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1.
Exocytotic exposure and recycling of membrane antigens of chromaffin granules: ultrastructural evaluation after immunolabeling 总被引:24,自引:12,他引:12
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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. 相似文献
2.
The recycling of a secretory granule membrane protein 总被引:2,自引:0,他引:2
We have used N-hydroxysuccinimido-d-biotin as a reagent for labeling proteins exposed at the surface of cultured bovine adrenal chromaffin cells during Ba2+-stimulated secretion. A specific secretory granule membrane constituent, dopamine-beta-hydroxylase (DBH), has been investigated using immunoprecipitation followed by electrophoresis. Within 30 min of stimulation, exposed DBH had been cleared from the cell surface. Nevertheless, quantitation of labeled DBH using [125I] streptavidin suggested that it remained undegraded over a period of 24 h, a time during which secretory granule stores of catecholamines were being replenished. Subcellular fractionation of the cultured cells suggested that, after 3 or 4 h, the biotinylated DBH, which was still membrane-bound, was located in particulate material that also contained cytochrome b561, another major secretory granule membrane component. 相似文献
3.
Selective recapture of secretory granule components after full collapse exocytosis in neuroendocrine chromaffin cells 总被引:1,自引:0,他引:1
Ceridono M Ory S Momboisse F Chasserot-Golaz S Houy S Calco V Haeberlé AM Demais V Bailly Y Bader MF Gasman S 《Traffic (Copenhagen, Denmark)》2011,12(1):72-88
In secretory cells, calcium-regulated exocytosis is rapidly followed by compensatory endocytosis. Neuroendocrine cells secrete hormones and neuropeptides through various modes of exo-endocytosis, including kiss-and-run, cavicapture and full-collapse fusion. During kiss-and-run and cavicapture modes, the granule membrane is maintained in an omega shape, whereas it completely merges with the plasma membrane during full-collapse mode. As the composition of the granule membrane is very different from that of the plasma membrane, a precise sorting process of granular proteins must occur. However, the fate of secretory granule membrane after full fusion exocytosis remains uncertain. Here, we investigated the mechanisms governing endocytosis of collapsed granule membranes by following internalization of antibodies labeling the granule membrane protein, dopamine-β-hydroxylase (DBH) in cultured chromaffin cells. Using immunofluorescence and electron microscopy, we observed that after full collapse, DBH remains clustered on the plasma membrane with other specific granule markers and is subsequently internalized through vesicular structures composed mainly of granule components. Moreover, the incorporation of this recaptured granule membrane into an early endosomal compartment is dependent on clathrin and actin. Altogether, these results suggest that after full collapse exocytosis, a selective sorting of granule membrane components is facilitated by the physical preservation of the granule membrane entity on the plasma membrane. 相似文献
4.
《Neurochemistry international》1986,9(3):391-399
Cultured chromaffin cells were stimulated with either Ba2+ or nicotine to secrete catecholamines. This resulted in the appearance of the chromaffin granule membrane protein, dopamine β-hydroxylase (DBH), on the cell surface. The DBH exposed on the cell surface was labeled using fluorescently tagged anti-DBH Fab fragments and the cell surface was simultaneously labeled with fluorescently tagged concanavalin A. Immediately after labeling, both fluorescent markers were localized on or near the cell surface; anti-DBH fluorescence was distributed as patches, but Con A fluorescence was uniformly distributed. Approximately 30 min after labeling, anti-DBH fluorescence appeared to be almost completely internalized without apparent redistribution on the surface whereas much of the Con A fluorescence remained on the cell surface.The rate of DBH endocytosis was quantified using 125I labeled anti-IgG to measure surface bound anti-DBH. Following stimulation of catecholamine secretion, DBH and DBH/anti-DBH complexes both disappeared from the cell surface at similar rates. The half-life on the cell surface was approximately 7 min. These results demonstrate that DBH was rapidly and selectively retrieved from the cell surface, probably from the site of exocytosis. 相似文献
5.
