Vesicle pools and Ca2+ microdomains: new tools for understanding their roles in neurotransmitter release.

Classical cell biology teaches that exocytosis causes the membrane of exocytic vesicles to disperse into the cell surface and that a cell must later retrieve by molecular sorting whatever membrane components it wishes to keep inside. We have tested whether this view applies to secretory granules in intact PC-12 cells. Three granule proteins were labeled with fluorescent proteins in different colors, and two-color evanescent-field microscopy was used to view single granules during and after exocytosis. Whereas neuro-peptide Y was lost from granules in seconds, tissue plasminogen activator (tPA) and the membrane protein phogrin remained at the granule site for over 1 min, thus providing markers for postexocytic granules. When tPA was imaged simultaneously with cyan fluorescent protein (CFP) as a cytosolic marker, the volume occupied by the granule appeared as a dark spot where it excluded CFP. The spot remained even after tPA reported exocytosis, indicating that granules failed to flatten into the cell surface. Phogrin was labeled with GFP at its luminal end and used to sense the pH in granules. When exocytosis caused the acidic granule interior to neutralize, GFP-phogrin at first brightened and later dimmed again as the interior separated from the extracellular space and reacidified. Reacidification and dimming could be reversed by application of NH(4)Cl. We conclude that most granules reseal in <10 s after releasing cargo, and that these empty or partially empty granules are recaptured otherwise intact.

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Secretory granules are recaptured largely intact after stimulated exocytosis in cultured endocrine cells

Fluid and HCO3− secretion is a vital function of all epithelia and is required for the survival of the tissue. Aberrant fluid and HCO3− secretion is associated with many epithelial diseases, such a...

/pdf/molecular-mechanism-of-pancreatic-and-salivary-gland-fluid-4m4vg56pio.pdf

Molecular Mechanism of Pancreatic and Salivary Gland Fluid and HCO3− Secretion

Neurotransmitter release: fusion or 'kiss-and-run'?

Rapid endocytosis (RE) occurs immediately after an exocytotic burst in adrenal chromaffin cells. Capacitance measurements of endoocytosis reveal that recovery of membrane is a biphasic process that is complete within 20 sec. The ultimate extent of membrane retrieval is precisely controlled and capacitance invariably returns to its prestimulation value. The mechanism of RE specifically requires intracellular Ca2+; Sr2+ and Ba2+ do not substitute, although all three cations support secretion. Thus the divalent cation receptors for RE and exocytosis must be distinct molecules. RE is dependent on GTP hydrolysis; it is blocked by GTP removal or replacement with guanosine 5'-[gamma-thio]triphosphate. In the presence of GTP, multiple rounds of secretion followed by RE could be elicited from the same cell. RE requires participation of dynamin, a guanine nucleotide binding protein, as revealed by intracellular immunological antagonism of this protein. Intact microtubules may be essential, as nocodazole also blocked RE. Whereas anti-dynamin antibodies blocked RE, anti-clathrin antibodies did not, suggesting that clathrin-coated vesicles are not involved in this form of endocytosis. RE may represent the initial step in the rapid recycling of secretory granules in the chromaffin cell.

https://www.pnas.org/content/pnas/92/18/8328.full.pdf

Rapid endocytosis coupled to exocytosis in adrenal chromaffin cells involves Ca2+, GTP, and dynamin but not clathrin

Exocytosis implies the fusion of the membrane of secretion granules with, and the insertion into, the plasmalemma. In non-growing systems such an insertion is temporary in that the inserted membrane is eventually removed. Turnover results indicate that the removed membrane is not destroyed but recycled within the cell and reused. In some systems exocytosis occurs over the entire plasmalemma, while in others it is restricted to discrete regions, characterized by peculiar morphology and composition. Thus the fusion of the two membranes is probably preceded by a recognition step. Structural specializations were detected in interacting granule and plasma membranes by freeze-fracture and surface labelling techniques: arrays of intramembrane particles in protozoans and nerve terminals; clearing of particles and surface antigens in other systems. Direct evidence, obtained in some secretory systems, indicates that after exocytosis the granules and plasma membranes do not intermix, but remain segregated. The subsequent recapture of membrane patches of the granule type (in many systems by means of coated pits and vesicles) could then account for the striking specificity of the recycling process, documented by both composition and structural studies. In different systems the recycling of granule membranes is carried out at greatly different rates. Recent results in the parotid gland and neuromuscular junction indicate that this process is Ca2+-dependent.

Exocytosis and membrane recycling.

In pancreatic acinar cells the intracellular transport of secretory proteins occurs through the interconnexion of distinct membrane-bounded compartments: in series, the rough surfaced endoplasmic reticulum (r.e.r.), the Golgi complex (g.c.) and the secretory (zymogen) granules (z.g.). The latter organelles are able to fuse their membrane with the plasmalemma (pm.) and discharge their content by exocytosis. In order to investigate the mechanisms by which the intracellular transport occurs we have investigated the composition as well as the rate of synthesis and turnover of the various membranes involved in the process. We found that these membranes have distinct differences in chemical composition and in the distribution of enzyme activities and that their rate of turnover is much slower than that of secretory proteins. Furthermore, SDS polyacrylamide gel electrophoresis of doubly labelled membrane proteins revealed that in each of these membranes there is a marked heterogeneity of turnover rates. These data indicate (a) that the membranes participating in the intracellular transport interact with one another in a non-random fashion, (b) that they are not synthesized concomitantly with the secretion products, (c) that membranes are synthesized independently from one another, and (d) that they are re-utilized in several secretory cycles. Consistent with these results, a model explaining the role of cellular membranes in protein secretion is described. It is proposed that the intracellular transport is effected through the specific non-random interaction (fusion-fission) and recycling of the various participating membranes. The possible relevance of these findings to other secretory systems is discussed.

J. Meldolesi

Papers

Exocytosis and membrane recycling.

Membranes and membrane surfaces: Dynamics of cytoplasmic membranes in pancreatic acinar cells