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First published online 20 February 2007
doi: 10.1242/jcs.03406

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Differential dynamics of Rab3A and Rab27A on secretory granules

The Physiological Laboratory, School of Biomedical Sciences, University of Liverpool, Crown Street, Liverpool, L69 3BX, UK

View larger version (60K): [in this window] [in a new window]   Fig. 1. Recruitment of EGFP-Rab3A and EGFP-Rab27A to newly synthesised granules in PC12 cells. The cells were examined 18 hours after transfection. (A-C) Cells after fixation co-immunostained with anti-Rab3A and anti-secretogranin II (anti-SGII). (D-F) Cells co-transfected with plasmids encoding ECFP-Rab27A and EGFP-Rab3A. (G-I) Cells transfected with a plasmid encoding ECFP-Rab27A, which after fixation, were immunostained with anti-SGII. (J-L) Cells cotransfected with plasmids encoding ECFP-Rab27A and prepro-atrial natriuretic factor-EGFP (ppANF-EGFP). Each overlay image on the right of the figure is a composite of green from the left image and red from the centre image. Areas of overlap appear in yellow. Magnified views of areas of colocalisation are shown at the bottom right. Bars, 4 µm.

View larger version (63K): [in this window] [in a new window]   Fig. 2. Improved colocalisation between ppANF-EGFP/EGFP-Rab3A/EGFP-Rab27A and secretory granules over time post transfection. PC12 cells were transfected with ppANF-EGFP (A-C,J-L), EGFP-Rab3A (D-F,M-O) or EGFP-Rab27A (G-I,P-R) and fixed 18 hours or 30 hours following transfection as indicated. After fixation, cells were immunostained with anti-secretogranin II (anti-SGII). Images are composites of green EGFP fluorescence and red anti-SGII immunofluorescence. Areas of overlap appear in yellow and increase from 18 to 30 hours post transfection. Bars, 4 µm. (S) Quantification of the colocalisation of EGFP and SGII. Data are shown for the correlation coefficient as mean ± s.e.m. (n=5).

View larger version (70K): [in this window] [in a new window]   Fig. 3. Appearance of Rab3A and Rab27A on immature secretory granules during granule biogenesis and maturation. (A) PC12 cells were transfected with ppANF-EGFP, EGFP-Rab3A or EGFP-Rab27A and cultured for 18 hours at 20°C to block granule budding at the TGN and then fixed (time 0) or incubated for 30 minutes at 37°C to allow budding of immature granules. (B) PC12 cells were co-transfected with ppANF-EGFP and mRFP-Rab3A or mRFP-Rab27A, cultured for 18 hours at 20°C and then fixed at the indicated times after incubation at 37°C. The images show overlays of ppANF-EGFP fluorescence in green and mRFP fluorescence in red. Expanded inserts are shown for the 10- and 30-minute time points to show lack of colocalisation at 10 minutes and colocalisation at 30 minutes. Bars, 4 µm.

View larger version (34K): [in this window] [in a new window]   Fig. 4. Dynamics of EGFP-Rab3A and EGFP-Rab27A on newly synthesised secretory granules in PC12 cells. (A-D) PC12 cells were transfected to express the indicated EGFP-tagged protein and examined after 18 hours. Regions of interest (ROIs) in each cell were bleached with high intensity laser (arrows) and the fluorescence recovery of these areas was followed over time. (E) Fluorescence in the ROI was recorded over time during low-intensity imaging. The data are shown as mean ± s.e.m. of 14 ARF1-EGFP, 13 ppANF-EGFP, 21 EGFP-Rab27 and 21 EGFP-Rab3A expressing cells. The data were corrected for general photobleaching for each cell at each time point and normalised by setting the initial fluorescence value for each cell to 100. (F) The data were replotted with the initial post-bleach data point set to 0. Bars, 2 µm.