Digitonin-Permeabilized Cells Are Exocytosis Competent 总被引:6,自引:3,他引:3
Theo Schäfer Urs O. Karli Eugen K.-M. Gratwohl Felix E. Schweizer Max M. Burger 《Journal of neurochemistry》1987,49(6):1697-1707
Release of norepinephrine from PC12 cells can be stimulated by free Ca2+ in micromolar concentrations after permeabilization with 10 micrograms/ml of digitonin. This release is time and temperature dependent, half-maximal at 0.3 microM Ca2+, and, after washing out of endogenous ATP, half-maximal at about 0.5 mM MgATP when exogenously added. Similar results were obtained with bovine adrenal chromaffin cells using the same protocol. Support for the idea that the mechanism of release from both permeabilized cell types is still exocytosis is demonstrated at the electron microscopic level by immunolabeling chromaffin granule membrane antigens that were introduced into the plasma membrane following stimulation. Electron micrographs furthermore demonstrate that chromaffin granules retain typical dense cores after permeabilization, indicating that leakiness of catecholamines from the granules was not a major factor. Pores, formed by digitonin in the plasma membranes, were utilized to introduce antibodies into such exocytosis-competent cells. Anti-actin and anti-chromaffin granule membrane antibodies show a staining pattern similar to conventionally fixed and stained preparations. Our results demonstrate that pores formed by digitonin do not impair the process of exocytosis although they are big enough to allow macromolecules to pass in both directions. The digitonin-permeabilized cell is therefore an ideal in vitro system with which to study the fusion process between chromaffin granules and the plasma membrane. 相似文献
6.
Effects of changes in osmolality on the stability and function of cultured chromaffin cells and the possible role of osmotic forces in exocytosis 总被引:3,自引:1,他引:2
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Recent evidence indicates that osmotic forces may play a role in exocytosis. To examine this possibility and to investigate the osmotic properties of storage granules within cells, we investigated the effects of changes of osmolality on stability and function of cultured bovine chromaffin cells. Cell volume measurements indicated that the cells behaved as osmometers and that the intracellular osmolality rapidly equilibrated with the osmolality of the extracellular medium. Hyperosmotic solutions strongly inhibited nicotinic agonist-stimulated secretion but did not alter nicotinic agonist-stimulated Ca(2+) uptake. Hyperosmotic solutions also strongly inhibited elevated potassium- stimulated secretion but only weakly inhibited elevated K(+)-stimulated Ca(2+) uptake. Thus, hyperosmotic solutions inhibited secretion at a step after calcium entry. Cells exposed to 165 mOs(1) solutions did not lyse and retained their capacity to store and secrete catecholamine upon stimulation. Significant intracellular lysis of chromaffin granules occurred within cells exposed to lower osmolalities. In contrast, 75 percent of the catecholamine was released from granules from cultured cells or from fresh adrenal medulla incubated in vitro at 210 mOs. The data provide evidence for a role for osmotic forces in exocytosis and suggest that if osmotic stress of the granule occurs during exocytosis, then water influx into chromaffin granules increases granule volume by at least 70 percent. The results also indicate that the osmotic properties of the granules are altered upon homogenization and subcellular fractionation of the cells. 相似文献
7.
Holz RW Hlubek MD Sorensen SD Fisher SK Balla T Ozaki S Prestwich GD Stuenkel EL Bittner MA 《The Journal of biological chemistry》2000,275(23):17878-17885
Kinetically distinct steps can be distinguished in the secretory response from neuroendocrine cells with slow ATP-dependent priming steps preceding the triggering of exocytosis by Ca(2+). One of these priming steps involves the maintenance of phosphatidylinositol 4, 5-bisphosphate (PtdIns-4,5-P(2)) through lipid kinases and is responsible for at least 70% of the ATP-dependent secretion observed in digitonin-permeabilized chromaffin cells. PtdIns-4,5-P(2) is usually thought to reside on the plasma membrane. However, because phosphatidylinositol 4-kinase is an integral chromaffin granule membrane protein, PtdIns-4,5-P(2) important in exocytosis may reside on the chromaffin granule membrane. In the present study we have investigated the localization of PtdIns-4,5-P(2) that is involved in exocytosis by transiently expressing in chromaffin cells a pleckstrin homology (PH) domain that specifically binds PtdIns-4, 5-P(2) and is fused to green fluorescent protein (GFP). The PH-GFP protein predominantly associated with the plasma membrane in chromaffin cells without any detectable association with chromaffin granules. Rhodamine-neomycin, which also binds to PtdIns-4,5-P(2), showed a similar subcellular localization. The transiently expressed PH-GFP inhibited exocytosis as measured by both biochemical and electrophysiological techniques. The results indicate that the inhibition was at a step after Ca(2+) entry and suggest that plasma membrane PtdIns-4,5-P(2) is important for exocytosis. Expression of PH-GFP also reduced calcium currents, raising the possibility that PtdIns-4,5-P(2) in some manner alters calcium channel function in chromaffin cells. 相似文献
8.
