayer culture were compared. Normal mammary epithelial cells exhibited different morphology and agglutinability by plant lectins, slower growth rate, and lower saturation density and cloning efficiency. In addition, the normal cells were sensitive to the toxic effect of DMBA, and were unable to grow in soft agar or to form tumors, when inoculated into newborn Sparague-Dawley rats. The converse was true in each case for the adenocarcinoma cells. Supported by Public Health Service Research Grant CA 01237603 from the National Cancer Institute Portions of this paper were presented at the 65th Annual Meeting of the American Association for Cancer Research at Houston, Texas, 1974.     

Tridimensional analysis of the formation of secretory vesicles in the Golgi apparatus of absorptive columnar cells of the mouse colon

The tridimensional structure of the Golgi apparatus has been studied in the absorptive cells of the mouse colon by means of reduced osmium postfixation and phosphatase cytochemistry. In thick sections of tissue impregnated with osmium tetroxide or treated with a technique to demonstrate TPPase activity, the Golgi formed a continuous ribbon-like structure capping the upper pole of the nucleus. Along the longitudinal axis of this ribbon, compact zones made up of superposed flattened saccules alternated with less compact zones which consisted of highly perforated saccules or bridging anastomosed tubules. In the cis-trans axis, the following elements were observed: (1) a cis element consisting of a continuous osmiophilic tubular network; (2) two or three subjacent elements selectively perforated by wells; (3) a trans compartment made up of two or three TPPase-reactive sacculotubular elements, some showing a "peeling-off" configuration. In some regions, the first flattened saccule of this trans compartment displayed discrete ovoid dilatations, located in compact zones and containing a dense granulofibrillar material; in the subjacent elements this material was seen concentrated in nodular swellings, at the intersection of the meshes of anastomosed membranous tubules. 100-300 nm vesicles containing a similar dense granulofilamentous material were observed in the trans Golgi zone and interspersed in the supranuclear cytoplasm between the Golgi zone and the apical surface of the cell. Smaller vesicles 80-100 nm in diameter containing a fine dusty material were also seen in proximity. These morphological observations suggested that at least two kinds of material were segregated in the saccules of the trans compartment and packaged in vesicles of two class sizes that detached from the Golgi stack on its trans aspect.     

Distribution of chromaffin secretory vesicles, acetylcholinesterase, and lysosomal enzymes in sucrose and Percoll gradients

Crude chromaffin secretory vesicles, obtained by differential centrifugation, were further purified on isotonic (Percoll) gradients. The chromaffin vesicle fractions recovered from the gradients contain acetylcholinesterase as well as lysosomal enzymes. With the aid of a subsequent sucrose gradient lysosomal enzymes could be removed from chromaffin vesicle fractions, but not acetylcholinesterase. This suggests that lysosomal enzymes do not pass through the chromaffin vesicles during the biogenesis of lysosomes but acetylcholinesterase does.     

Collagen secretion screening in Drosophila supports a common secretory machinery and multiple Rab requirements

Collagens are large secreted trimeric proteins making up most of the animal extracellular matrix. Secretion of collagen has been a focus of interest for cell biologists in recent years because collagen trimers are too large and rigid to fit into the COPII vesicles mediating transport from the endoplasmic reticulum (ER) to the Golgi. Collagen-specific mechanisms to create enlarged ER-to-Golgi transport carriers have been postulated, including cargo loading by conserved ER exit site (ERES) protein Tango1. Here, we report an RNAi screening for genes involved in collagen secretion in Drosophila. In this screening, we examined distribution of GFP-tagged Collagen IV in live animals and found 88 gene hits for which the knockdown produced intracellular accumulation of Collagen IV in the fat body, the main source of matrix proteins in the larva. Among these hits, only two affected collagen secretion specifically: PH4αEFB and Plod, encoding enzymes known to mediate posttranslational modification of collagen in the ER. Every other intracellular accumulation hit affected general secretion, consistent with the notion that secretion of collagen does not use a specific mode of vesicular transport, but the general secretory pathway. Included in our hits are many known players in the eukaryotic secretory machinery, like COPII and COPI components, SNAREs and Rab-GTPase regulators. Our further analysis of the involvement of Rab-GTPases in secretion shows that Rab1, Rab2 and RabX3, are all required at ERES, each of them differentially affecting ERES morphology. Abolishing activity of all three by Rep knockdown, in contrast, led to uncoupling of ERES and Golgi. We additionally present a characterization of a screening hit we named trabuco (tbc), encoding an ERES-localized TBC domain-containing Rab-GAP. Finally, we discuss the success of our screening in identifying secretory pathway genes in comparison to two previous secretion screenings in Drosophila S2 cells.     

