Granule Matrices Research Articles

Imaging viscosity of intragranular mucin matrix in cystic fibrosis cells

Abnormalities of mucus viscosity play a critical role in the pathogenesis of several respiratory diseases, including cystic fibrosis. Currently, there are no approaches to assess the rheological properties of mucin granule matrices in live cells. This is the first example of the use of a molecular rotor, a BODIPY dye, to quantitatively visualize the viscosity of intragranular mucin matrices in a large population of individual granules in differentiated primary bronchial epithelial cells using fluorescence lifetime imaging microscopy.

Expression and subcellular localization of the Qa-SNARE syntaxin17 in human eosinophils

BackgroundSNARE members mediate membrane fusion during intracellular trafficking underlying innate and adaptive immune responses by different cells. However, little is known about the expression and function of these proteins in human eosinophils, cells involved in allergic, inflammatory and immunoregulatory responses. Here, we investigate the expression and distribution of the Qa-SNARE syntaxin17 (STX17) within human eosinophils isolated from the peripheral blood. MethodsFlow cytometry and a pre-embedding immunonanogold electron microscopy (EM) technique that combines optimal epitope preservation and secondary Fab-fragments of antibodies linked to 1.4nm gold particles for optimal access to microdomains, were used to investigate STX17. ResultsSTX17 was detected within unstimulated eosinophils. Immunogold EM revealed STX17 on secretory granules and on granule-derived vesiculotubular transport carriers (Eosinophil Sombrero Vesicles-EoSVs). Quantitative EM analyses showed that 77.7% of the granules were positive for STX17 with a mean±SEM of 3.9±0.2 gold particles/granule. Labeling was present on both granule outer membranes and matrices while EoSVs showed clear membrane-associated labeling. STX17 was also present in secretory granules in eosinophils stimulated with the cytokine tumor necrosis factor alpha (TNF-α) or the CC-chemokine ligand 11 CCL11 (eotaxin-1), stimuli that induce eosinophil degranulation. The number of secretory granules labeled for STX17 was significantly higher in CCL11 compared with the unstimulated group. The level of cell labeling did not change when unstimulated cells were compared with TNF-α-stimulated eosinophils. ConclusionsThe present study clearly shows by immunanonogold EM that STX17 is localized in eosinophil secretory granules and transport vesicles and might be involved in the transport of granule-derived cargos.

Effect of low temperatures on compound 48/80-induced intracellular Ca2+ changes and exocytosis of rat peritoneal mast cells.

It has been well documented that compound 48/80-induced exocytosis of mast cells is accompanied by changes in intracellular Ca2+ concentration ([Ca2+]i) showing a biphasic pattern: an initial phase which constitutes an abrupt increase, followed by a plateau phase. The former is caused by Ca2+ release from intracellular Ca2+ stores, and the latter is the result of secondary Ca2+ influx. Low temperatures lead to the inhibition of exocytosis, but the precise mechanism remains unclear. The present study aims to reveal whether [Ca2+]i changes are affected by the environmental temperature. To this end, we developed a novel imaging method to record [Ca2+]i changes and exocytotic processes simultaneously. Rat peritoneal mast cells were loaded by Indo-1/AM or Fluo-3/AM for measuring [Ca2+]i, and the exocytosed granule matrices were stained by sulforhodamine-B. Cells were stimulated by compound 48/80, and [Ca2+]i changes and exocytosis were recorded by means of a real-time confocal microscope. At 37 degrees C, [Ca2+]i changes in stimulated mast cells showed a sustained plateau phase. Granule discharge was observed at the cell surface, and, in addition, most of the intracellular granule matrices were involved in compound exocytosis. The granule discharge and compound exocytosis proceeded over a period of a few minutes. At 4 degrees C, the plateau phase of [Ca2+]i changes declined rapidly, although the initial phase was not suppressed. Granule discharge occurred at the cell surface, but compound exocytosis ceased within a few minutes. These findings indicate that a low temperature inhibits compound exocytosis which can be caused by Ca2+ influx. The present imaging method represents a powerful tool for investigating the stimulus-secretion coupling of mast cells.

Ca increase in secretory granules of stimulated mast cells

The elemental content of rat peritoneal mast-cell secretory granules has been measured by X-ray microanalysis. Two distinct categories of granules were analyzed: intact granules, seen in control samples, and spumous granules, corresponding to exocytosed granule matrices. The average Ca content of intact granules was found to be approximately equal to cytosolic concentration, and to increase up to 40-fold in spumous granules. A significant increase was also observed for Na and Cl. These changes were not observed (for Ca) or weaker (for Na and Cl) if the cells had been challenged in the absence of nominal extracellular Ca; in this case, there was also a significant decrease in the sulphur content, suggesting a partial dispersion of the organic matrix components.In exocytosed granule matrices, in the presence but not in the absence of extracellular Ca, a slow and long-lasting increase of intragranular free Ca was monitored by changes in the fluorescence of the Ca-sensitive probes Fluo-3 and Calcium Green-5N, accumulated within rat mast-cell secretory granules. These findings are discussed along two lines:• It is proposed that the calcium uptake by the exocytosed mast-cell granule matrices can have a physiological relevance for the surrounding tissue.• Mast-cell granules do not disperse after exocytosis. The major uptake of Ca which is seen after opening of the exocytotic pore could be responsible for the exceptional stability of the externalized matrices.

