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1.
Summary The presence of excess membrane has been observed in the secretory granules of mast cells activated via the physiological mechanism of IgE receptor-mediated exocytosis. This excess membrane is the result of ade novo assembly from phospholipid, cholesterol, and other membrane components stored in the quiescent granule. Following receptor stimulation, membrane bilayer structures of varying size and shape can be seen in the subperigranular membrane space where the perigranular membrane has lifted away from the granule matrix. Vesicles as small as 25 nm in outer diameter are frequently found beneath the perigranular membrane at the site of granule fusion. Membrane in the form of elongated vesicles, tubes, or sheets has also been observed. The wide variation in size and shape of the newly assembled membrane may reflect the spontaneity of the entropy-driven membrane generation process and the fluid characteristic of the biological membrane in general. Fusion of the newly assembled membrane with the perigranular membrane enables the activated granule to enlarge. This rapid expansion process of the perigranular membrane may be the principal mechanism by which an activated granule can achieve contact with the plasma membrane in order to generate pore formation. The fact that new membrane assembly also occurs in the IgE receptor-mediated granule exocytosis, supports our observation thatde novo membrane generation is an inherent step in the mechanism of mast cell granule exocytosis. Whether new membrane assembly is a common step in the mechanism of secretory granule exocytosis in general, must await careful reinvestigation of other secretory systems.  相似文献   

2.
By combining ultrastructural techniques with a biochemical approach to study the mechanism of mast cell stimulus-secretion coupling and by using purified secretory granules to confirm those early biochemical events which originate from within the secretory granule, a new model for the mechanism of secretory granule exocytosis has emerged. This model not only provides the mechanism by which an activated granule can achieve fusion with the plasma membrane, but it also provides the rationale for the linking of the various early biochemical events to the process of granule activation and thus to exocytosis. Although we still do not understand how the 'activating signal', which results from the stimulation of cell surface receptors, can be conveyed to the granule to cause its activation, we are certain that this 'signal' must cause an influx of water into the matrix of the target granule. This influx of water is what initiates the granule activation process. The major intragranular events which are triggered by this water influx include: (i) de novo membrane assembly; (ii) protein proteolysis; (iii) release of arachidonic acid from matrix-bound phospholipid by phospholipase A2; (iv) initiation of the arachidonic acid cascade and the synthesis of eicosanoids; (v) rapid phospholipid turnover; and (vi) the discharge of matrix materials into the cytoplasm of the activated cell via the fusion of de novo generated vesicles with the perigranular membrane. The ejection of some matrix contents which may include histamine, Ca2+, calmodulin, protease, the products of the arachidonic acid cascade and the products of phospholipid turnover into the cytosole, may serve to turn on the various metabolic machineries needed to initiate a cellular recovery phase.  相似文献   

3.
MATURATION OF RAT MAST CELLS : An Electron Microscope Study   总被引:9,自引:8,他引:1       下载免费PDF全文
Electron microscope study of rat mast cell maturation corroborates certain interpretations of features of mast cell differentiation based on light microscope studies. In addition, the ultrastructural variation observed in the granules of differentiating mast cells suggests that granule formation begins with the elaboration of dense granules about 70 mµ in diameter inside Golgi vacuoles. These progranules appear to aggregate inside a membrane and fuse to form dense cords 70 to 100 mµ in diameter. These dense cords are embedded in a finely granular material possibly added to the developing granule by direct continuity between perigranular membranes and cisternae of rough endoplasmic reticulum. The dense cords and finely granular material then appear to be replaced by a mass of strands about 30 mµ in diameter, thought to be a reorganization product of the two formerly separate components. A process interpreted as compaction of the strands completes the formation of the dense, homogeneous granules observed in mature rat mast cells. The similarity between mast cell granule formation and the elaboration of other granules is considered, with special reference to rabbit polymorphonuclear leukocyte azurophil granules. The relationships between the ultrastructural, histochemical, and radioautographic characteristics of mast cell granule formation are considered, and the significance of the perigranular membrane is discussed.  相似文献   

4.
Secretory granules have been observed to swell during the process of exocytosis. Swelling is an indication of osmotic stress. The probable role of osmotic pressure in facilitating membrane fusion makes it necessary to determine whether granule membrane 'swelling' can occur prior to its fusion with the plasma membrane (pore formation) in the process of exocytosis. By subjecting adjacent thin and semi-thin sections of an activated granule to ultrastructural examination for membrane enlargement, and to metachromatic staining for verification of pore formation it is concluded that the perigranular membrane can indeed enlarge prior to pore formation. However, the degree of membrane enlargement can far exceed the limit of 2-3% stretching allowed under normal osmotic stress for a membrane bilayer. Such an extensive membrane enlargement, which takes place in the mechanism of exocytosis, cannot be achieved without being accompanied by the insertion of additional membrane.  相似文献   

