Exocytotic exposure and recycling of membrane antigens of chromaffin granules: ultrastructural evaluation after immunolabeling.

Abstract

Abstract. 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 rain 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. SECRETION from adrenal medulla occurs by exocylosis (2, 21,26). This process, which adds membranes of chromaffin granules to the plasma membrane, is followed by endocytosis, as shown in early studies (l l, 15). In 1972 we demonstrated (34) that the synthesis rate of the membrane proteins of chromaffin granules was significantly lower than that of the secretory proteins (chromogranins) of the contents (for similar results in exocrine pancreas see reference 4). Based on this study we proposed that the membranes of chromaffin granules are reused for several secretory cycles (see also the discussion in reference 31). This concept gained considerable support when it was shown that in stimulated endocrine cells exogenous markers were taken up by endocytosis and were subsequently found in newly formed secretory vesicles (see references 5, 8, 9, and 24). These experiments demonstrated recycling of membranes but did not yet establish the specific nature of the recycled membranes. To achieve this, specific antigens of the secretory vesicle membrane have to be followed throughout the exo/endocytosis cycle. Two antigens of the chromaffin granules, i.e., dopamine, 13-hydroxylase and glycoprotein III, proved suitable for such an approach. In a first series of immunofluorescent studies it was established that these granule antigens become exposed on the cell surface of isolated chromaffin cells during exocytotic activity (3,13,18, 19,27). By quantitative evaluation we were able to show that after stimulati...

A Nibbling Mechanism for Clathrin-mediated Retrieval of Secretory Granule Membrane after Exocytosis

“…These data are consistent with a freeze-fracture study that suggested the fused chromaffin granule membrane remains a distinct entity in the plasma membrane (11). Immunoelectron microscopy detected formation of clathrin-coated pits and vesicles containing glycoprotein III within 15 min of stimulation, but the spatial relationship to exocytotic sites could not be determined (8).…”

“…After 6 h, glycoprotein III was again found in large dense core granules. The antigen was also detected in vesicles close to but not within Golgi stacks (8). Second, biotinylation of exposed DBH upon chromaffin granule fusion demonstrated that DBH had not degraded after 24 h and was associated with another granule membrane, protein cytochrome b561 (39).…”

“…The development of methods to isolate gram quantities of bovine chromaffin granule membranes enabled purification of chromaffin granule membrane proteins and the development of complementary antibodies that permitted tracking of specific granule components after fusion. Primary antibodies against the lumenal domain of either dopamine-␤-hydroxylase (7), a major chromaffin granule membrane protein, and glycoprotein III protein (8,9) provided a means to label protein exposed to the extracellular environment upon secretion and then follow its fate. Both proteins appear as puncta in the plasma membrane upon stimulation; dopamine-␤-hydroxylase (DBH) 2 puncta were shown to reflect fusion of individual granules and not aggregation of the protein (10).…”

A Leucine-based Motif Mediates the Endocytosis of Vesicular Monoamine and Acetylcholine Transporters

“…However, endocytosis presumably functions to retrieve LDCV membrane proteins after exocytosis. Indeed, the LDCV proteins glycoprotein III and ICA512 reappear in secretory granules after exposure at the cell surface (13,14).…”

Retrieval and Reuse of Pituitary Secretory GranuleProteins

“…Sporadic evidence of functional recycling (retrieval and reuse) of secretory granule membrane proteins has been collected over the last 2 decades. In chromaffin cells, stimulation of secretion causes internalization of glycoprotein III/clusterin and dopamine ␤-hydroxylase from the plasma membrane and their subsequent re-entry into secretory granules (2)(3)(4). In insulinoma cells, stimulated secretion resulted in the endocytosis of two secretory granule membrane proteins, phogrin and ICA512, and their re-entry into newly forming insulin granules (5,6).…”