scholarly journals Calcium-dependence of synexin binding may determine aggregation and fusion of lamellar bodies

1997 ◽  
Vol 322 (1) ◽  
pp. 103-109 ◽  
Author(s):  
Namita SEN ◽  
Alan R. SPITZER ◽  
Avinash CHANDER
Keyword(s):  
Arachidonic Acid ◽  
Membrane Fusion ◽  
Plasma Membranes ◽  
Secretory Granules ◽  
Lamellar Body ◽  
Lipid Vesicles ◽  
Dependent Manner ◽  
Lamellar Bodies ◽  
Calcium Dependent

Synexin (annexin VII) is a member of the annexin family of calcium and phospholipid binding proteins that promote calcium-dependent aggregation and fusion of lipid vesicles or secretory granules. We have previously suggested that synexin may be involved in membrane fusion processes during exocytosis of lung surfactant since it promotes fusion in vitro of lamellar bodies with plasma membranes. In this study, we characterized calcium-dependency of synexin binding to lamellar bodies and plasma membranes, since such binding is the initial, and, therefore, may be the rate-limiting step in membrane aggregation and fusion. The binding of biotinylated synexin to lamellar bodies and plasma membranes increased in a calcium-dependent manner reaching a maximum at approx. 200 ƁM Ca2+. Binding to lamellar bodies was completely inhibited by unlabelled synexin. Gel-overlay analysis showed that synexin bound to an approx. 76 kDa protein in the lamellar body and plasma membrane fractions. The calcium kinetics were noticeably similar for synexin binding to lamellar bodies and plasma membranes, aggregation of lamellar bodies, and fusion of lamellar bodies with lipid vesicles. At low calcium concentrations, aggregation of lamellar bodies could be increased with increasing synexin concentration, and arachidonic acid increased all three parameters (binding, aggregation, and fusion) in a similar manner. The effects of calcium and arachidonic acid on these three parameters suggest that synexin binding to lamellar bodies may be a rate-determining step for fusion during surfactant secretion. Furthermore, at near physiological calcium levels, the membrane fusion may be enhanced by elevated concentrations of synexin and polyunsaturated fatty acids.

scholarly journals Pancreatic plasma membranes: promiscuous partners in membrane fusion

1994 ◽  
Vol 298 (3) ◽  
pp. 599-604 ◽  
Author(s):  
E G Lee ◽  
S J Marciniak ◽  
C M MacLean ◽  
J M Edwardson
Keyword(s):  
Membrane Fusion ◽  
Plasma Membranes ◽  
Secretory Granules ◽  
The Other ◽  
Zymogen Granules ◽  
Other Hand

We have developed a system in which the fusion of pancreatic plasma membranes with zymogen granules can be studied in vitro. We show here that pancreatic plasma membranes fuse not only with pancreatic zymogen granules but also with parotid secretory granules. In contrast, parotid membranes fuse only with parotid granules and not with pancreatic granules. The extent of fusion is insensitive to Ca2+ for all combinations of plasma membranes and granules. Guanosine 5′-[gamma-thio]triphosphate (GTP[S]), on the other hand, stimulates fusion of pancreatic membranes with both pancreatic granules and parotid granules, but inhibits fusion between parotid membranes and parotid granules.

scholarly journals Ca2+-independent fusion of secretory granules with phospholipase A2-treated plasma membranes in vitro

1995 ◽  
Vol 307 (2) ◽  
pp. 563-569 ◽  
Author(s):  
T Nagao ◽  
T Kubo ◽  
R Fujimoto ◽  
H Nishio ◽  
T Takeuchi ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Linoleic Acid ◽  
Phospholipase A2 ◽  
Plasma Membranes ◽  
Secretory Granules ◽  
Fusion Process ◽  
Amylase Release ◽  
Fusogenic Activity ◽  
Rat Parotid

The fusion of secretory granules with plasma membranes prepared from rat parotid gland was studied in vitro to clarify the mechanism of exocytosis. Fusion of the granules with plasma membranes was measured by a fluorescence-dequenching assay with octadecyl rhodamine B, and release of amylase was also measured to confirm the fusion as a final step of the secretory process. Plasma membranes that had been pretreated with porcine phospholipase A2 (PLA2) in the presence of 20 microM Ca2+ fused with the granules within 30 s, and induced amylase release by reacting with the membranes of granules, whereas without this pretreatment they had no significant effect. The fusion process accompanied by amylase release was induced in the presence of 10 mM EGTA, and therefore was apparently Ca(2+)-independent. On the other hand, the presence of EGTA or 100 microM quinacrine, an inhibitor of PLA2, during treatment of plasma membranes with PLA2 inhibited their fusogenic activity, suggesting the importance of activation of PLA2. Arachidonic acid and linoleic acid were released from the plasma membranes during the PLA2 treatment. The presence of albumin, an adsorbent of fatty acids, during the treatment also inhibited the activity. Pretreatment of the membranes with arachidonic acid or linoleic acid did not have any effect, but the presence of exogenously added arachidonic acid during PLA2 treatment enhanced the membrane-fusion-inducing effect of PLA2. Pretreatment of the membranes with lysophosphatidylcholine induced fusogenic activity. These findings suggest that the conformational change in the plasma-membrane phospholipids induced by PLA2 and the presence of arachidonic acid or linoleic acid produced by PLA2 are important in the process of fusion of secretory granules with the plasma membranes of rat parotid acinar cells and that the fusion process itself is independent of Ca2+.