5.
The mechanism of secretory granule formation and exocytosis in the endocrine cells of normal and transplanted rat pancreas was studied using electron microscopy. On the one hand, formation of secretory granules starts with the dilatation of the 2 ends or the vesicularization of the middle parts of rough endoplasmatic reticulum (RER). On the other hand, prohormone ribosomes condense into the vesicles of the GOLGI apparatus. This probably indicates that the GOLGI complex is not the only source of formation of secretory granules. Exocytosis occurs with the formation of an electron dense streak between the perigranular membrane and the apical cell membrane. This is followed by the rupture of the streak at this midpoint allowing the granule to extrude into the space between the cell membrane and the parenchymal basal membrane. This fusion-rupture-extrusion mechanism repeats itself at the parenchymal and capillary basal membranes and also at the endothelium until it gets into the capillary lumen, showing that hormones of pancreatic endocrine cells may be actively transported into circulation as intact secretory granules. There is no significant morphological difference between the mechanism of secretory granule formation in normal and transplanted pancreatic tissue.  相似文献   

6.
Large amounts of membranes enriched either in perigranular membranes or in plasma membranes have been successfully isolated from rat peritoneal mast cells. A cycle consisting of a single sonication pulse to disrupt the mast cells followed by centrifugation to separate the released granules was repeated until 90% of the mast cells were disrupted. This technique resulted in a high yield of intact granules since the released granules were only exposed to the single sonication pulse. The intact granules were separated from plasma membrane fragments by centrifugation through a Percoll gradient. The perigranular membranes were then obtained by osmotic lysis of the purified intact granules. The plasma membrane fraction was enriched 4.5-fold (range, 4.1-6.1) in 5'-nucleotidase activity, a plasma membrane marker enzyme. No suitable marker enzyme activity was found for the perigranular membrane fraction. An important aspect of this procedure is its potential for obtaining both a plasma and perigranular membrane preparation in high yield and purity from the same mast cell preparation.  相似文献   

7.
In vitro degranulation of rat mast cells was studied at different intervals ranging from 10 to 60 sec after adding the histamine liberator, compound 48/80 (0.4 µg/ml, 17°C). The ultrastructural changes were followed by electron microscopy, and parallel assays were made to determine the histamine released. In addition, the extracellular tracers lanthanum and hemoglobin (demonstrated by its peroxidative activity) were applied to mast cells to follow communication of the extracellular space with the cavities formed during degranulation. After a lag period of 10 sec, degranulation started in the most peripherally located granules. The perigranular membrane fused with the plasma membrane, resulting in a pore bridged by a thin diaphragm. This was followed by rupture of the diaphragm and extrusion of the granule matrix (exocytosis). The process advanced towards the cell interior by fusion and opening of the deeper situated granules to the formerly opened granule cavities. At the end of the process, the cell was filled by a system of complicated cavities containing a number of altered granules. Extracellular tracers have shown that these intracellular cavities were in unbroken communication with the extracellular space from the very beginning of their formation. Both lanthanum and hemoglobin were found to be adsorbed to the limiting membrane of the cavities and bound to altered mast cell granules. In contrast, no tracer substance was present in nondegranulating mast cells. Degranulation of mast cells by compound 48/80 is regarded as a sequential exocytosis, a process similar to that described for some exocrine gland cells. All the "intracellular" cavities, formed by degranulation, were shown to communicate with the extracellular space; consequently, granules lying in these cavities must be considered as biologically extracellular. The present findings support the view that histamine is released from the granule matrix by the extracellular ionic milieu.  相似文献   

8.
1. The accessibility of phospholipids in the membrane of the adrenomedullary storage vesicles (chromaffin granules) has been studied. 2. The reaction of 2,4,6-trinitrobenzenesulphonic acid with both intact granules and their ghosts, results in the labelling of 70% of the phosphatidylethanolamine. 3. The action of phospholipase A2 (from bee venom), phospholipase C (from Bacillus cereus) and sphingomyelinase C (from Staphylococcus aureus) on granules and their ghosts was followed as a function of time. No significant difference was observed between the intact granules and their ghosts. 4. In the intact granules the various treatments led to varying amounts of lysis although again no evidence was obtained that such lysis in any way increased the amount of accessible phospholipid. 5. Highly purified granule preparations were also compared with the so-called "large granule" fraction and no significant differences were detected. 6. Approx. 67% of phosphatidylethanolamine + phosphatidic acid, 50% of phosphatidylserine + phosphatidylinositol, 65% of phosphatidylcholine and 20% of sphingomyelin is accessible to enzymatic degradation. In total, approx. 50% of all the phospholipids reacted. 7. It is also shown that, unlike in enzymatic treatment, all the phosphatidylcholine can be exchanged in the presence of a phospholipid exchange protein (prepared from beef liver). 8. It is concluded that transmembrane movement of phosphatidylcholine is slow in isolated membranes of chromaffin granules. The presence of the exchange protein, however, in conjunction with membrane proteins and specific phospholipid arrangements may catalyse this transmembrane movement.  相似文献   