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. 相似文献
9.
We have recently developed a combination of polarization and total internal reflection fluorescence microscopy (pTIRFM) to
monitor changes in plasma membrane topology occurring after fusion of chromaffin granules. In this report, pTIRFM is further
exploited to reveal two major findings in regards to the secretory pathway in bovine chromaffin cells. First, we show that
changes in membrane topology are sometimes detected even prior to fusion. This occurs with high probability in a small subset
of granules that appear in the evanescent field during the experiment. On these occasions, the plasma membrane invaginates
with the movement just preceding the appearance of a granule in the evanescent field. Such events may represent a direct interaction
of the granule with the plasma membrane. Second, we show that the topological fate of the post-fusion, granule/plasma membrane
intermediate is regulated by divalent cation. When Sr2+ is used instead of Ca2+ to trigger exocytosis, membrane topology in the exocytotic region is stabilized with significant curvature and indentation. 相似文献
10.
Xu J Tang KS Lu VB Weerasinghe CP Tse A Tse FW 《American journal of physiology. Cell physiology》2005,289(5):C1122-C1133
Glucocorticoid is reported to regulate catecholamine synthesis and storage. However, it is not clear whether the actual amount of catecholamine released from individual granules (quantal size, Q) in mature chromaffin cells is affected by glucocorticoid. Using carbon fiber amperometry, we found that dexamethasone did not affect mean cellular Q or the proportional release from different populations of granules in rat chromaffin cells cultured for 1 day in a serum-free defined medium. After two extra days of culture in the defined medium, there was a rundown in mean cellular Q, and it was associated with a shift in the proportional release from the different granule populations. This phenomenon could not be rescued by serum supplementation but could be prevented by dexamethasone via an action that was independent of changes in voltage-gated Ca2+ channel (VGCC) density. Using simultaneous measurements of membrane capacitance and cytosolic Ca2+ concentration, we found that for cells cultured in defined medium dexamethasone enhanced the exocytotic response triggered by a brief depolarization (50 ms) without affecting the VGCC density or the fast exocytotic response triggered via flash photolysis of caged Ca2+. Thus glucocorticoid may regulate the number of immediately releasable granules that are in close proximity to a subset of VGCC. Because chromaffin cells in vivo are exposed to high concentrations of glucocorticoid, our findings suggest that the paracrine actions of glucocorticoid maintain the mean catecholamine content in chromaffin cell granules as well as the colocalization of releasable granules with VGCCs. catecholamines; paracrine action; exocytosis; calcium channels 相似文献
11.
Exocytotic exposure and retrieval of membrane antigens of chromaffin granules: quantitative evaluation of immunofluorescence on the surface of chromaffin cells 总被引:15,自引:7,他引:8
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《The Journal of cell biology》1984,98(5):1817-1824
The exocytotic exposure of antigens of chromaffin granule membranes was studied with chromaffin cells isolated from bovine adrenal medulla. Antigens on the cell surface were visualized by indirect membrane immunofluorescence employing antisera against glycoprotein III and dopamine beta-hydroxylase. With unstimulated cells, only weak immunofluorescence on the cell surface was observed, whereas stimulated cells (with carbachol or Ba2+) exhibited much stronger reactions. In all cases the staining appeared as dots and patches. To quantitatively prove these observations, we analyzed the immunostained cells using a fluorescence-activated cell sorter. After stimulation, the average fluorescence intensity of the cell population was enhanced. This increase correlated with the degree of catecholamine secretion. The fluorescence intensity of stimulated cells varied over a broad range indicating that individual cells reacted variably to the secretagogues. When stimulated cells were incubated at 37 degrees C for up to 45 min after stimulation, a decrease of membrane immunofluorescence approaching that of unstimulated control cells was observed. Apparently, the membranes of chromaffin granules, which had been incorporated into the plasma membrane, were retrieved by a specific and relatively fast process. This retrieval of the antigen from the cell surface was blocked by sodium azide, but not influenced by colchicine, cytochalasin B, and trifluoperazine. The quantitative methods established in this paper should prove useful for further study of the kinetics of the exo-endocytotic cycle in secretory tissues. 相似文献
12.