Synexin and GTP increase surfactant secretion in permeabilized alveolar type II cells

2001 ◽  
Vol 280 (5) ◽  
pp. L991-L998 ◽  
Author(s):  
Avinash Chander ◽  
Namita Sen ◽  
Alan R. Spitzer
Keyword(s):  
Membrane Fusion ◽  
Plasma Membranes ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Dependent Manner ◽  
Type Ii ◽  
Lamellar Bodies ◽  
Direct Role ◽  
Concentration Dependent Manner ◽  
Surfactant Secretion

We have previously suggested that synexin (annexin VII), a Ca2+-dependent phospholipid binding protein, may have a role in surfactant secretion, since it promotes membrane fusion between isolated lamellar bodies (the surfactant-containing organelles) and plasma membranes. In this study, we investigated whether exogenous synexin can augment surfactant phosphatidylcholine (PC) secretion in synexin-deficient lung epithelial type II cells. Isolated rat type II cells were cultured for 20–22 h with [3H]choline to label cellular PC. The cells were then treated with β-escin, which forms pores in the cell membrane and releases cytoplasmic proteins including synexin. These cells, however, retained lamellar bodies. The permeabilized type II cells were evaluated for PC secretion during a 30-min incubation. Compared with PC secretion under basal conditions, the presence of Ca2+(up to 10 μM) did not increase PC secretion. In the presence of 1 μM Ca2+, synexin increased PC secretion in a concentration-dependent manner, which reached a maximum at ∼5 μg/ml synexin. The secretagogue effect of synexin was abolished when synexin was inactivated by heat treatment (30 min at 65°C) or by treatment with synexin antibodies. GTP or its nonhydrolyzable analog β:γ-imidoguanosine-5′-triphosphate also increased PC secretion in permeabilized type II cells. The PC secretion was further increased in an additive manner when a maximally effective concentration of synexin was added in the presence of 1 mM GTP, suggesting that GTP acts by a synexin-independent mechanism to increase membrane fusion. Thus our results support a direct role for synexin in surfactant secretion. Our study also suggests that membrane fusion during surfactant secretion may be mediated by two independent mechanisms.

Chlorobutanol, a Preservative of Desmopressin, Inhibits Human Platelet Aggregation and Release In Vitro

1990 ◽  
Vol 64 (03) ◽  
pp. 473-477 ◽  
Author(s):  
Shih-Luen Chen ◽  
Wu-Chang Yang ◽  
Tung-Po Huang ◽  
Shiang Wann ◽  
Che-ming Teng
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Atp Release ◽  
Free Calcium ◽  
Dependent Manner ◽  
Antiplatelet Effect ◽  
Human Platelet Aggregation ◽  
Acid Pathway ◽  
Arachidonic Acid Pathway

SummaryTherapeutic preparations of desmopressin for parenteral use contain the preservative chlorobutanol (5 mg/ml). We show here that chlorobutanol is a potent inhibitor of platelet aggregation and release. It exhibited a significant inhibitory activity toward several aggregation inducers in a concentration- and time-dependent manner. Thromboxane B2 formation, ATP release, and elevation of cytosolic free calcium caused by collagen, ADP, epinephrine, arachidonic acid and thrombin respectively were markedly inhibited by chlorobutanol. Chlorobutanol had no effect on elastase- treated platelets and its antiplatelet effect could be reversed. It is concluded that the antiplatelet effect of chlorobutanol is mainly due to its inhibition on the arachidonic acid pathway but it is unlikely to have a nonspecitic toxic effect. This antiplatelet effect of chlorobutanol suggests that desmopressin, when administered for improving hemostasis, should not contain chlorobutanol as a preservative.

scholarly journals Sec1p directly stimulates SNARE-mediated membrane fusion in vitro

