scholarly journals Freeze-fracture of membrane fusions during exocytosis in pancreatic B-cells.

1977 ◽  
Vol 75 (1) ◽  
pp. 23-30 ◽  
Author(s):  
L Orci ◽  
A Perrelet ◽  
D S Friend
Keyword(s):  
Plasma Membrane ◽  
B Cells ◽  
Secretory Granule ◽  
Mammalian Cells ◽  
Freeze Fracture ◽  
Total Clearance ◽  
Close Apposition ◽  
Fracture Appearance ◽  
Pancreatic B Cells ◽  
Glucose Stimulated Insulin Secretion

To examine the freeze-fracture appearance of membrane alterations at sites of exocytosis in mammalian cells, we studied the secretory granule and plasma membrane of rat pancreatic B-cells during glucose-stimulated insulin secretion. Constant features observed were the scarcity of particles in secretory-granule P-fracture faces and the almost total clearance of intramembranous particles in P-and E fracture faces of the plasma membrane in areas of close apposition of these two membranes preceding fusion; also observed was the temporary persistence of particle-cleared regions after the fusion was completed. Our observations thus support the concept that membranes fuse at sites of closely apposed, particle-free regions and that the physiologically created clear areas found in freeze-fracture replicas of the plasma membrane are the hallmarks of incipient or recent membrane fusion.

scholarly journals The adherence of human neutrophils and eosinophils to schistosomula: evidence for membrane fusion between cells and parasites.

1980 ◽  
Vol 86 (1) ◽  
pp. 46-63 ◽  
Author(s):  
J P Caulfield ◽  
G Korman ◽  
A E Butterworth ◽  
M Hogan ◽  
J R David
Keyword(s):  
Plasma Membrane ◽  
Outer Membrane ◽  
Mammalian Cells ◽  
Surface Membrane ◽  
Freeze Fracture ◽  
Spatial Relationship ◽  
Hybrid Membrane ◽  
Human Neutrophils ◽  
Inner Membrane ◽  
Edge Zone

Human neutrophils and eosinophils adhere to the surface of schistosomula of Schistosoma mansoni that have been preincubated with antischistosomular sera with or without complement. Neutrophils are seen to form small (< 0.5 micrometer), heptalaminar and large (5-8 micrometer), pentalaminar fusions with the normal pentalaminar parasite surface membrane. By freeze-fracture techniques, attachment areas 5-8 micrometer in diameter are seen to form between neutrophils and schistosomula. These areas have three zones--an edge and two centrally located areas, one of which is rich and one of which is poor in intramembrane particles (IMPs). The edge zone is continuous around the attachment areas and is usually composed of a skip-fracture that passes out of the schistosomular outer membrane into the inner membrane. In some cases, the edge zone is made up of a string of IMPs. The IMP-rich central areas have an IMP concentration similar to that of unattached neutrophil membranes, are raised off of the surface of the schistosomulum, and have two normal schistosomular membranes underneath indicating that they are indeed unattached. the IMP-poor central areas are composed of a fused or hybrid membrane that is continuous with the neutrophil plasma membrane but that bears the same spatial relationship to the schistosomular inner membrane that the normal outer membrane does. Similar changes are seen in samples prepared with glycerination. Eosinophils generally do not fuse with the schistosomular outer membrane but, instead, discharge their granular contents onto the surface of the schistosomula and appear to adhere to the parasite through this discharged material. It is suggested that schistosomula have a capability to fuse with mammalian cells and that this fusion proceeds from a fusion of the outer leaflets to a fusion of the bilayers, as appears also to be the case in other systems.

scholarly journals Membrane particle changes attending the acrosome reaction in guinea pig spermatozoa

1977 ◽  
Vol 74 (2) ◽  
pp. 561-577 ◽  
Author(s):  
DS Friend ◽  
L Orci ◽  
A Perrelet ◽  
R Yanagimachi
Keyword(s):  
Plasma Membrane ◽  
Guinea Pig ◽  
Acrosome Reaction ◽  
Plasma Membranes ◽  
Freeze Fracture ◽  
Phosphatidylcholine Liposomes ◽  
Acrosomal Membrane ◽  
Large Particles ◽  
Fracture Appearance ◽  
Preparatory Stage