9.
Beta-adrenergic receptors were characterized by measuring the specific binding of 3H-dihydroalprenolol (DHA) on intact isolated rat peritoneal mast cells (RPMC) and on perigranular membranes derived from purified RPMC granules. The specific binding of 3H-DHA reaches an equilibrium within 30 min at 5 degrees C and is linear with cell number. Scatchard analysis reveals two populations of binding sites on intact cells: with KD = 10.6 +/- 2.6 and 129 +/- 4.7 nM and Bmax of 186 +/- 38 and 1200 +/- 415 fmol/10(6) cells, respectively. Each cell contains 120 X 10(3) high-affinity binding sites and 720 X 10(3) low-affinity binding sites. There appears to be neither alpha-adrenergic nor muscarinic cholinergic receptors on the RPMC. Specific binding of 3H-DHA also occurred to isolated granules with perigranular membranes. The binding was saturable with a single population of binding sites with an affinity (KD) of 7.0 +/- 0.45 nM. Maximum binding (Bmax) was calculated at 56.6 +/- 1.9 fmol/10(9) granules. Subfractionation of granule components demonstrated that the specific binding sites appear to be localized exclusively on the perigranular membrane.  相似文献   

10.
Adenosine triphosphate (ATP) induces the release of catecholamines, endogenous ATP, and soluble protein from chromaffin granules isolated from the adrenal medulla. When ATP exerts this action, it is hydrolyzed by enzymes present in the granule membrane, and part of the Pi liberated from ATP is transferred to the protein and lipid of the granule membrane. The phosphorylated lipid component, which was identified by thin-layer and ion-exchange chromatography as diphosphatidylinositol, was formed from ATP and monophosphatidylinositol. This latter phospholipid was the substrate for the enzyme phosphatidylinositol kinase. Both substrate and enzyme are components of the granule membranes, because they have a similar subcellular distribution as dopamine beta-hydroxylase (a granule membrane marker). The formation of diphosphatidylinositol was Mg(2 plus)-dependent, it was further stimulated by Mn(2 plus), it was inhibited by N-ethylmaleimide and the reaction had an optimal pH of 5. The synthesis of diphosphatidylinositol was also shown to occur in chromaffin granules "in situ". during the stimulation of the adrenal medulla by acetylcholine.  相似文献   

11.
Summary Using an in situ tannic acid perfusion technique, this study presents evidence that the removal of membrane components from the rat atrial secretory granule membrane after granule exocytosis is mediated by coated vesicles. When tannic acid is used to arrest the post-fusion stages of granule release, coated pit formation occurs on granule membrane, which, although continuous with the sarcolemma, is easily recognised by the membrane omega profile and the continued presence of the granule core. Tannic acid perfusion, before aldehyde fixation, allows a degree of continued cell function, and granule fusions can persist after tannic acid has reached the cell. This results in an increase in the numbers of fusion profiles and the appearance of coated pits on granule membrane at these sites. The proportion of granules with coats increases with perfusion time, suggesting that endocytotic, as well exocytotic events, may be arrested by the action of tannic acid. Coated vesicles are also involved at earlier stages of the release pathway. In other types of secretory system this is considered to represent recycling of membrane proteins as part of the maturation process of the granule. Although arrested granules exhibiting this clathrin coat could have had the coat prior to fusion, as part of the maturation process, our results show that it is more likely to represent a second stage of membrane protein recycling; the postfusion reclamation of proteins from the sarcolemma. This facet of the tannic acid perfusion procedure suggests a general method for quantifying coated pit formation during secretory granule release.  相似文献   

