Punctate appearance of dopamine-beta-hydroxylase on the chromaffin cell surface reflects the fusion of individual chromaffin granules upon exocytosis

A secretion from cultured bovine chromaffin cells was stimulated to examine the pattern of exocytotic fusion on the plasma membrane. Confocal microscopy revealed that dopamine-beta-hydroxylase immunofluorescence in intact cells stimulated for 20s with the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium was almost entirely punctate and evenly distributed on the cell surface. The basis for the fine, punctate appearance of dopamine-beta-hydroxylase was investigated. Dopamine-beta-hydroxylase presentation on the surface of permeabilized cells stimulated with 1-30 mu M Ca2+ was punctate and similar to that on the plasma membrane of intact cells. The fluorescence intensities of both surface dopamine-beta-hydroxylase sites and internal chromaffin granules were estimated by computerized digital image analysis. The surface area of punctate surface dopamine-beta-hydroxylase (0.218+/-0.013 mu m(2), mean+/-S.E.M.) is similar to the surface area of a 0.28 mu m diameter chromaffin granule (0.25 mu m(2)). The average fluorescence intensity integrated over the area of the surface spots was 25-30% of the average chromaffin granule intensity, a fraction that is similar to the published values of 40-50% of the dopamine-beta-hydroxylase in the chromaffin granule being membrane bound. The surface density of the spots is consistent with the number of granules undergoing exocytosis. The spots do not tend to be clumped. The key conclusions from this work are that each individual punctate site of dopamine-beta-hydroxylase represents the fusion of a single chromaffin granule and that the distribution of dopamine-beta-hydroxylase spots over the cell surface is extensive and random, suggesting that each individual granule associates with its own release site.