To examine the freeze-fracture appearance of membrane alterations accompanying the preparation of sperm membranes for fusions-the first preparatory stage occurring before physiological release of the acrosomal content, the second afterward-we induced the acrosome reaction in capacitated guinea pig spermatozoa by adding calcium to the mixture. The most common features observed before fusion of the acrosomal and plasma membranes were the deletion of fibrillar intramembranous particles from the E-fracture faces of both membranes, and the clearance of globular particles from the P face of the plasma membrane-events taking place near the terminus of the equatorial segment. Large particles, >12nm, remained not far from the cleared E-face patches. The P face of the outer acrosomal membrane is virtually clear from the outset. In addition, when fusion was completed, occasional double lines of large particles transiently embossed the P face of the plasma membrane (postacrosomal) side of the fusion zone. Behind the line of fusion, another series of particle-cleared foci emerged. We interpreted these postfusion membrane clearances as a second adaptation for sperm-egg interaction. Induction of the acrosome reaction in media containing phosphatidylcholine liposomes resulted in their apparent attachment, incorporation, or exchange in both the originally and secondarily cleared regions. Our observations support the concepts that membranes become receptive to union at particle- deficient interfaces, and that the physiologically created barren areas in freeze-fracture replicas may herald incipient membrane fusion.

scholarly journals Melanophilin accelerates insulin granule fusion without predocking to the plasma membrane

2020 ◽  
Author(s):  
Ada Admin ◽  
Hao Wang ◽  
Kouichi Mizuno ◽  
Noriko Takahashi ◽  
Eri Kobayashi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Secretory Granule ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Granule Exocytosis ◽  
Synaptic Vesicle Exocytosis ◽  
Granule Membrane ◽  
Myosin Va ◽  
Vesicle Exocytosis ◽  
Glucose Stimulated Insulin Secretion

Direct observation of fluorescence-labeled secretory granule exocytosis in living pancreatic β cells has revealed heterogeneous prefusion behaviors: some granules dwell beneath the plasma membrane before fusion, while others fuse immediately once they are recruited to the plasma membrane. Although the former mode seems to follow sequential docking-priming-fusion steps as found in synaptic vesicle exocytosis, the latter mode, which is unique to secretory granule exocytosis, has not been explored well. Here, we show that melanophilin, one of the effectors of the monomeric GTPase Rab27 on the granule membrane, is involved in such an accelerated mode of exocytosis. Both melanophilin-mutated <i>leaden</i> mouse and melanophilin-downregulated human pancreatic β cells exhibit impaired glucose-stimulated insulin secretion, with a specific reduction in fusion events that bypass stable docking to the plasma membrane. Upon stimulus-induced [Ca<sup>2+</sup>]<sub>i</sub> rise, melanophilin mediates this type of fusion by dissociating granules from myosin-Va and actin in the actin cortex and by associating them with a fusion-competent, open form of syntaxin-4 on the plasma membrane. These findings provide the hitherto unknown mechanism to support sustainable exocytosis by which granules are recruited from the cell interior and fuse promptly without stable predocking to the plasma membrane.

scholarly journals Activation of alpha-2-adrenoceptors results in an increase in F-actin formation in HIT-T15 pancreatic B-cells

1995 ◽  
Vol 307 (1) ◽  
pp. 169-174 ◽  
Author(s):  
H C Cable ◽  
A el-Mansoury ◽  
N G Morgan
Keyword(s):  
Plasma Membrane ◽  
Insulin Secretion ◽  
B Cells ◽  
Fluorescence Microscopy ◽  
Pertussis Toxin ◽  
Actin Polymerization ◽  
Secretory Granules ◽  
Rhodamine Phalloidin ◽  
Late Step ◽  
Pancreatic B Cells

