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First published online May 4, 2004
doi: 10.1242/10.1242/jcs.01093

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Retention and stimulus-dependent recycling of dense core vesicle content in neuroendocrine cells

Department of Biological Sciences, University of Denver, Denver, CO 80208, USA

View larger version (51K): [in a new window]   Fig. 1. Stimulation-dependent surface labeling and internalization of non-secreted PRL. (A-D) Lactotrophs were stimulated for 5 min at room temperature in high [K+] external solution containing anti-PRL antibodies, then rinsed, and fixed at the indicated time point, permeablized and processed for indirect immunofluorescence. (A) t=0; (B) t=10; (C) t=20; (D) t=80 minutes. (E,F) Surface labeling of non-internalized anti-PRL. Cells were labeled with anti-PRL as above and then fixed and processed for indirect immunofluorescence without permeablization of the plasma membrane. (E) t=0; (F) t=10 minutes. Deconvolved images from the middle of a z-series are shown. DAPI staining shown in blue. Scale bar is 10 µm for all fluorescence images. (G) Gold-labeled secondary antibody was applied to non-permeablized cells fixed at t=0 to reveal anti-PRL in surface-exposed dense cores. Bar, 400 nm.

View larger version (17K): [in a new window]   Fig. 2. Intracellular fusion of vesicles containing internalized anti-PRL. Quantitative fluorescence analysis of data from experiments described in Fig. 1 suggest fusion of the structures containing the internalized anti-PRL. Analysis was performed on complete z-series of images as described in Materials and Methods. (A) The number of structures containing internalized anti-PRL decreased over time. The graph shows the mean (±s.e.m.) number of stained puncta per cell at each time point. The difference between the means at t=0 (100.6±7.0) and t=20 (38.6±4.3) and between t=0 and t=80 (42.0±5.0) were significant (P<0.005; Student's t-test). (B). Relative size of the structures containing internalized anti-PRL over time was estimated from the fluorescence intensity of individual puncta. Histograms of the integrated fluorescence of individual puncta in cells at each time point demonstrate a clear increase in the proportion of brightly stained (large) structures at the 20 and 80 minute time points compared to the 0 time point (means were significantly different; P<0.001; Mann-Whitney U-test). Data are pooled from 10-12 randomly selected cells per time point.

View larger version (118K): [in a new window]   Fig. 3. Internalized anti-PRL was located in structures with multiple dense cores. (A-C) Electron micrographs of cells stimulated in the presence of anti-PRL antibodies and then fixed at the 20 minute time point as described in Fig. 1, were then stained with gold-labeled secondary antibodies. (A) Low power image acquired at 4000 magnification of a section containing six gold-labeled clusters (location highlighted with boxes, box marked `b' is displayed at high magnification in B) demonstrates that the internalized anti-PRL was throughout the cell. (B) The gold-labeled dense cores often clustered together or (C) appeared to have begun to coalesce. (D,E) Control cells processed as for above except that anti-PRL primary antibody was omitted from the stimulation solution. Note the presence of multi-granule clusters containing small cores (arrows). For B-E micrographs were acquired at 30,000 magnification. Bars, 300 nm (A); 200 nm (B-E).

View larger version (75K): [in a new window]   Fig. 4. Ultrastructural identification of internalized FM1-43 stained dense cores. Photoconversion revealed that internalized FM1-43 was located in dense cores that were clustered within a membrane. Dark FM1-43-DAB reaction product (arrows) is clearly distinguished from unlabeled dense core vesicles (arrowheads). FM1-43 was found in vesicles containing cores that appeared to coalesce (A) and in vesicles with multiple distinct cores (B). Bar, 200 nm for both images.

View larger version (90K): [in a new window]   Fig. 5. Vesicles containing internalized anti-PRL recycle in a stimulus-dependent manner. To measure recycling, cells that had internalized anti-PRL in response to a first stimulation were sequentially incubated with Cy2-labeled secondary antibodies in standard external solution for 30 minutes and then Cy3-labeled secondary antibodies in high [K+] external solution for 5 minutes. Anti-PRL that recycled to the cell surface in a basal manner was labeled with Cy2 (green). Granules that recycled back to the cell surface in a stimulus-dependent manner were labeled with Cy3 only (red). (A) Basal recycling (green) measured from t=50 to 80 minutes; stimulus-dependent recycling measured at t=80. (B) Basal recycling (green) measured from t=80 to 110 minutes; stimulus-dependent recycling measured at t=110. (See Materials and Methods for details of the assay and Table 1 for quantitation.) DAPI (blue) was included in the mounting media. The images shown are projection images from the entire z-series. Bar, 10 µm.

View larger version (24K): [in a new window]   Fig. 6. Characterization of the anti-PRL recycling pathway. Cells were stained for internalized anti-PRL at the 0, 10, 20 and 80 min time points as described for Fig. 1. All images are from the t=20 min time point. (A,B) Following fixation and permeabilization, cells were also stained with anti-TGN38 or anti-syntaxin 6. (C) Cell fixed at the t=20 min time point, without permeablization, showing internalized Alexa 488 labeled anti-PRL (green) and DiI-LDL (red) that had been chased into the lysosomes. (D). Cell fixed at the t=20 minute time point that had been internalized Alexa 488 anti-PRL (green) and Alexa 546 transferrin (red). Bar, 10 µm. DAPI (blue) was included in the mounting media. (E) The extent of colocalization between internalized anti-PRL and the respective markers was determined from complete z-series of cells and is expressed as the percent of pixels that were positive for anti-PRL and positive for marker out of the number of pixels that were positive for anti-PRL. Data given are means (±s.e.m.) from 12 to 20 cells per condition and 30-50 z-sections per cell.

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