Imaging direct, dynamin-dependent recapture of fusing secretory granules on plasma membrane lawns from PC12 cells - PubMed (original) (raw)

Imaging direct, dynamin-dependent recapture of fusing secretory granules on plasma membrane lawns from PC12 cells

Phillip Holroyd et al. Proc Natl Acad Sci U S A. 2002.

Abstract

During exocytosis, secretory granules fuse with the plasma membrane and discharge their content into the extracellular space. The exocytosed membrane is then reinternalized in a coordinated fashion. A role of clathrin-coated vesicles in this process is well established, whereas the involvement of a direct retrieval mechanism (often called kiss and run) is still debated. Here we report that a significant population of docked secretory granules in the neuroendocrine cell line PC12 fuses with the plasma membrane, takes up fluid-phase markers, and is retrieved at the same position. Fusion allows for complete discharge of small molecules, whereas GFP-labeled neuropeptide Y (molecular mass approximately equal 35 kDa) is only partially released. Retrieved granules were preferentially associated with dynamin. Furthermore, recapture is inhibited by guanosine 5'-[gamma-thio]triphosphate and peptides known to block dynamin function. We conclude that secretory granules can be recaptured immediately after formation of an exocytotic opening by an endocytic reaction that is spatially and temporally coupled to soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent fusion, but is not a reversal of the fusion reaction.

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Figures

Fig 1.

Fig 1.

Ca2+-dependent exocytosis of secretory granules in a cell-free preparation, monitored by video microscopy using either acridine orange or NPY-GFP as content marker. Membrane sheets with attached secretory granules labeled by either the acidophilic dye acridine orange (A) or expression of the secretory granule marker NPY-GFP (B) were incubated in a solution containing 500 nM free calcium, 0.5 mg/ml rat brain cytosol, and 2 mM MgATP to stimulate exocytosis. Images were taken every 30 s for 15 min, and the fluorescence intensity of individual granules was measured (see Materials and Methods). (C and D) Exemplary intensity traces of those granules shown in A and B. Intensity values were corrected for local background, normalized to initial intensity, and plotted against time. (C) When acridine orange was used, granules either lost their fluorescence (_F_lost) or were slowly bleached (_F_const). (D) Changes in fluorescence intensity of granules labeled with NPY-GFP. Granules disappeared (_F_lost), became brighter (_F_up), became dimmer (_F_down), or did not change in fluorescence intensity (_F_const). Orange bars, fluorescence intensity after addition of 20 mM (NH4)2SO4 that abolishes the pH gradient across the granule membrane. (E and F) Relative abundance (percent of total) of granules classified according to their fluorescence intensity changes as described above. For acridine orange four membrane sheets were analyzed, and for NPY-GFP 10 membrane sheets were analyzed. (G) Exocytosis of NPY-GFP-labeled secretory granules from membrane sheets derived from intact cells pretreated with high K+ or control buffers for 2 min at 37°C in the presence of 20 μM sulforhodamine. Membrane sheets were prepared immediately after such treatment or after a 30-min recovery at 37°C. Membrane sheets were then stimulated as described above. Values are mean ± SEM.

Fig 2.

Fig 2.

Endocytic capture of the fluid-phase marker sulforhodamine by secretory granules after stimulation of exocytosis and its dependence on calcium. (A) Membrane sheets with docked NPY-GFP-labeled secretory granules were stimulated for exocytosis (as in Fig. 1) in the presence of 5 μM sulforhodamine and imaged as before. Exemplary images from the sequence in the GFP channel are shown. After 15 min, sulforhodamine was washed out, and images were acquired in the red and the green channel. Two red spots are visible that are concentric with secretory granules that have undergone exocytosis (_F_up+lost and _F_up+down in A), whereas an inactive granule (_F_const in A) has no corresponding signal. (B) Calcium dependence of exocytosis, endocytosis, and the individual release modes. For each calcium concentration, five to nine membrane sheets were analyzed. Rates of endocytosis were corrected for random overlap (ranging between 3% and 5%) as described (20). The shaded area indicates calcium concentrations at which the assay allows no evaluation of the data because secretory granules not associated with the membrane sheets but attached to the glass also display changes in fluorescence intensity (triangles, Upper). Values are mean ± SEM. Effect of (C) GTP and GTPγS or (D) a peptide corresponding to the proline-rich domain of dynamin (dynamin PRD peptide) and a GST fusion protein corresponding to the Src homology 3 domain of amphiphysin on endocytosis of secretory granules. Note that experiments shown in D were performed in the absence of cytosol to avoid interference with cytosolic proteins. Values are mean ± SEM (n = 6–11 membrane sheets for each condition).

Fig 3.

Fig 3.

Gallery of electron micrographs showing organelles sequestering HRP after 2 min of high K+ stimulation of intact PC12 cells. Organelles that have taken up HRP appear more electron dense. To quantitate HRP uptake, line scans were performed through the center of the labeled structure, measuring its average intensity (gran int) and the intensity of the surrounding cytosol (cyt int). Secretory granules were identified because of their round shape, size (a diameter of ≈120 nm in this clone; ref. 51), and the presence of a clearly visible dense core devoid of internal membrane. The density was calculated according to: percentage density = (1 − gran int/cyt int) × 100. (A_–_D) Organelles classified as secretory granules (numbers indicate percentage density; see text). (E and F) Double labeling of secretory granules from NPY-GFP-transfected cells containing GFP (ImmunoGold labeling) and trapped HRP after stimulation of intact cells. (G and H) HRP-labeled organelles probably representing endosomes. (Scale bar, 100 nm.) (I and J) Histograms showing the percentage density distribution of secretory granules in stimulated cells in the absence (I, n = 45 granules) and presence (J, n = 101 granules) of HRP.

Fig 4.

Fig 4.

Association of secretory granules with dynamin. (A) Triple-labeling of membrane sheets that were stimulated for exocytosis as in Fig. 1 and then fixed. The position of secretory granules was visualized by NPY-GFP (green channel). Granule recapture was monitored by sulforhodamine uptake (red channel, see Fig. 2_A_). Dynamin (far-red channel, displayed as yellow) was localized by immunocytochemistry using a standard procedure (17) except that all incubation times were shortened by ≈70%. (B) Percentage of granules colocalized with dynamin immunoreactivity. The first column is derived from experiments in which membrane sheets were prepared, immediately fixed, and stained for dynamin. For the second and third columns membrane sheets were prepared and stimulated for exocytosis with 0.5 μM [Ca2+]free, 2 mM MgATP, and 0.5 mg/ml rat brain cytosol in the presence of 5 μM sulforhodamine, followed by washing, fixation, immunolabeling, and imaging. The last two columns are derived from experiments in which intact PC12 cells were prestimulated by high K+ for 2 min in the presence of 20 μM sulforhodamine. This process was immediately followed by generation of membrane sheets, fixation, and immunostaining for dynamin. In all cases association of granules with dynamin was corrected for channel crosstalk as well as for random association. For every condition 12 membrane sheets were analyzed. Values are mean ± SEM.

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