1. Alpha-2-adrenoceptor agonists, such as noradrenaline, are potent inhibitors of insulin secretion, and it has been suggested that they control a late step in the pathway of exocytosis. We have investigated whether this could be related to a change in the extent of actin polymerization in the pancreatic B-cell, since actin microfilaments are implicated in regulating the access of secretory granules to the plasma membrane prior to exocytosis. 2. Cultured HIT-T15 pancreatic B-cells responded to noradrenaline with an increase in F-actin content, as judged by a rise in the fluorescence output after probing of the cells with phalloidin (a toxin which binds specifically to F-actin) conjugated to rhodamine. The response to noradrenaline was rapid, dose-dependent and sustained and could be reproduced by the highly selective alpha-2-agonist UK14,304. Examination of HIT-T15 cells by fluorescence microscopy after treatment with rhodamine-phalloidin, revealed a significant localization of F-actin immediately adjacent to the plasma membrane. The pattern of F-actin distribution in the cells was not altered dramatically by noradrenaline, although the intensity of staining close to the plasma membrane appeared to be slightly reduced. 3. The increase in F-actin content induced by noradrenaline and UK14,304 was inhibited significantly by the alpha-2-antagonist idazoxan but not by the alpha-1-selective antagonist prazosin. Pretreatment of HIT-T15 cells with pertussis toxin did not lead to any direct alteration in F-actin content, although the toxin significantly modified the responses induced by noradrenaline and UK14,304. In each case, cells incubated for 24 h with pertussis toxin responded to the alpha-2-agonist with an enhanced fluorescence output, indicating that F-actin levels had increased still further. This did not correlate with any gross change in the distribution of F-actin as judged by fluorescence microscopy. 4. The results demonstrate that alpha-2-adrenoceptors are coupled to control of actin polymerization in HIT-T15 cells. They suggest that regulation of F-actin formation could be a component of the mechanism by which alpha-2-agonists mediate inhibition of insulin secretion.

scholarly journals Increase of gap junctions between pancreatic B-cells during stimulation of insulin secretion.

1979 ◽  
Vol 82 (2) ◽  
pp. 441-448 ◽  
Author(s):  
P Meda ◽  
A Perrelet ◽  
L Orci
Keyword(s):  
Insulin Secretion ◽  
B Cells ◽  
Gap Junctions ◽  
Freeze Fracture ◽  
Secretory Activity ◽  
Isolated Rat Islets ◽  
Pancreatic B Cells ◽  
Stimulation Of

The development of gap junctions between pancreatic B-cells was quantitatively assessed in freeze-fracture replicas of isolated rat islets under different conditions of insulin secretion. The results show that in resting B-cells, gap junctions are small and scarce but that these junctions increase when insulin secretion is stimulated. Both a short (90 min) stimulation by glucose in vitro and a prolonged (2.5 d) stimulation by glibenclamide in vivo raise the number of gap junctions; in addition, the glibenclamide stimulation causes an increase in the size of individual gap junctions. As a consequence, the total area occupied by gap junctions on the B-cell membrane and the ratio of this area to the cell volume were found significantly increased in the latter condition. The slight increase of these values observed after the glucose stimulation did not reach significance. These data indicate a change of gap junctions during the secretory activity of the pancreatic B-cells. The possibility that the coupling of the cells is affected by the treatment is discussed.

Topology of morphologically detectable protein and cholesterol in membranes of polypeptide-secreting cells

1981 ◽  
Vol 296 (1080) ◽  
pp. 47-54 ◽  
Keyword(s):  
Plasma Membrane ◽  
Secretory Granule ◽  
Plasma Membranes ◽  
Secretory Pathway ◽  
Freeze Fracture ◽  
Fracture Morphology ◽  
Cholesterol Content ◽  
Intramembrane Particles ◽  
Exocrine Cells ◽  
Luminal Membrane