Summary 1. To understand better the mechanisms which govern the sensitivity of secretory vesicles to a calcium stimulus, we compared the abilities of injected chromaffin granule membranes and of endogenous cortical granules to undergo exocytosis inXenopus laevis oocytes and eggs in response to cytosolic Ca2+. Exocytosis of chromaffin granule membranes was detected by the appearance of dopamine--hydroxylase of the chromaffin granule membrane in the oocyte or egg plasma membrane. Cortical granule exocytosis was detected by release of cortical granule lectin, a soluble constituent of cortical granules, from individual cells.2. Injected chromaffin granule membranes undergo exocytosis equally well in frog oocytes and eggs in response to a rise in cytosolic Ca2+ induced by incubation with ionomycin.3. Elevated Ca2+ triggered cortical granule exocytosis in eggs but not in oocytes.4. Injected chromaffin granule membranes do not contribute factors to the oocyte that allow calcium-dependent exocytosis of the endogenous cortical granules.5. Protein kinase C activation by phorbol esters stimulates cortical granule exocytosis in bothXenopus laevis oocytes andX. laevis eggs (Bement, W. M., and Capco, D. G.,J. Cell Biol. 108, 885–892, 1989). Activation of protein kinase C by phorbol ester also stimulated chromaffin granule membrane exocytosis in oocytes, indicating that although cortical granules and chromaffin granule membranes differ in calcium responsiveness, PKC activation is an effective secretory stimulus for both.6. These results suggest that structural or biochemical characteristics of the chromaffin granule membrane result in its ability to respond to a Ca2+ stimulus. In the oocytes, cortical granule components necessary for Ca2+-dependent exocytosis may be missing, nonfunctional, or unable to couple to the Ca2+ stimulus and downstream events. 相似文献
13.
Svend Kirkeby 《Cell and tissue research》1995,283(1):85-92
Adrenal medullary chromaffin cells secrete catecholamines through exocytosis of their intracellular chromaffin granules. Osmotic granule swelling has been implicated to play a role in the generation of membrane stress associated with the fusion of the granule membrane. However, controversy exists as to whether swelling occurs before or after the actual fusion event. Using morphometric methods we have determined the granule diameter distributions in rapidly frozen, freeze-substituted chromaffin cells. Our measurements show that intracellular chromaffin granules increase in size from an average of 234 nm to 274 nm or 277 nm in cells stimulated to secrete with nicotine or high external K+, respectively. Granule swelling occurs before the formation of membrane contact. Ammonium chloride, an agent which inhibits stimulated catecholamine secretion by approximately 50% by altering the intragranular pH, also inhibits granule swelling. In addition, ammonium chloridetreated secreting cells show more granule-plasma membrane contacts than untreated secreting cells. Sodium propionate induces granule swelling in the absence of secretagogue and has been shown to enhance nicotine- and high K+- induced catecholamine release. These results indicate that in adrenal chromaffin cells granule swelling is an essential step in exocytosis before fusion pore formation, and is related to the pH of the granule environment. 相似文献
14.
Yik JH Saxena A Weigel JA Weigel PH 《Biochemical and biophysical research communications》2002,292(4):980-986
The examination of insulin exocytosis at the single cell level by conventional electrophysiologic and amperometric methods possesses inherent limitations, and may not accurately reflect the morphologic events of exocytosis of the insulin granule. To overcome some of these limitations, we show by epifluorescent microscopy of a fluorescent dye, FM1-43, its incorporation into the plasma membrane and oncoming insulin granules undergoing exocytosis, and their core proteins. Using this method, we tracked exocytosis in real-time in insulinoma INS-1 and single rat islet beta cells in response to KCl and glucose. We observed both single transient and multi-stepwise increases in membrane FM1-43 fluorescence, suggesting single granule exocytosis as well as sequential and compound exocytosis, respectively. Confocal microscopy of nonpermeabilized cells shows that some of the exocytosed insulin granules labeled by the FM1-43 dye could also be labeled with insulin antibodies, suggesting prolonged openings of the fusion pores and slow dissolution of the granule core proteins on the membrane surface. 相似文献
15.