12.
The formation of dense core secretory granules is a multistage process beginning in the trans Golgi network and continuing during a period of granule maturation. Direct interactions between proteins in the membrane and those in the forming dense core may be important for sorting during this process, as well as for organizing membrane proteins in mature granules. We have isolated two mutants in dense core granule formation in the ciliate Tetrahymena thermophila, an organism in which this pathway is genetically accessible. The mutants lie in two distinct genes but have similar phenotypes, marked by accumulation of a set of granule cargo markers in intracellular vesicles resembling immature secretory granules. Sorting to these vesicles appears specific, since they do not contain detectable levels of an extraneous secretory marker. The mutants were initially identified on the basis of aberrant proprotein processing, but also showed defects in the docking of the immature granules. These defects, in core assembly and docking, were similarly conditional with respect to growth conditions, and therefore are likely to be tightly linked. In starved cells, the processing defect was less severe, and the immature granules could dock but still did not undergo stimulated exocytosis. We identified a lumenal protein that localizes to the docking-competent end of wildtype granules, but which is delocalized in the mutants. Our results suggest that dense cores have functionally distinct domains that may be important for organizing membrane proteins involved in docking and fusion.  相似文献   

13.
Local actin assembly is associated with sites of exocytosis in processes ranging from phagocytosis to compensatory endocytosis. Here, we examine whether the trigger for actin-coat assembly around exocytosing Xenopus egg cortical granules is 'compartment mixing'--the union of the contents of the plasma membrane with that of the secretory granule membrane. Consistent with this model, compartment mixing occurs on cortical granule-plasma membrane fusion and is required for actin assembly. Compartment mixing triggers actin assembly, at least in part, through diacylglycerol (DAG), which incorporates into the cortical granule membranes from the plasma membrane after cortical granule-plasma membrane fusion. DAG, in turn, directs long-term recruitment of protein kinase Cbeta (PKCbeta) to exocytosing cortical granules, where it is required for activation of Cdc42 localized on the cortical granules. The results demonstrate that mixing of two membrane compartments can direct local actin assembly and indicate that this process is harnessed during Xenopus egg cortical granule exocytosis to drive compensatory endocytosis.  相似文献   

14.
Summary We have examined the cortex of the teleost (Brachydanio rerio) egg before and during exocytosis of cortical granules by scanning, transmission, and freeze-fracture electron microscopy. In the unactivated egg, the P-face of the plasma membrane exhibits a random distribution of intramembranous particles, showing a density of 959/m2 and an average diameter of 8 nm. Particles over P- and E-faces of the membranes of cortical granules are substantially larger and display a significantly lower density. An anastomosing cortical endoplasmic reticulum forms close associations with both the plasma membrane of the egg and the membranes of cortical granules. Exocytosis begins with cortical granules pushing up beneath the plasma membrane to form domeshaped swellings, coupled with an apparent clearing of particles from the site of contact between the apposed membranes. A depression in the particle-free plasma membrane appears to mark sites of fusion and pore formation between cortical granules and plasma membranes. Profiles of exocytotic vesicles undergo a predictable sequence of morphological change, but maintain their identity in the egg surface during this transformation. Coated vesicles form at sites of cortical granule breakdown. Differences in particle density between cortical granules and egg plasma membranes persist during transformation of the exocytotic profiles. This suggests that constituents of the 2 membrane domains remain segregated and do not intermix rapidly, lending support to the view that the process of membrane retrieval is selective (i.e., cortical granule membrane is removed).  相似文献   

15.
Activation of the teleost (Brachydanio) fish egg includes the exocytosis of cortical granules, the construction of a mosaic surface consisting of the unfertilized egg plasma membrane and the limiting membranes of the cortical granules, and the appearance of coated and smooth vesicles in the cytoplasm (Donovan and Hart, '82). Unfertilized and activated eggs were incubated in selected extracellular tracers to (1) determine experimentally if cortical granule exocytosis was coupled with the endocytosis of membrane during the cortical reaction, and (2) establish the intracellular pathway(s) by which internalized vesicles were processed. Unfertilized eggs incubated in dechlorinated tap water or Fish Ringer's solution containing either horseradish peroxidase (HRP; 10 mg/ml), native ferritin (12.5 mg/ml), or cationized ferritin (12.5 mg/ml) were activated as judged by cortical granule breakdown and elevation of the chorion. Cells treated with HRP and native ferritin exhibited a delay in cortical granule exocytosis when compared with water-activated eggs lacking the tracer. Each tracer was internalized through the formation of a coated vesicle from a coated pit. Since coated pits appeared to be topographically restricted to the perigranular membrane domain of the mosaic egg surface, their labeling, particularly with cationized ferritin, strongly suggested that the retrieved membrane was of cortical granule origin. Cationized ferritin and concanavalin A (Con A) coupled with either hemocyanin or ferritin labeled the surface of the unactivated egg and both domains of the mosaic egg surface. Transformation of the deep evacuated cortical granule crypt into later profiles of exocytosis was accompanied by increased Con A binding. Within activated egg cortices, HRP reaction product, native ferritin, and cationized ferritin were routinely localized in smooth vesicles, multivesicular bodies, and autophagic vacuoles. Occasionally, each tracer was found in small coated vesicles adjacent to the Golgi and within Golgi cisternae. The intracellular distribution of HRP, native ferritin, and cationized ferritin suggests that internalized membrane is primarily processed by organelles of the lysosomal compartment. A second and less significant pathway is the Golgi complex.  相似文献   