The freeze-fracture morphology of intracellular and plasma membranes in endocrine and exocrine polypeptide-secreting cells has been studied to detect changes while these membranes interact during secretion. A qualitative and quantitative evaluation of intramembrane particles and filipin binding as indicators of protein and cholesterol content of the membranes, respectively, reveals the following changes. From the forming of the maturing pole of the Golgi complex, membranes lose morphologically detectable protein and gain morphologically detectable cholesterol. The protein-poor, cholesterol-rich secretory granule membrane then interacts with a richly particulate plasma membrane in endocrine cells and with a moderately particulate luminal membrane in exocrine cells. The site of interaction between secretory granule and plasma membrane is characterized by a local clearing of intramembrane particles; by contrast, filipin-binding sites revealing cholesterol are present in this area. In exocrine cells, the fused secretory granule, which is initially rich in filipin-cholesterol complexes and poor in particles, appears to lose progressively its filipin labelling to resemble the poorly labelled luminal membrane. These findings, although they cannot be interpreted definitely at present, clearly show impressive changes of membrane structure along the secretory pathway and suggest that a corresponding degree of functional specialization is needed for proper interaction to occur.

scholarly journals Effects of glucose and glucagon on the fructose 2,6-bisphosphate content of pancreatic islets and purified pancreatic B-cells. A comparison with isolated hepatocytes

1984 ◽  
Vol 221 (3) ◽  
pp. 759-764 ◽  
Author(s):  
A Sener ◽  
E Van Schaftingen ◽  
M Van de Winkel ◽  
D G Pipeleers ◽  
F Malaisse-Lagae ◽  
...  
Keyword(s):  
B Cells ◽  
Pancreatic Islets ◽  
Isolated Hepatocytes ◽  
Saturation Curve ◽  
Isolated Pancreatic Islets ◽  
Early Increase ◽  
Glucose Phosphorylation ◽  
Pancreatic B Cells ◽  
Glucose Stimulated Insulin Secretion ◽  
Related Increase

Glucose caused a sustained and dose-related increase in the fructose 2,6-bisphosphate content of isolated pancreatic islets, as well as of purified pancreatic B-cells. With isolated B-cells, the glucose saturation curve was sigmoidal and superimposable on that obtained with hepatocytes isolated from unfed rats. However, the response to glucose was notably faster in purified B-cells than in isolated hepatocytes. In contrast again with the situation prevailing in the liver, glucagon failed to decrease significantly the concentration of fructose 2,6-bisphosphate in either islets or purified B-cells. It is proposed that, in the process of glucose-stimulated insulin secretion, an early increase in fructose 2,6-bisphosphate formation may, by causing activation of 6-phosphofructo-1-kinase, allow glycolysis to keep pace with the rate of glucose phosphorylation.

scholarly journals Melanophilin accelerates insulin granule fusion without predocking to the plasma membrane

2020 ◽  
Author(s):  
Ada Admin ◽  
Hao Wang ◽  
Kouichi Mizuno ◽  
Noriko Takahashi ◽  
Eri Kobayashi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Secretory Granule ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Granule Exocytosis ◽  
Synaptic Vesicle Exocytosis ◽  
Granule Membrane ◽  
Myosin Va ◽  
Vesicle Exocytosis ◽  
Glucose Stimulated Insulin Secretion

Direct observation of fluorescence-labeled secretory granule exocytosis in living pancreatic β cells has revealed heterogeneous prefusion behaviors: some granules dwell beneath the plasma membrane before fusion, while others fuse immediately once they are recruited to the plasma membrane. Although the former mode seems to follow sequential docking-priming-fusion steps as found in synaptic vesicle exocytosis, the latter mode, which is unique to secretory granule exocytosis, has not been explored well. Here, we show that melanophilin, one of the effectors of the monomeric GTPase Rab27 on the granule membrane, is involved in such an accelerated mode of exocytosis. Both melanophilin-mutated <i>leaden</i> mouse and melanophilin-downregulated human pancreatic β cells exhibit impaired glucose-stimulated insulin secretion, with a specific reduction in fusion events that bypass stable docking to the plasma membrane. Upon stimulus-induced [Ca<sup>2+</sup>]<sub>i</sub> rise, melanophilin mediates this type of fusion by dissociating granules from myosin-Va and actin in the actin cortex and by associating them with a fusion-competent, open form of syntaxin-4 on the plasma membrane. These findings provide the hitherto unknown mechanism to support sustainable exocytosis by which granules are recruited from the cell interior and fuse promptly without stable predocking to the plasma membrane.

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