Journal: Proceedings of the National Academy of Sciences of the United States of America
Article Title: Imaging direct, dynamin-dependent recapture of fusing secretory granules on plasma membrane lawns from PC12 cells
Figure Lengend Snippet: Ca 2+ -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 (NH 4 ) 2 SO 4 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.
Article Snippet: Suitable preparations were then stimulated by using the indicated [Ca2+ ]free in the presence of 2 mM MgATP, 0.5 mg/ml rat brain cytosol , omitted in the experiments shown in Fig. D , and 5 μM sulforhodamine 101 (Molecular Probes).
Techniques: Microscopy, Marker, Labeling, Expressing, Incubation, Fluorescence, Derivative Assay