Ronald W. Holz 《Cellular and molecular neurobiology》1988,8(3):259-268
1. Calcium-dependent exocytosis of catecholamines from intact and digitonin-permeabilized bovine adrenal chromaffin cells was investigated. 2. 45Ca2+ uptake and secretion induced by nicotinic stimulation or depolarization in intact cells were closely correlated. The results provide strong support for Ca2+ entry being the trigger for exocytosis. 3. Experiments in which the H+ electrochemical gradient across the intracellular secretory granule (chromaffin granule) membrane was altered indicated that the gradient does not play an important role in exocytosis. 4. Ca2+ entry into the cells is associated with activation of phospholiphase C and a rapid translocation of protein kinase C to membranes. 5. The plasma membrane of chromaffin cells was rendered permeable to Ca2+, ATP, and proteins by the detergent digitonin without disruption of the intracellular secretory granules. In this system in which the intracellular milieu can be controlled, micromolar Ca2+ directly stimulated catecholamine secretion. 6. Treatment of the cells with phorbol esters and diglyceride, which activate protein kinase C, enhanced phosphorylation and subsequent Ca2+-dependent secretion in digitonin-treated cells. 7. Phorbol ester-induced secretion could be specifically inhibited by trypsin. The experiments indicate that protein kinase C modulates but is not necessary for Ca2+-dependent secretion. 相似文献
16.
Low molecular mass GTP-binding proteins of adrenal chromaffin cells are present on the secretory granule 总被引:8,自引:0,他引:8
Adrenal medullary homogenates and chromaffin granule membranes were separated by SDS-polyacrylamide gel electrophoresis and GTP-binding proteins detected using [alpha-32P]GTP binding to nitrocellulose blots. Four GTP-binding polypeptides of 24, 22, 20 and 18 kDa were routinely found in medullary homogenates and all were also found in isolated chromaffin granule membranes. The GTP-binding polypeptides co-sedimented with granule membrane markers following separation on sucrose gradients. On the basis of trypsin sensitivity and resistance to extraction, the GTP-binding proteins appeared to be tightly bound to the cytoplasmic surface of the granules. One or more of the secretory granule GTP-binding proteins could be involved in exocytosis in adrenal chromaffin cells. 相似文献
17.
An investigation of the molecular properties of glycoprotein III has shown this to be a major component of molecular aggregates present in the membrane and soluble fractions of secretory vesicles from bovine adrenal medulla. These aggregates also contain components identified as glycoproteins H, J, and K which are molecular forms of Kex2-related proteases (glycoprotein H) and carboxypeptidase H (glycoprotein components J and K) and which have functions concerned with the processing of prohormones. A number of experiments indicated that these glycoproteins were associated. These components were coimmunoprecipitated from the soluble and membrane fractions of chromaffin granules. Purification of soluble glycoprotein III using wheat germ agglutinin-Sepharose resulted in the recovery of similar proportions of glycoproteins H, J, and K and gel filtration of the eluted material in combination with immunoprecipitation revealed the presence of heteroaggregates containing all of the glycoproteins. Similar results were obtained following octylglucoside solubilization of chromaffin granule membranes. Glycoprotein components III, H, J, and K were also found to have identical distributions following fractionation of chromaffin granule membranes with Triton X-114. It was concluded that the aggregates seen in the soluble fraction reflect an association of these components in the chromaffin granule membrane. This raises the possibility that these interactions are important for the targetting of these glycoproteins to secretory granules. 相似文献
18.