Ribonuclease-gold ultrastructural localization of heparin in isolated human lung mast cells stimulated to undergo anaphylactic degranulation and recovery in vitro.

Human mast cells are a rich and unique source of heparin, which is stored in cytoplasmic secretory granules and accounts for metachromasia, a staining property used to identify mast cells by light microscopy. The sub-cellular locations of heparin in secretory human mast cells and human mast cells recovering from secretion are not known. Acquisition of this knowledge requires ultrastructural imaging of well-preserved cells with a visible probe which binds to heparin. We sought to develop this knowledge regarding human mast cell secretion by using a labelling method for heparin that depends on the well-known property of ribonuclease inhibition by heparin. Human lung mast cells were isolated, partially purified, either stimulated or not stimulated to secrete with anti-IgE, and recovered 20 min or 6 h later for routine electron microscopy. Histamine secretion was also determined. A previously developed post-embedding, enzyme-affinity-gold electron microscopic technique to image ribonucleic acid (RNA) with ribonuclease-gold (R-G), which also binds to the enzyme inhibitor, heparin, was employed to determine the sub-cellular locations of heparin in non-secretory and secretory mast cells as well as in mast cells recovered from short-term cultures after secretion. Specificity controls for the novel use of this method and quantification of granule labelling in these controls were performed. Heparin was labelled by R-G in electron-dense granules within non-secretory human lung mast cells (HLMCs), in electron-dense granules that persisted in secretory HLMCs at the maximum histamine secretion time (20 min), and in electron-dense granules within recovering HLMCs. Specificity controls showed that gold alone did not label HLMCs and that absorption with heparin significantly reduced or abrogated HLMC granule staining with R-G, but that RNA absorption did not. Heparin stores were absent in newly formed, electron-lucent intracytoplasmic degranulation channels in secretory HLMCs. Electron-dense granule matrices in the process of extrusion to the cell exterior still retained heparin at the instant of cellular secretion. Non-granule heparin stores bound R-G in recovering HLMCs. These locations included resolving degranulation channels, as newly emergent granules partitioned and condensed within them, and electron-dense content-containing vesicles and progranules within synthetic mast cells. Ultimately, all known ultrastructural patterns of HLMC granules developed in recovering cells, and each of them contained heparin. Heparin was secreted from HLMCs which were stimulated by anti-IgE, and heparin was recovered by a combination of conservative and synthetic mechanisms in HLMCs after a secretory event.

Neurotransmitter release through the exocytotic fusion pore

The exocytotic fusion pore transiently connects the secretory granule and plasma membrane during secretory vesicle fusion. Recent evidence on the properties of the fusion pore measured electrophysiologically suggest that the fusion pore is a lipid structure formed by a tension-driven fusion mechanism. We propose that a macromolecular scaffold of proteins directs and regulates the fusion process by drawing a characteristic plasma membrane dimple into close proximity with the secretory granule. Although the identity of the proteins making up the scaffold are largely unknown, studies on the regulation of exocytosis implicate the GTP binding protein Rab3 and unknown Ca2+ binding proteins as part of the scaffold, which appears to behave as a coincidence detector. Simultaneous measurement of fusion and the release of secretory products, along with some unusual properties of secretory granule matrices, have led to the unexpected conclusion that the release of secretory products from the matrix may also be regulated.

Lectinocytochemical specificity in human eosinophils and neutrophils: a reexamination.

As with secretory granules in other cell types, many of the protein components that make up the cytoplasmic granules of human leukocytes are glycoproteins. It is not unexpected, therefore, that lectins specific for various carbohydrate moieties can be localized in these granules following appropriate protocols for specimen preparation and thin-section labeling. In this study, isolated human eosinophils and neutrophils were prepared for lectin-gold electron microscopic localization by procedures that involve no exposure to aqueous fixatives, buffers, or solvents (rapid cryofixation and molecular distillation drying), thus removing the potential problem of constituent extraction or translocation during so-called "wet chemical" processing. In contrast to other reports, data presented illustrate the specific binding of soybean agglutinin (SBA) to eosinophil granule matrices (not the crystalline cores), as well as to a population of granules in neutrophils. A similar labeling pattern for wheat germ agglutinin (WGA) was also seen, confirming the presence of N-acetyl-D-galactosamine and N-acetyl-D-glucosamine residues in eosinophil and neutrophil granule matrices. These studies emphasize the need for carefully designed specimen preparation as well as subsequent thin-section labeling procedures in lectinocytochemical studies.