16.
PADGEM (platelet activation dependent granule-external membrane protein) is an integral membrane protein of the alpha granules of platelets and Weibel-Palade bodies of endothelial cells that is expressed on the plasma membrane upon cell activation and granule secretion. Activated platelets, but not resting platelets, bind to neutrophils, monocytes, HL60 cells, and U937 cells. This interaction is inhibited by anti-PADGEM antibodies, PADGEM, and EDTA; anti-GPIIb-IIIa, anti-thrombospondin, anti-GPIV, and thrombospondin produce no effect. Neutrophils and U937 cells, in contrast to Jurkatt cells, contain PADGEM recognition sites, as shown by binding of PADGEM contained in phospholipid vesicles. These results indicate that PADGEM mediates adhesion of activated platelets to monocytes and neutrophils. Therefore, PADGEM shares not only structural but also functional homology with ELAM-1 and MEL-14, members of a new family of vascular cell adhesion molecules.  相似文献   

17.
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.  相似文献   

18.
The delivery of newly-formed secretory content to the granule inventory occurs through direct fusion of recently formed granules and mature granules. The introduction of knockout mice allowed us to investigate the characteristics of the delivery process and to determine the core protein machinery required for granule growth. The SNARE machinery mediates membrane fusion and is essential for the granule lifecycle. In the current work, we use VAMP8 knockout mice to show that the SNARE machinery plays a critical role in the process of granule homotypic fusion. Consistent with this, the mutated mouse pancreatic acinar secretory granules are significantly smaller compared to the control group, demonstrating few granule profiles that might be the result of homotypic fusion.  相似文献   

19.
Total internal reflection fluorescence microscopy was used to monitor changes in individual granule motions related to the secretory response in chromaffin cells. Because the motions of granules are very small (tens of nanometers), instrumental noise in the quantitation of granule motion was taken into account. ATP and Ca2+, both of which prime secretion before fusion, also affect granule motion. Removal of ATP in permeabilized cells causes average granule motion to decrease. Nicotinic stimulation causes a calcium-dependent increase in average granule motion. This effect is more pronounced for granules that undergo exocytosis than for those that do not. Fusion is not preceded by a reduction in mobility. Granules sometimes move 100 nm or more up to and within a tenth of a second before fusion. Thus, the jittering motion of granules adjacent to the plasma membrane is regulated by factors that regulate secretion and may play a role in secretion. Motion continues until shortly before fusion, suggesting that interaction of granule and plasma membrane proteins is transient. Disruption of actin dynamics did not significantly alter granule motion.  相似文献   

20.
Killing of virally infected cells or tumor cells by cytotoxic T lymphocytes requires targeting of lytic granules to the junction between the CTL and its target. We used whole-cell patch clamp to measure the cell capacitance at fixed intracellular [Ca2+] to study fusion of lytic granules in human CTLs. Expression of a fluorescently labeled human granzyme B construct allowed identification of lytic granule fusion using total internal reflection fluorescence microscopy. In this way capacitance steps due to lytic granule fusion were identified. Our goal was to determine the size of fusing lytic granules and to describe their behavior at the plasma membrane. On average, 5.02 ± 3.09 (mean ± s.d.) lytic granules were released per CTL. The amplitude of lytic granule fusion events was ~ 3.3 fF consistent with a diameter of about 325 nm. Fusion latency was biphasic with time constants of 15.9 and 106 seconds. The dwell time of fusing lytic granules was exponentially distributed with a mean dwell time of 28.5 seconds. Fusion ended in spite of the continued presence of granules at the immune synapse. The mobility of fusing granules at the membrane was indistinguishable from that of lytic granules which failed to fuse. While dwelling at the plasma membrane lytic granules exhibit mobility consistent with docking interspersed with short periods of greater mobility. The failure of lytic granules to fuse when visible in TIRF at the membrane may indicate that a membrane-confined reaction is rate limiting.  相似文献   

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