Granule and plasma membranes do not intermingle during exocytosis. (A) Simultaneously acquired images showing fluorescence of CaG-C18 (green, stains plasma membrane) and SRB (red, stains extracellular spaces). Some preexisting fused granules are apparent in the SRB image, but not in the CaG-C18 image; arrow, acinar lumen. (B) Corresponding images obtained after uncaging CCh. Several newly fused granules are apparent in the SRB image (arrowheads show examples), but are not labeled by CaG-C18. (C) Traces show a representative single event (Inset), and mean measurements (± 1 SEM) of SRB (red trace) and CaG-C18 fluorescence (green) from 18 events (main plot). SRB traces were normalized to the peak, and CaG-C18 traces were normalized to fluorescence measured in the plasma membrane. Asterisks indicate time points where significance was tested for the CaG-C18 data. (D) Control experiment demonstrating mobility of CaG-C18 in the plasma membrane. Images were obtained before (a) and ≈80 s after (b) photobleaching a small region of membrane (arrowed box in a). Graph shows fluorescence recovery monitored from the bleached area, fitted by an exponential (τ = 11.46 s).

Exocytotic events in pancreatic acini induced by photorelease of CCh. (A Left) A group of three cells on the edge of a pancreatic lobule, imaged by two-photon excitation of an extracellular marker dye (SRB). Fluorescence is apparent in the solution surrounding the cells, in the intercellular spaces, and in the acinar lumen (a “tristar” shape in the center). (Right) The same cells a few seconds after a flash of UV light to photolyse caged CCh (≈1 μM). Bright fluorescent spots in the apical domains of the cells mark granules that fused during exocytosis. (B) Superimposed traces show measurements of average fluorescence records over time from five regions of interest placed over event locations. Individual traces were normalized to the peak fluorescence. (C) Distribution of event latencies after CCh photorelease (nine independent preparations and 274 events) expressed as the average number of events per cell, per 10-s time bin, per optical slice. (D) Distribution of granule diameters, measured at full width at half maximum (0.51 ± 0.02 μm, mean ± SEM; n = 41). (E) Example of compound exocytosis. (Upper) A time series of successive events involving successive granule-granule fusion, leading to a chain of three fused granules extending deep into the cell. (Scale bar, 2 μm.) (Lower) A line scan image derived from this record by displaying fluorescence intensity along a line (vertical axis; scale bar, 2 μm) through the three vesicles (Right Upper) as a function of time (horizontal axis; scale bar, 10 s). Graph shows distribution of latencies between first- and second-granule events (n = 36 paired events). Curve is a single exponential with τ = 22s.

Proposed cycle of exocytosis and endocytosis in acinar cells. Cartoon illustrates the following: (A) Granule before fusion; (B) Fused granule, with the fusion pore structure allowing continued aqueous interchange but acting as a barrier to intermingling of granule and plasma membranes; (C) Fusion of a secondary granule onto the persistent ghost of a primary vesicle by means of granule-associated fusion pore proteins; and (D) recycling of granule membrane by pinching back of patches from a primary ghost.

Protracted fusion pore openings. (A) Trace shows the (OG) fluorescence signal during a representative vesicle-fusion event, demonstrating a biphasic signal with a protracted plateau, lasting several minutes. (Inset) Images were derived from two-photon z sections recorded during the plateau phase by deconvolution and 3D reconstruction. Orthogonal views show the vesicle apparently connected to an associated duct by a narrow neck. (scale bar, 0.5 μm) (B) Photobleaching of dye in the granule reveals maintained aqueous continuity between the granule lumen and duct during the plateau phase. The trace illustrates OG fluorescence monitored from a single-fusion event, and corresponding images were captured at times indicated on the trace. A flash of intense light (440-480 nm) was applied (horizontal bar) to photobleach dye in the granule and adjacent duct. The curve is an exponential fit, showing almost complete fluorescence recovery to the prebleach plateau level. (C) Another example, showing fluorescence recovery after bleaching later during the plateau phase of an exocytotic event.

Control experiments using the lipophilic dye FM1-43 demonstrate labeling of granule membrane. (A) Simultaneously acquired images showing fluorescence of the lipophilic dye FM1-43 (green, stains plasma membrane) and SRB (red, stains extracellular space). SRB and FM1-43 were present at all times in the extracellular medium; arrow, acinar lumen. (B) Entry of SRB into vesicles after CCh-induced exocytosis was accompanied by a rise in FM1-43 fluorescence. (C) Traces show a representative single event (Inset) and mean measurements (± SEM: main graph) of SRB and FM1-43 fluorescence from 24 events. Asterisks indicate times when significance was tested for the FM1-43 data. SRB traces were normalized to the peak, and FM1-43 traces were normalized to fluorescence measured in the plasma membrane. (D) Profiles of SRB and FM1-43 fluorescence measured along a scan line drawn through an averaged image of 28 granules. (E) A cluster of fixed, permeabilized acinar cells showing FM1-43 staining of intracellular membranes, but no staining of granule contents (dark spots in the apical domains).

Granule lifetimes and termination of fluorescence signals. (A) Superimposed traces show fluorescence records from nine representative events, displaying diverse granule lifetimes. Traces are normalized to the plateau amplitude and are aligned in time to their rising phases. Owing to the slow imaging rate, the initial peaks were not well resolved. (B) The decline in fluorescence at the end of the plateau phase is associated with shrinkage of the granule. The larger image (SRB fluorescence) is a low-magnification view of an acinar lumen showing a number of CCh-stimulated granule events. The sequence of images on the right show high-magnification views of the granule arrowed at left, at various times (indicated in s from end of plateau) during its lifetime. The graph shows measurements derived from the regions of interest shown in the image at 0 s. Traces are shown beginning from the time when fluorescence began to decline from the plateau and are normalized to the same initial intensities. (C) Lack of lipid interchange at the end of the granule lifetime. (Inset) Graph shows simultaneous measurements of SRB (dotted trace) and CaG-C18 fluorescence (solid trace) obtained as in Fig. 3 during the entire lifetime of an individual vesicle. The main graph shows mean measurements from six events, aligned relative to the onset of decay of SRB fluorescence from the plateau phase. Fluorescence intensities are scaled relative to the SRB fluorescence at the end of the plateau phase, CaG-C18 fluorescence intensities were scaled to the fluorescence measured at the plasma membrane.