View larger version (28K): [in this window] [in a new window]   Fig. 5. Dynamics of EGFP-Rab3A on newly synthesised and old secretory granules in PC12 cells. PC12 cells were transfected to express the indicated EGFP-tagged protein and examined 18, 30 or 54 hours post transfection. Regions of interest (ROIs) in each cell were bleached with high intensity laser and the fluorescence recovery of these areas was followed over time. Fluorescence in the ROI was recorded over time during low-intensity imaging. The data are shown as mean ± s.e.m. for 32 EGFP-Rab3A expressing cells at 18 hours, 10 at 30 hours and 27 at 54 hours. The data were corrected for general photobleaching for each cell at each time point and normalised by setting the initial fluorescence value for each cell to 100 and with the initial post-bleach data point set to 0.

View larger version (33K): [in this window] [in a new window]   Fig. 6. Inhibition of Hsp90 does not affect EGFP-Rab3A dynamics or exocytosis in PC12 cells. (A) Cells were transfected with 0.5 µg EGFP-Rab3A and at 18 hours post transfection small regions of interest (ROIs) in each cell were bleached with high intensity laser. Fluorescence recovery was recorded over time and corrected to account for bleaching sustained during low-intensity imaging, summed and normalised to the first data point post-bleach. Data are shown from control cells and cells recorded in parallel that had been treated with 10 µM geldanamycin (GA) for 1 hour before bleaching (n=11 for each condition). (B) Secretion of exogenous human growth hormone (hGH) from cells treated with varying concentrations of GA for 1 hour before stimulation was assayed. Stimulated cells were exposed to 300 µM ATP, hGH release over 15 minutes was assayed and expressed as a percentage of total hGH. (C) Representative image of control cells expressing glucocorticoid receptor-EGFP (GCR-EGFP) 18 hours after transfection with 0.5 µg GCR-EGFP showing two adjacent cells in which the GCR-EGFP is either predominantly cytosolic or alternatively nuclear. Bar, 2 µm. (D) Effect of geldanamycin on GCR-EGFP translocation to the nucleus. After transfection with GCR-EGFP, treated cells were exposed to 1 µM dexamethasone on ice for 1 hour or 1 µM dexamethasone on ice for 1 hour with the addition of 10 µM geldanamycin for the final 30 minutes. Following a 20-minute incubation at 37°C and fixation, 100 cells were scored for each condition according to the cytosolic or nuclear localisation of GCR-EGFP Data are expressed as cytoplasmic:nuclear ratios and are representative of two independent experiments.

View larger version (29K): [in this window] [in a new window]   Fig. 7. Effect of activation of exocytosis on secretory granules labelled with EGFP-ppANF, EGFP-Rab3A or EGFP-Rab27A in live cells. Cells were transfected to express EGFP-ppANF, EGFP-Rab3A or EGFP-Rab27A and imaged 18 hours after transfection. Cells showing low levels of fusion protein expression were selected for observation and stimulated by perfusion with 300 µM ATP. (A) Example of the time course of Ca2+ changes from monitoring of X-Rhod fluorescence following ATP stimulation (n=11 cells). (B) Disappearance of a ppANP-EGFP-labelled granule during stimulation. The images shown are sequential frames in which the sudden disappearance of a granule (arrow) can be observed. (C) Effect of stimulation with 300 µM ATP on secretory granules labelled with EGFP-ppANF, EGFP-Rab3A or EGFP-Rab27A as indicated. Images were taken at 19 seconds and 76 seconds (after the elevation in cytosolic Ca2+). Overlay images are shown with t=19 seconds in green and t=76 seconds in red so that granules that disappear between the images appear green. Granules that disappeared in the ppANF-EGFP cell are indicated with arrowheads. Some granules did not disappear but moved during the recording and so appear green in the overlay; an example is indicated by the arrow in the EGFP-Rab3A-transacted cell. Bars, 4 µm. (D) The number of granules that disappeared between the before and after images was identified by the comparison of images at 19 and 76 seconds, quantified and shown as mean ± s.e.m. for eight cells expressing each construct.