The fate of the surplus membrane following exocytosis of mast cell granules was studied by the extracellular tracer Ruthenium red (Ru red). Isolated rat peritoneal mast cells were stimulated with 4 micrograms/ml polylysine, washed and maintained in a culture medium for 80 min. Mast cells were observed both with the light microscope after adding Ru red in physiological solution and with the electron microscope after fixation in Ru red-containing fixatives. Whereas all exocytotic cavities were found to be stained with Ru red immediately after stimulation, a gradual lack of staining was observed in the subsequent period. The exits of the cavities were sealed by membrane fusions which resulted in closed vacuoles containing exocytosed granule remnants. These vacuoles often fused with each other to form a few giant vacuoles. The overwhelming majority of the vacuoles were observed to be closed 30 to 80 min after stimulation. In one experiment a quantitative analysis was performed to assess the degree of membrane recapture by sealing of the exocytotic cavities. A considerable portion of the plasma membrane area was retrieved in this way as early as between 15 and 30 min after stimulation. We conclude that the dominant mechanism of membrane retrieval in the early period following exocytosis is the recapture of large membrane areas by sealing of exocytotic cavities. 相似文献
19.
Exocytosis in bovine chromaffin cells: studies with patch-clamp capacitance and FM1-43 fluorescence
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Kilic G 《Biophysical journal》2002,83(2):849-857
In response to physiological stimuli, neuroendocrine cells secrete neurotransmitters through a Ca(2+)-dependent fusion of secretory granules with the plasma membrane. We studied insertion of granules in bovine chromaffin cells using capacitance as a measure of plasma membrane area and fluorescence of a membrane marker FM1-43 as a measure of exocytosis. Intracellular dialysis with [Ca(2+)] (1.5-100 microM) evoked massive exocytosis that was sufficient to double plasma membrane area but did not swell cells. In principle, in the absence of endocytosis, the addition of granule membrane would be anticipated to produce similar increases in the capacitance and FM1-43 fluorescence responses. However, when endocytosis was minimal, the changes in capacitance were markedly larger than the corresponding changes in FM1-43 fluorescence. Moreover, the apparent differences between capacitance and FM1-43 fluorescence changes increased with larger exocytic responses, as more granules fused with the plasma membrane. In experiments in which exocytosis was suppressed, increasing membrane tension by osmotically induced cell swelling increased FM1-43 fluorescence, suggesting that FM1-43 fluorescence is sensitive to changes in the membrane tension. Thus, increasing membrane area through exocytosis does not swell chromaffin cells but may decrease membrane tension. 相似文献
20.
Meyer MZ Déliot N Chasserot-Golaz S Premont RT Bader MF Vitale N 《The Journal of biological chemistry》2006,281(12):7919-7926
Neuroendocrine cells release hormones and neuropeptides by exocytosis, a highly regulated process in which secretory granules fuse with the plasma membrane to release their contents in response to a calcium trigger. Using chromaffin and PC12 cells, we have recently described that the granule-associated GTPase ARF6 plays a crucial role in exocytosis by activating phospholipase D1 at the plasma membrane and, presumably, promoting the fusion reaction between the two membrane bilayers. ARF6 is activated by the nucleotide exchange factor ARNO following docking of granules to the plasma membrane. We show here that GIT1, a GTPase-activating protein stimulating GTP hydrolysis on ARF6, is the second molecular partner that turns over the GDP/GTP cycle of ARF6 during cell stimulation. Western blot and immunofluorescence experiments indicated that GIT1 is cytosolic in resting cells but is recruited to the plasma membrane in stimulated cells, where it co-localizes with ARF6 at the granule docking sites. Over-expression of wild-type GIT1 inhibits growth hormone secretion from PC12 cells; this inhibitory effect was not observed in cells expressing a GIT1 mutant impaired in its ARF-GTPase-activating protein (GAP) activity or in cells expressing other ARF6-GAPs. Conversely reduction of GIT1 by RNA interference increased the exocytotic activity. Using a real time assay for individual chromaffin cells, we found that microinjection of GIT1 strongly reduced the number of exocytotic events. These results provide the first evidence that GIT1 plays a function in calcium-regulated exocytosis in neuroendocrine cells. We propose that GIT1 represents part of the pathway that inactivates ARF6-dependent reactions and thereby negatively regulates and/or terminates exocytotic release. 相似文献