2004 ◽  
Vol 167 (1) ◽  
pp. 75-85 ◽  
Author(s):  
Brenton L. Scott ◽  
Jeffrey S. Van Komen ◽  
Hassan Irshad ◽  
Song Liu ◽  
Kirilee A. Wilson ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Fusion ◽  
Membrane Trafficking ◽  
Snare Complex ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Bud Neck ◽  
Precise Role

Sec1 proteins are critical players in membrane trafficking, yet their precise role remains unknown. We have examined the role of Sec1p in the regulation of post-Golgi secretion in Saccharomyces cerevisiae. Indirect immunofluorescence shows that endogenous Sec1p is found primarily at the bud neck in newly budded cells and in patches broadly distributed within the plasma membrane in unbudded cells. Recombinant Sec1p binds strongly to the t-SNARE complex (Sso1p/Sec9c) as well as to the fully assembled ternary SNARE complex (Sso1p/Sec9c;Snc2p), but also binds weakly to free Sso1p. We used recombinant Sec1p to test Sec1p function using a well-characterized SNARE-mediated membrane fusion assay. The addition of Sec1p to a traditional in vitro fusion assay moderately stimulates fusion; however, when Sec1p is allowed to bind to SNAREs before reconstitution, significantly more Sec1p binding is detected and fusion is stimulated in a concentration-dependent manner. These data strongly argue that Sec1p directly stimulates SNARE-mediated membrane fusion.

scholarly journals Arachidonic acid promotes the binding of 5-lipoxygenase on nanodiscs containing 5-lipoxygenase activating protein in the absence of calcium-ions

2020 ◽  
Author(s):  
Ramakrishnan B. Kumar ◽  
Pasi Purhonen ◽  
Hans Hebert ◽  
Caroline Jegerschöld
Keyword(s):  
Arachidonic Acid ◽  
Complex Formation ◽  
Calcium Ions ◽  
Dependent Manner ◽  
Present Communication ◽  
Nuclear Membranes ◽  
His Tag ◽  
C Terminus ◽  
Transmission Electron

AbstractAmong the first steps in inflammation is the conversion of arachidonic acid (AA) stored in the cell membranes into leukotrienes. This occurs mainly in leukocytes and depends on the interaction of two proteins: 5-lipoxygenase (5LO), stored away from the nuclear membranes until use and 5-lipoxygenase activating protein (FLAP), a transmembrane, homotrimeric protein, constitutively present in nuclear membrane. We could earlier visualize the binding of 5LO to nanodiscs in the presence of Ca2+-ions by the use of transmission electron microscopy (TEM) on samples negatively stained by sodium phosphotungstate. In the absence of Ca2+-ions 5LO did not bind to the membrane. In the present communication, FLAP reconstituted in the nanodiscs which could be purified if the His-tag was located on the FLAP C-terminus but not the N-terminus. Our aim was to find out if 1) 5LO would bind in a Ca2+-dependent manner also when FLAP is present? 2) Would the substrate (AA) have effects on 5LO binding to FLAP-nanodiscs? TEM was used to assess the complex formation between 5LO and FLAP-nanodiscs along with, sucrose gradient purification, gel-electrophoresis and mass spectroscopy. It was found that presence of AA by itself induces complex formation in the absence of added calcium. This finding corroborates that AA is necessary for the complex formation and that a Ca2+-flush is mainly needed for the recruitment of 5LO to the membrane. Our results also showed that the addition of Ca2+-ions promoted binding of 5LO on the FLAP-nanodiscs as was also the case for nanodiscs without FLAP incorporated. In the absence of added substances no 5LO-FLAP complex was formed. Another finding is that the formation of a 5LO-FLAP complex appears to induce fragmentation of 5LO in vitro.

Characterization of a cDNA encoding a novel band 4.1-like protein in zebrafish

1997 ◽  
Vol 75 (5) ◽  
pp. 623-632 ◽  
Author(s):  
Gregory M Kelly ◽  
Bruno Reversade
Keyword(s):  
Cell Communication ◽  
Membrane Skeleton ◽  
Adult Brain ◽  
Dependent Manner ◽  
Midblastula Transition ◽  
Protein 4.1 ◽  
Calcium Dependent ◽  
Calcium Calmodulin Dependent ◽  
Integral Role