Extracellular Localization of Human Connective Tissue Mast Cell Granule Contents

In early phases of cutaneous inflammation, connective tissue mast cell degranulation is associated with apparent secretion and externalization of immunoreactive chymotryptic serine proteinase. To determine whether this event is associated with structural evidence of granule externalization, we studied the sequential evolution of IgE-mediated hypersensitivity in vivo, as well as mast cell degranulation provoked by a variety of stimuli in cultured explants of human skin. By 1 min after intradermal antigen challenge with ragweed extract, mast cell degranulation was associated with apparent extrusion of intragranule constituents into the pericellular connective tissue. Similar features typified cultured skin explants exposed for 45 min to anti-IgE and other mast cell secretagogues (morphine sulfate, calcium ionophore A23187, compound 48/80, and substance P). Once externalized, granule constituents could be identified within the dermal matrix by their rounded contour and structural similarity to solubilized granule matrices remaining within actively secreting cells. These data indicate that externalization of connective tissue mast cell granule contents occurs early after secretagogue exposure, potentially accounting for infrequent documentation of this event in naturally occurring dermatoses. The ability to recognize externalized granule products at a morphologic level should facilitate the understanding of interactions between mast cell-derived mediators and target structures of the dermal microvasculature.

Ultrastructural localization of lysozyme in human colon eosinophils using the protein A-gold technique: effects of processing on probe distribution.

The distribution of lysozyme (muramidase) within eosinophil leukocytes situated in the lamina propria of human colon was studied by immunoelectron microscopy using a range of standard techniques. Tissue processed in a variety of glutaraldehyde- or paraformaldehyde-based fixatives was partially dehydrated and embedded in the acrylic resin LR White. Tissue thus treated showed lysozyme in pale cytoplasmic granules and the matrix of specific granules, but not in their crystalloids. Trypsinization of sections had little effect on this result, and tissues fixed in glutaraldehyde and embedded in Araldite showed a low level of reactivity with a similar distribution. After etching LR or Araldite sections with sodium metaperiodate, the pale granules and specific granule matrices became negative for lysozyme and the crystalloids became positive. Because crystalloids also were labeled with normal rabbit immunoglobulin after etching, this apparent redistribution of label could be due to nonspecific binding rather than exposure of masked epitope.

Proton nuclear magnetic resonance studies of mast cell histamine.

The state of histamine in mast cells was studied by 1H NMR spectroscopy. Spectra were measured for histamine in situ in intact mast cells, for histamine in suspensions of mast cell granule matrices that had been stripped of their membranes, and for histamine in solutions of heparin. The 1H NMR spectrum of intact mast cells is relatively simple, consisting predominantly of resonances for intracellular histamine superimposed on a weaker background of resonances from heparin and proteins of the cells. All of the intracellular histamine contributes to the NMR signals, indicating it must be relatively mobile and not rigidly associated with the negatively charged granule matrix. Spectra for intracellular histamine and for histamine in granule matrices are similar, indicating the latter to be a reasonable model for the in situ situation. The dynamics of binding of histamine by granule matrices and by heparin are considerably different; exchange of histamine between the bulk water and the granule matrices is slow on the 1H NMR time scale, whereas exchange between the free and bound forms in heparin solution is fast. The chemical shifts of resonances for histamine in mast cells are pH dependent, decreasing as the intragranule pH increases without splitting or broadening. The results are interpreted to indicate that histamine in mast cells is relatively labile, with rapid exchange between bound histamine and pools of free histamine in water compartments confined in the granule matrix.

Sorting of three secretory proteins to distinct secretory granules in acidophilic cells of cow anterior pituitary.

The distribution of three proteins discharged by regulated exocytosis--growth hormone (GH), prolactin (PRL), and secretogranin II (SgII)--was investigated by double immunolabeling of ultrathin frozen sections in the acidophilic cells of the bovine pituitary. In mammotrophs, heavy PRL labeling was observed over secretory granule matrices (including the immature matrices at the trans Golgi surface) and also over Golgi cisternae. In contrast, in somatotrophs heavy GH labeling was restricted to the granule matrices; vesicles and tubules at the trans Golgi region showed some and the Golgi cisternae only sparse labeling. All somatotrophs and mammotrophs were heavily positive for GH and PRL, respectively, and were found to contain small amounts of the other hormone as well, which, however, was almost completely absent from granules, and was more concentrated in the Golgi complex, admixed with the predominant hormone. Mixed somatomammotrophs (approximately 26% of the acidophilic cells) were heavily positive for both GH and PRL. Although admixed within Golgi cisternae, the two hormones were stored separately within distinct granule types. A third type of granule was found to contain SgII. Spillage of small amounts of each of the three secretory proteins into granules containing predominantly another protein was common, but true intermixing (i.e., coexistence within single granules of comparable amounts of two proteins) was very rare. It is concluded that in the regulated pathway of acidophilic pituitary, cell mechanisms exist that cause sorting of the three secretory proteins investigated. Such mechanisms operate beyond the Golgi cisternae, possibly at the sites where condensation of secretion products into granule matrices takes place.

