A nibbling mechanism for clathrin-mediated retrieval of secretory granule membrane after exocytosis

Abstract

Clathrin-mediated endocytosis is the major pathway for recycling of granule membrane components after strong stimulation and high exocytotic rates. It resembles "classical" receptor-mediated endocytosis but has a trigger that is unique to secretion, the sudden appearance of the secretory granule membrane in the plasma membrane. The spatial localization, the relationship to individual fusion events, the nature of the cargo, and the timing and nature of the nucleation events are unknown. Furthermore, a size mismatch between chromaffin granules (∼300-nm diameter) and typical clathrin-coated vesicles (∼90 nm) makes it unlikely that clathrin-mediated endocytosis internalizes as a unit the entire fused granule membrane. We have used a combination of total internal reflection fluorescence microscopy of transiently expressed proteins and time-resolved quantitative confocal imaging of endogenous proteins along with a fluid-phase marker to address these issues. We demonstrate that the fused granule membrane remains a distinct entity and serves as a nucleation site for clathrin- and dynamin-mediated endocytosis that internalizes granule membrane components in small increments.

FIGURE 1.

Co-localization of chromaffin granule membrane proteins after fusion. Cultured bovine chromaffin cells were stimulated for 30 s with 56 mm K⁺ at 34 °C. The solution was replaced with buffer containing 5.6 mm K⁺ and either immediately placed on ice (0 °C, A–C) or incubated for an additional 5 min at 34 °C (D–F) before being placed on ice (Protocol 1, see “Experimental Procedures”). All cells were then incubated with antibodies to the lumenal domains of three granule proteins (A and D, DBH; B and E, VMAT2; C and F, synaptotagmin 1 (syn)) for 60 min on ice and then processed and imaged by confocal microscopy. Yellow arrowheads indicate instances of co-localization of the three proteins. G, unstimulated cells were processed for DBH as in A. Scale bars = 2 μm.

FIGURE 2.

Clathrin light chain-GFP accumulates at sites of fusion within 20 s. Chromaffin cells transiently expressing NPY-mCherry and clathrin light chain-GFP were stimulated with 56 mm K⁺ and imaged by TIRFM at 10 Hz. Shown are surface plots (rendered by ImageJ) for a region of interest indicating a secretory granule immediately before (A) and after (B) fusion. Surface plots for clathrin fluorescence are shown just before fusion (C) and 25 s later (D), when clathrin accumulated near the site of granule disappearance. E, shown are plot profiles of fluorescence intensities through the clathrin spot immediately before granule fusion (as seen in C), 6 s post-fusion (when clathrin begins to accumulate), and 25 s post-fusion (as in D).

FIGURE 3.

Multiple rounds of clathrin light chain-GFP accumulation and disappearance after fusion. Multiple rounds of clathrin accumulation and disappearance were often observed at fusion sites during live-cell TIRF imaging. An example of such a cycle is shown here. Chromaffin cells transiently expressing NPY-mCherry and clathrin light chain-GFP were stimulated with 56 mm K⁺ and imaged by TIRFM at 10 Hz. Plot profiles of fluorescence intensities through an accumulating clathrin punctum were generated in ImageJ. Panel A shows the initial accumulation of clathrin immediately after granule fusion. The center of the fusing granule is indicated by the line at 0.5 μm. Clathrin begins to accumulate (arrows) in the punctum on the right at 5.8 s and reaches an initial peak 11.8 s post-fusion. B, after peaking, clathrin declines (downward arrows), reaches a minimum at 14.8 s, and then regains peak values 2 s later (upward arrows). In C, the clathrin intensity of the punctum on the right peaked at 16.8 s post-fusion (last time point in B) and was maintained for an additional 3.4 s before its disappearance 500 ms later (downward arrow). Clathrin again began to accumulate within 100 ms (first upward arrow) and had regained much of its previous intensity 4.1 s later. Note that there were clathrin intensity changes at 0.25 μm that were not well synchronized with the clathrin punctum at 0.75 μm.

FIGURE 4.

Co-localization of clathrin light chain, DBH, and dynamin2. Chromaffin cells were incubated ± 56 mm K⁺ for 15 s at 34 °C. The incubation solution was removed, and cells were placed on ice and incubated with antibody to the lumenal domain of DBH for 15 min. Cells were subsequently permeabilized with ice-cold methanol before incubation with antibodies to clathrin light chain and dynamin2. Scale bar = 2 μm.

FIGURE 5.

Time course of loss of exposed DBH and VMAT2 from the plasma membrane. Cells were incubated with ± 56 mm K⁺ for 15 s at 34 °C. The solution was replaced with buffer containing 5.6 mm K⁺, and the cells were maintained at 34 °C for times ranging from 0 to 30 min, after which they were placed on ice, incubated with antibodies to the lumenal domains of DBH (A) and VMAT2 (B) for 60 min, fixed, and then imaged. A, 12–14 cells, averaging 600–800 DBH puncta, were analyzed for each time point. For unstimulated cells, there were 15 cells with 417 puncta. B, 10–12 cells, averaging 126–166 VMAT2 puncta, were analyzed for each time point. C, average number of DBH puncta/10 μm plasma membrane for the time course shown in panel A.

FIGURE 6.

