Spatial organization of Ca2+ entry and exocytosis in mouse pancreatic beta-cells

Secretion from single pancreatic beta -cells was imaged using a novel techn ique in which Zn2+, costored in secretory granules with insulin, was detect ed by confocal fluorescence microscopy as it was released from the cells. U sing this technique, it was observed that secretion from beta -cells was li mited to an active region that comprised similar to 50% of the cell perimet er. Using ratiometric imaging with indo-1, localized increases in intracell ular Ca2+ concentration ([Ca2+](i)) evoked by membrane depolarization were also observed. Using sequential measurements of secretion and [Ca2+](i) at single cells, colocalization of exocytotic release sites and Ca2+ entry was observed when cells were stimulated by glucose or K+. Treatment of cells w ith the Ca2+ ionophore 4-Br-A23187 induced large Ca2+ influx around the ent ire cell circumference. Despite the non-localized increase in [Ca2+](i), se cretion evoked by 4-Br-A23187 was still localized to the same region as tha t evoked by secretagogues such as glucose. It is concluded that Ca2+ channe ls activated by depolarization are localized to specific membrane domains w here exocytotic release also occurs; however, localized secretion is not ex clusively regulated by localized increases in [Ca2+](i), but instead involv es spatial localization of other components of the exocytotic machinery. (C ) 2001 Academic Press.