Membrane skeleton protein 4.1 and other members of a family of proteins that link the cytoskeleton to the plasma membrane may play an integral role in cell communication during development. The polymerase chain reaction and degenerate oligodeoxynucleotide primers to consensus sequences in the putative membrane-binding domain of the protein 4.1 superfamily were used to isolate cDNAs encoding members of the zebrafish protein 4.1 family. Zebrafish stage- and tissue-specific first strand cDNA was used in the PCR. After the reaction, amplicons of the predicted size were sequenced to confirm their relationship to the protein 4.1 superfamily. One cDNA, with a high degree of similarity to a mouse novel band 4.1-like cDNA, was used to probe a zebrafish adult brain library. A 2.4-kb cDNA was isolated and found to encode a 619 amino acid polypeptide homologous to mouse novel band 4.1-like protein 4. Zebrafish nbl4 mRNA is maternally supplied and is expressed throughout embryogenesis. In adults, nbl4 is found in the ovary, eye, heart, and brain, but not in gut or skeletal muscle. When synthetic nbl4 mRNA is translated in vitro it binds calmodulin in a calcium-dependent manner. These data indicate that zebrafish nbl4 is a maternal transcript owing to its presence before the midblastula transition, and it is present later on in specific adult structures. The ability to bind calmodulin would suggest that the function of nbl4 protein may be potentially regulated via a calcium-calmodulin dependent mechanism.

scholarly journals Matrix-transmitted paratensile signaling enables myofibroblast–fibroblast cross talk in fibrosis expansion

2020 ◽  
Vol 117 (20) ◽  
pp. 10832-10838 ◽  
Author(s):  
Longwei Liu ◽  
Hongsheng Yu ◽  
Hui Zhao ◽  
Zhaozhao Wu ◽  
Yi Long ◽  
...  
Keyword(s):  
Cross Talk ◽  
Growth Models ◽  
Tissue Level ◽  
Dependent Manner ◽  
Cell Level ◽  
Fibrous Matrix ◽  
Calcium Dependent ◽  
Spatiotemporal Feature ◽  
Fibroblast Foci

While the concept of intercellular mechanical communication has been revealed, the mechanistic insights have been poorly evidenced in the context of myofibroblast–fibroblast interaction during fibrosis expansion. Here we report and systematically investigate the mechanical force-mediated myofibroblast–fibroblast cross talk via the fibrous matrix, which we termed paratensile signaling. Paratensile signaling enables instantaneous and long-range mechanotransduction via collagen fibers (less than 1 s over 70 μm) to activate a single fibroblast, which is intracellularly mediated by DDR2 and integrin signaling pathways in a calcium-dependent manner through the mechanosensitive Piezo1 ion channel. By correlating in vitro fibroblast foci growth models with mathematical modeling, we demonstrate that the single-cell-level spatiotemporal feature of paratensile signaling can be applied to elucidate the tissue-level fibrosis expansion and that blocking paratensile signaling can effectively attenuate the fibroblast to myofibroblast transition at the border of fibrotic and normal tissue. Our comprehensive investigation of paratensile signaling in fibrosis expansion broadens the understanding of cellular dynamics during fibrogenesis and inspires antifibrotic intervention strategies targeting paratensile signaling.

scholarly journals The Receptor for Parathyroid Hormone and Parathyroid Hormone-Related Peptide Is Hydrolyzed and Its Signaling Properties Are Altered by Directly Binding the Calpain Small Subunit

Endocrinology ◽  
2005 ◽  
Vol 146 (5) ◽  
pp. 2336-2344 ◽  
Author(s):  
Masako Shimada ◽  
Matthew J. Mahon ◽  
Peter A. Greer ◽  
Gino V. Segre
Keyword(s):  
Parathyroid Hormone ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Small Subunit ◽  
Hek293 Cells ◽  
Calpain Inhibitor ◽  
Dependent Manner ◽  
Intact Cells ◽  
Calcium Dependent

Abstract We show calcium-dependent, direct binding between the N-terminal portion of the PTH/PTHrP receptor (PTH1R) C-terminal intracellular tail and the calpain small subunit. Binding requires, but may not be limited to, amino acids W474, S475, and W477. The wild-type, full-length rat (r) PTH1R, but not rPTH1R with W474A/W477A substitutions, copurifies with the endogenous calpain small subunit in HEK293 cells. Calpain hydrolyzes ΔNt-rPTH1R, a receptor with a 156-amino acid N-terminal deletion, in a calcium-dependent manner in vitro and in intact cells. Most importantly, PTH stimulation increases the cleavage of ΔNt-rPTH1R and rPTH1R-yellow fluorescent protein in HEK293 cells, and of talin in HEK293 cells expressing rPTH1R-yellow fluorescent protein and in ROS17/2.8 osteoblast-like cells that express rPTH1R endogenously. The absence of calpain in Capn4-null embryonic fibroblasts and the lowered calpain activity in MC3T3-E1 osteoblastic cells due to stable expression of the calpain inhibitor, calpastatin, reduce PTH-stimulated cAMP accumulation. The calpain small subunit is the second protein, in addition to the sodium-hydrogen exchanger regulatory factor, and the first enzyme that binds the PTH1R; PTH1R bound to both of these proteins results in altered PTH signaling.

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