Model of a nibbling mechanism for clathrin-mediated endocytosis. Upon stimulation, the membrane of fused chromaffin granules inserts into the plasma membrane. Clathrin adaptors are then rapidly recruited to sites of fusion by phosphatidylinositol-4,5-diphosphate (PIP₂) and granule membrane proteins (e.g. synaptotagmin-1, VMAT2) with adaptor binding motifs. Subsequent binding of clathrin permits the formation of clathrin-coated vesicles, whose scission requires dynamin GTPase activity. Because the typical clathrin-coated vesicle (90-nm diameter, 0.025-μm² surface area) is too small to internalize the entire chromaffin granule membrane patch (0.282 μm² surface area) as a unit, the process is repeated (nibbling), and the patch is internalized incrementally.

FIGURE 7.

Dynamin inhibitor dyngo 4a, but not its inactive congener dyngo8a, blocks nibbling. Chromaffin cells were preincubated for 30 min with 20 μm dyngo 4a, 20 μm dyngo8a, or vehicle (DMSO, 0.2%) in serum-free medium before incubation ± 56 mm K⁺ for 20 s at 34 °C. The solutions were replaced with 5.6 mm K⁺, and the cells were incubated for 15 min at 0 or 34 °C in the continuing presence of inhibitors. All the cells were then placed on ice and incubated with antibody to the lumenal domain of DBH for 60 min, fixed, and then imaged. There were 19–33 cells analyzed per group with 1150–1250 puncta averaged per time point. The active dynamin GTPase inhibitor dyngo 4a (A) completely blocked the loss of DBH from the surface, whereas the inactive congener dyngo8a (B) had no effect on DBH internalization.

FIGURE 8.

Internalized puncta contain much less DBH immunoreactivity than do initial surface puncta. Chromaffin cells were incubated ± 56 mm K⁺ for 30 s at 34 °C. The cells were then chilled on ice and incubated with anti-DBH for 30 min at 0 °C (Protocol 2). After the labeling with anti-DBH, the cells were rinsed, and one group was warmed to 34 °C for 15 min to permit endocytosis (B), whereas the other was maintained on ice (A). The cells were then fixed and imaged. Scale bars = 2 μm. C, histograms of the fluorescence intensities of the surface and endocytosed DBH are shown. Initial surface puncta (on ice): n = 11 cells, 620 puncta; internalized DBH at 34 °C: n = 8 cells, 634 puncta. The fluorescence intensities were unsaturated.

FIGURE 9.

Secretion increases co-localized uptake of Alexafluor^TM-cadaverine and DBH. A–F, chromaffin cells were incubated ± 56 mm K⁺ for 30 s at 34 °C followed by a 30-s incubation in 5.6 mm K⁺-containing solution to allow for rapid endocytosis. Cells were then incubated for 15 min at 34 °C in buffer with 5.6 mm K and 0.5 mm Alexafluor^TM-cadaverine (A and C) and anti-DBH (B and D) (Protocol 3). Cells were then placed on ice, fixed with 2.5% glutaraldehyde, and permeabilized for 7 min with ice-cold methanol before the addition of secondary antibody. Cells were imaged by confocal microscopy. Unstimulated (A and B); stimulated (C and D). Scale bars = 2 μm. E, the number of Alexafluor^TM-cadaverine puncta/1000 pixels (5625 μm²) was determined for unstimulated and stimulated cells. n = 14–20 cells/group. F–H, shown are frequency distributions of Alexafluor^TM-cadaverine-containing vesicles with or without DBH in stimulated and unstimulated cells. Unstimulated: n = 19 cells, 439 puncta; stimulated: n = 11 cells, and 695 puncta, 444 with DBH and 251 without DBH.

FIGURE 10.

Clathrin light chain is associated with a subset of Alexafluor^TM-cadaverine- and DBH-containing vesicles. Chromaffin cells were incubated ± 56 mm K⁺ for 30 s at 34 °C and incubated for 30 s with 5.6 mm K⁺ buffer followed by 5 min at 34 °C with 0.5 mm Alexafluor^TM-cadaverine and anti-DBH. The cells were then placed on ice, fixed with 2.5% glutaraldehyde, and permeabilized with ice-cold methanol before the addition of secondary antibody against clathrin light chain and imaging by confocal microscopy. n = 13 cells, all DBH- and Alexafluor^TM-cadaverine-containing puncta; n = 921, DBH-, Alexafluor^TM-cadaverine-, and clathrin light chain-containing puncta, n = 134.

FIGURE 11.

Size of clathrin-coated endocytic vesicles in resting and stimulated chromaffin cells. Chromaffin cells were incubated ± 56 mm K⁺ for 60 s at 34 °C and immediately placed on ice and rinsed before fixation for transmission electron microscopy. A, shown are diameters of vesicles with typical clathrin coats in unstimulated and stimulated cells. The mean measured diameters of the unstimulated (n = 24) and stimulated (n = 32) coated vesicles were 77 ± 3 and 87 ± 5 nm, respectively. More than 20 cells in each group were examined. See “Experimental Procedures” for an estimation of the true value of vesicle diameters in thin sections. B–D, shown are electron micrographs of a coated vesicle in an unstimulated cell (B), a budding coated vesicle in a stimulated cell (C), and a large coated vesicle in a stimulated cell (D), all indicated by arrows. CG identifies one of several chromaffin granules in the images. Scale bars in B–D = 100 nm.