Exocytosis, the fusion of a vesicle with the plasma membrane is the principal way a cell can release lipophobic substances to the outside environment. It is probable that the basic machinery of exocytosis is similar across different cell types. But recent studies have shown the process of exocytosis may be differently regulated in different cells. Here we describe novel characteristics of the prolonged (many min) exocytotic events in exocrine cells of the mouse pancreas.
Mice were humanely killed and the pancreas gland removed. The gland was then incubated in collagenase (Worthingto, n CLSPA) for 5-10 min at 37 °C. The tissue was then resuspended in extracellular solution (containing [mM] NaCl 135, KCL 5, MgCl2 1, CaCl2 1, Hepes 10 – pH 7.4 NaOH) and gently triturated to produce a preparation of large clusters of acinar cells. The clusters were then placed on Poly-l-lysine coated coverslips. The cell clusters were imaged using a custom-built 2-photon microscope. Images were then processed using Metamorph software (Universal Imaging).
We imaged lobules and smaller fragments of mouse pancreatic tissue that retained the typical morphology of the intact exocrine glands. Inclusion of a fluorescent probe (Sulphorhodamine B or Oregon Green, Molecular Porbes) in the extracellular bathing medium labelled acinar ducts and the extracellular space between cells, but dyes were excluded from the cell interior. Uncaging of caged CCh with a flash of UV light rapidly evoked fluorescence spots in the apical, but not basal, regions of cells. In many cases we observed examples of compound exocytosis, where secondary vesicles fuse with a primary vesicle and become filled with dye passing from the duct via the primary vesicle. Using fluorescence recovery after photobleaching (FRAP) techniques we show that the fusion pore remains open for protracted periods of time (minutes) to allow free exchange between the aqueous vesicle lumen and the outside. Using lipophillic dyes we show no evidence for interchange of lipid between the plasma membrane and the vesicle membrane during the lifetime of the vesicle. We propose that the unusually long fusion pore lifetime is an adaptation permitting compound exocytosis, whereby the lingering ghost of an empty primary vesicle acts as a conduit through which the contents of a secondary vesicle can be released. The lack of lipid intermixing across the fusion pore may then facilitate the selective recycling of vesicle membrane and preservation of apical membrane integrity.
This work was supported by The Wellcome Trust and NIH