Regeneration of rat mast cells after histamine secretion: changes in histidine decarboxylase activity and heparin synthesis.

Histidine decarboxylase activity in rat peritoneal mast cells is depressed temporarily after histamine secretion induced by antigen in sensitized cells or by compound 48/80. Recovery starts after 10 min. and the enzyme activity is completely restored in about 1-2 hours. There is no stimulation of the enzyme activity upto 4 hours after the exposure to the releaser. The enzyme activity shows more prolonged depression after histamine release induced by inonophore A23187 probably because of leakage of the cytoplasmic enzyme. Inspite of the loss of some granules from the mast cells, histamine uptake is enhanced during the first two hours apparently due to the uptake into the granule matrices in the compound exocytotic vacuoles. These granules remain within the cell, but the release of endogenous histamine to the extracellular fluid during secretion makes the binding sites available for histamine uptake. There is no indication of increased histamine production during the period of observation, viz. up to 6 hours after the secretion. The heparin content is reduced during the first two hours due to the loss of granules from the cells, but this is restored during 4-6 hours by increasing synthetic activity. Heparin synthesis is reduced to about 40% of the normal value during the first 4 hours, and is practically restored to normal during 4-6 hours.

Ultrastructural characterization of human corticotrophs (ACTH cells).

Ultrastructural characterization of human corticotrophs (ACTH cells) was performed by 'superimposition technique', which enabled detailed ultrastructural observation of immunoreactive ACTH cells in adjacent semi-thin light microscopic immunoperoxidase and routine electron microscopic sections. The human corticotrophs were large and round or polygonal and were not stellate. They had scanty rough endoplasmic membranes and were packed with numerous large secretory granules measuring from 250 to 500 nm in diameter. The sizes of secretory granules in 6 human pituitaries were 448 +/- 128, 344 +/- 86, 448 +/- 117, 244 +/- 65, 316 +/- 76, and 340 +/- 93 nm, respectively. The granules were not seen in a single row along the plasma membrane as is the case in the rat. They possessed somewhat irregular outlines with a rarely discernible halo. Different densities of granule matrices were occasionally found. The cells often contained a few large heterogeneous vacuoles. From these findings, the human ACTH cells were recognized to be remarkably different in cell shape and size, properties of secretory granules and cytoplamic inclusions from those of the rat pituitary gland. In respect to secretory granule properties, the human ACTH cells are similar to those of some other mammals (fox, young pig, and lerot). More data is required to elucidate the relationship between human ACTH cell morphology and functional state.

Fine structural localization of arylsulfatase B activity in the rabbit blood platelets.

Fine structural localization of arylsulfatase in the rabbit blood platelets has been investigated in this study. Among many cell organellae, reaction products were exclusively observed in the alpha granules of the platelets. Within the alpha granules, arylsulfatase activity appeared to localize in variable patterns, i.e. reaction products confined mainly at the peripheral region in many granules, while they deposited heavily throughout the granule matrices in some others. In a blood platelets, each alpha granule showed the different staining pattern which indicated more variable functional heterogeneity in the granules.

Structural features of histamine release in rat peritoneal mast cells. A study with toluidine blue.

Cellular events correlated to the histamine release process were studied in rat peritoneal mast cells applying the dye toluidine blue as histamine-discharging agent. The effects of toluidine blue were studied both in the presence and absence of calcium. Histamine release was clearly dose-dependent within the range 2X10<sup>––</sup><sup>5</sup> m toluidine blue to 4X10<sup>––4</sup> m. Whereas spontaneous histamine release values were in the order of a few percent, maximum release in the presence of calcium amounted to 65–80%. Histamine release was accompanied by characteristic morphologic changes in the cells: granule fine structure was markedly altered, perigranular vacuoles were formed and, through repeated fusions of the vacuole-delimiting membranes, large caveolae containing granule matrices arose. In fortuitous sections, these cavities were seen to communicate with the extracellular space through pores, often of substantial size. Granule extrusion from the cellular domain to the surrounding medium, although it did occur to a certain extent, was not a prominent feature of the histamine release process. The dose-response curve for histamine release was lowered by roughly one half in the absence of calcium. This was reflected in mast cell morphology by a decreased tendency towards granule alteration and vacuole formation. The present observations are viewed in the light of various suggested models for the mode of histamine release from mast cells.