Proteins are secreted by both constitutive and regulated secretory pathways in lactating mouse mammary epithelial cells

Lactating mammary epithelial cells secrete high levels of caseins and other milk proteins. The extent to which protein secretion from these cells occurs in a regulated fashion was examined in experiments on secretory acini isolated from the mammary glands of lactating mice at 10 d postpartum. Protein synthesis and secretion were assayed by following the incorporation or release, respectively, of [35S]methionine-labeled TCA-precipitable protein. The isolated cells incorporated [35S]methionine into protein linearly for at least 5 h with no discernible lag period. In contrast, protein secretion was only detectable after a lag of approximately 1 h, consistent with exocytotic secretion of proteins immediately after passage through the secretory pathway and package into secretory vesicles. The extent of protein secretion was unaffected by the phorbol ester PMA, 8-bromo-cAMP, or 8-bromo-cGMP but was doubled by the Ca2+ ionophore ionomycin. In a pulse-label protocol in which proteins were prelabeled for 1 h before a chase period, constitutive secretion was unaffected by depletion of cytosolic Ca2+ but ionomycin was found to give a twofold stimulation of the secretion of presynthesized protein in a Ca(2+)-dependent manner. Ionomycin was still able to stimulate protein secretion after constitutive secretion had terminated. These results suggest that lactating mammary cells possess both a Ca(2+)-independent constitutive pathway and a Ca(2+)-activated regulatory pathway for protein secretion. The same proteins were secreted by both pathways. No ultrastructural evidence for apocrine secretion was seen in response to ionomycin and so it appears that regulated casein release involves exocytosis. Ionomycin was unlikely to be acting by disassembling the cortical actin network since cytochalasin D did not mimic its effects on secretion. The regulated pathway may be controlled by Ca2+ acting at a late step such as exocytotic membrane fusion.     

Implantation in vitro: co-culture of rat blastocyst and epithelial cell vesicles

Implantation of blastocysts involves conversion of maternal and embryonic cell surfaces from a nonadhesive to an adhesive state in response to the internally driven developmental program or to externally generated factors. However, the intricacies of the cellular and subcellular changes that promote the attachment are not known, because these changes are difficult to determine in situ because of the nonaccessibility of the site. To overcome this, an in vitro model of implantation was developed by co-culturing rat blastocysts and uterine epithelial cells of the same gestational age (day 5 postcoitum; plug day as day 1) in drops hanging from the lid of a Petri dish. The system was used to study the changes on the surface membranes of the cells of the trophectoderm and uterine epithelium and to evaluate the antiadhesive activity of the newly designed test substances. The isolated epithelial cell vesicles were co-cultured with zona-free blastocysts in the microdrops (40–50 µl) hanging from the lid of a 60-mm Petri dish. The lid was placed over the lower dish, which was presaturated with the medium. The culture was examined 48 h later to determine the site of adhesion of epithelial cell vesicles with the trophoblasts lining the blastocyst. The cell-cell adhesion was monitored on a computerized image analyzer. To validate the adhesion of blastocysts and epithelial cell vesicles in co-culture, the expression of a cell adhesion molecule, uvomorulin, was studied using immunocytochemical technique after incubating with antiuvomorulin antibody. Intense staining was noted on the membrane surfaces at the site of attachment of the blastocyst and cell vesicles.The authors express their sincere thanks to the Ministry of Health and Family Welfare, Government of India, for their financial support     

Difference in distribution of membrane proteins between low- and high-density secretory granules in parotid acinar cells

Secretory granules (SGs) are considered to be generated as immature granules and to mature by condensation of their contents. In this study, SGs of parotid gland were separated into low-, medium-, and high-density granule fractions by Percoll-density gradient centrifugation, since it was proposed that the density corresponds to the degree of maturation. The observation with electron microscopy showed that granules in the three fractions were very similar. The average diameter of high-density granules was a little but significantly larger than that of low-density granules. Although the three fractions contained amylase, suggesting that they are all SGs, distribution of membrane proteins was markedly different. Syntaxin6 and VAMP4 were localized in the low-density granule fraction, while VAMP2 was concentrated in the high-density granule fraction. Immunoprecipitation with anti-syntaxin6 antibody caused coprecipitation of VAMP2 from the medium-density granule fraction without solubilization, but not from Triton X-100-solubilized fraction, while VAMP4 was coprecipitated from both fractions. Therefore, VAMP2 is present on the same granules, but is separated from syntaxin6 and VAMP4, which are expected to be removed from immature granules. These results suggest that the medium-density granules are intermediates from low- to high-density granules, and that the membrane components of SGs dynamically change by budding and fusion during maturation.     

Improved preservation of the form and contents of wall vesicles and the golgi apparatus in freeze substituted hyphae ofSaprolegnia

Summary Secretory vesicles involved in cell wall synthesis (wall vesicles) and the Golgi apparatus have been compared in conventionally fixed and freeze substituted hyphae of the oomycete fungusSaprolegnia ferax. Wall vesicles freeze substituted in various fluids range from spherical to tubular and contain an intensely staining, phosphorous rich matrix. In contrast diverse conventional fixations cause artefactual constrictions in most tubular vesicles and loss of their intensely staining contents. These data are interpreted to show the existence of an intravesicular skeletal system, with cellular regulation, to determine vesicle morphology and intravesicular synthesis of a hypothetical phosphorylated glycolipid cell wall precursor. Whilst freeze substitution gives superior preservation of wall vesicle morphology, it does not demonstrate any preferential association between wall vesicles and microtubules thus suggesting that microtubules are only indirectly involved in wall vesicle transport. Freeze substitution is superior to conventional fixation for analysis of the Golgi apparatus because it uniquely reveals both differentiation of a specific single cisterna in each Golgi body and greater differences in membrane thicknesses throughout the endomembrane system.