Fusion Of Chromaffin Granules Research Articles

Polarized TIRFM Reveals Changes in Plasma Membrane Topology Before and During Granule Fusion

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.

Chapter 21 Synexin (annexin VII) and membrane fusion during the process of exocytotic secretion

Publisher Summary Synexin is a cytosolic protein, which in the presence of increased calcium can undergo a conformational change that renders it highly hydrophobic. This chapter discusses some of the assumptions to model the fusion process in vitro, the ways in which synexin promotes different aspects of the model, and some of the physical and chemical evidence underlying the present understanding of synexin action during membrane fusion. The ability of synexin to fuse target membranes lies in the fact that once activated by calcium, the synexin monomers form oligomers, which are capable of interacting with two membranes simultaneously. While the hydrophobic bridge hypothesis has been developed to explain the process of fusion driven by synexin, it need not be limited in principle to just this fusion event. It may be anticipated that any fusion event in which a hydrophobic environment is introduced to the potential fusion site, may proceed by the hydrophobic bridge mechanism and is exemplified by studies on calcium-dependent, synexin driven membrane contact and fusion of chromaffin granules, chromaffin granule ghosts, and phospholipid bilayer liposomes.

In vitro fusion of lung lamellar bodies and plasma membrane is augmented by lung synexin

Lamellar bodies of lung epithelial type II cells undergo fusion with plasma membrane prior to exocytosis of surfactant into the alveolar lumen. Since synexin from adrenal glands promotes aggregation and fusion of chromaffin granules, we purified synexin-like proteins from bovine lung cytosolic fraction, and evaluated their effect on the fusion of isolated lamellar bodies and plasma membrane fractions. Synexin activity, which co-purified with an approx. 47 kDa protein (pI 6.8), was assessed by following calcium-dependent aggregation of liposomes prepared from a mixture of phosphatidylcholine:phosphatidylserine (PC:PS, 3:1, mol/mol). Lung synexin caused aggregation of liposomes approximating lung surfactant lipid-like composition, isolated lamellar bodies, or isolated plasma membrane fraction. Lung synexin promoted fusion only in the presence of calcium. It augmented fusion between lamellar bodies and plasma membranes, lamellar bodies and liposomes, or between two populations of liposomes. However, selectivity with regard to synexin-mediated fusion was observed as synexin did not promote fusion between plasma membrane and liposomes, or between liposomes of surfactant lipid-like composition and other liposomes. These observations support a role for lung synexin in membrane fusion between the plasma membrane and lamellar bodies during exocytosis of lung surfactant, and suggest that such fusion is dependent on composition of interacting membranes.

Immunogold labeling of the calcium binding protein synexin in isolated adrenal chromaffin granules and chromaffin cells

Exocytotic fusion of granules in the adrenal medulla chromaffin cell is triggered by a rise in the concentration of cytosolic Ca2+upon cell activation. The protein synexin, annexin VII, was originally found in the adrenal medulla and has been shown to cause aggregation and to support fusion of chromaffin granules in a Ca2+-dependent manner. We have previously suggested that synexin may there fore play a role in the exocytotic fusion process. In order to obtain more structural information on synexin, we performed immuno-electron microscopy on frozen ultrathin sections of both isolated chromaffin granules and chromaffin cells.Chromaffin granules were isolated from bovine adrenal medulla, and synexin was isolated from bovine lung. Granules were incubated in the presence or absence of synexin (24 μg per mg granule protein) and Ca2+(1 mM), which induces maximal granule aggregation, in 0.3M sucrose-40m MMES buffer(pH 6.0). Granules were pelleted, washed twice in buffer without synexin and fixed with 2% glutaraldehyde- 2% para formaldehyde in 0.1 M phosphate buffer (GA/PFA) for 30 min. Chromaffin cells were isolated and cultured for 3-5 days, and washed and incubated in Krebs solution with or without 20 uM nicotine. Cells were fixed 90 sec after on set of stimulation with GA/PFA for 30 min. Fixed granule or cell pellets were washed, infiltrated with 2.3 M sucrose in PBS, mounted and frozen in liquid N2.

Lipid modification of adrenal plasma membranes by phospholipase A2 and their fusion with chromaffin granules.

Secretion of adrenal catecholamines occurs by Ca-dependent exocytosis in which fusion of chromaffin granules with plasma membranes and subsequent lysis of granules are the most distinct steps. We investigated the involvement of phospholipase A2 in exocytosis in an in vitro model system in which chromaffin granules were allowed to interact with plasma membranes. Adrenal plasma membranes by themselves did not cause the release of catecholamines from the granules. By contrast, plasma membranes which were treated with phospholipase A2 caused a remarkable release of catecholamines. The fusogenic activity of phospholipase A2 treated plasma membranes was strongly dependent on a low concentration of Ca (10−6M order). Lyso-phosphatidylcholine caused the release of catecholamines, but its effect was not dependent on Ca. Lysophosphatidylcholine which was loaded to plasma membranes also showed fusogenic activity which was also dependent on Ca. From these results, we postulate that in stimulus-secretion coupling in adrenal medulla, endogenous phospholipase A2 conforms the plasma membrane into lysophospholipids enriched form which showed fusogenic activity and caused the lysis of chromaffin granules in a Ca-dependent manner.

Further characterization of the aggregation and fusion of chromaffin granules by synexin as a model for compound exocytosis.

Synexin was isolated from bovine liver and found to aggregate adrenal chromaffin granules in the same Ca2+-dependent manner as previously described for adrenal synexin. The chromaffin granule aggregating activity of liver synexin was blocked in vitro by the addition of an antibody prepared to the 47,000 molecular weight band extracted from an SDS gel of an adrenal medullary synexin preparation. Chromaffin granules aggregated by synexin fused when exposed to cis-unsaturated fatty acids at concentrations comparable to those released from phospholipids by stimulated secretory cells. The synexin-induced aggregation reaction was blocked by Erythrosin B, a common food coloring, and by the phenothiazine antipsychotic trifluoperazine and promethazine. The aggregation and fusion of chromaffin granules thus appears to be a useful model system for studying synexin from diverse tissues and for testing pharmacologically or toxicologically active substances for effects on secretory systems.

A Cell-free Model for Protein-Lipid Interactions in Exocytosis: Aggregation and Fusion of Chromaffin Granules in the Presence of Calcium, Synexin, and CIS-Unsaturated Fatty Acids

A Cell-free Model for Protein-Lipid Interactions in Exocytosis: Aggregation and Fusion of Chromaffin Granules in the Presence of Calcium, Synexin, and CIS-Unsaturated Fatty Acids