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First published online 20 January 2004
doi: 10.1242/jcs.00934

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Dense-core granules: a specific hallmark of the neuronal/neurosecretory cell phenotype

¹ Department of Neuroscience, DIBIT, Vita-Salute San Raffaele University, Via Olgettina, 58, 20132 Milan, Italy
² Immunology of Diabetes Unit, IRCCS San Raffaele, Via Olgettina 60, 20132 Milan, Italy
³ Department of Pharmacology, University of Innsbruck, Peter-Mayr-Strasse 1a, A-6020 Innsbruck, Austria

View larger version (10K): [in a new window]   Fig. 1. CgA and CgB levels in clones isolated from wild-type PC12 (A) and in subclones isolated from the PC12-15 clone after stable transfection of empty vector (A) or antisense CgA sequences (B). Scatter plot analysis of CgA and CgB western blot bands decorated by the specific polyclonal antisera, GE-19 and PE-11, respectively. The gels were loaded (25 µg of protein/lane) with total cell lysates. The densitometric CgA and CgB signals, normalised to tubulin, are indicated as percentages of the values in the reference PC12-15 clone. (A) Open squares illustrate the CgA/CgB ratios of 9 PC12 clones isolated from wild-type PC12 (Clementi et al., 1992); closed squares the ratios of 3 subclones isolated from the PC12-15 clone after transfection with the empty vector. (B) Twelve subclones of the reference PC12-15 clone, isolated after stable transfection with antisense sequences against rat CgA. A,L,O are the subclones illustrated in the corresponding panels of Fig. 2.

View larger version (64K): [in a new window]   Fig. 2. Distribution of CgA and CgB in the cells of subclones isolated from the PC12-15 clone after stable transfection with CgA antisense sequences. The confocal images of CgA (GE-19 antiserum, red) are shown in the left panels; those of CgB (monoclonal CIRO, green) are in the middle; the merged images are to the right. Subclone V was transfected with the empty vector; V' is a close-up view of cells showing non-parallel distribution of CgA and CgB in cytoplasmic granules or groups of them. A,L,O were transfected with CgA antisense sequences. Arrows indicate cells very low in CgA where CgB is in contrast at control levels or above. A cell with CgA levels similar to controls and poor in CgB is indicated by the short arrow. Scale bar: 8 µm.

View larger version (67K): [in a new window]   Fig. 3. Intracellular distribution of CgA, CgB, lamp1 and lamp3 in CV-1 fibroblasts (A,B,C); expression of CgA in PC12-64 and CV-1 cells (D). A-C show merged images of CV-1 cells stably transfected with human CgA and dually immunodecorated for the latter granin (monoclonal LK2H10, red) together with (green) lamp1, lamp3 or CgB (monoclonal CIRO antibody), respectively. Notice that CgA and the two lysosomal markers, lamp1 and lamp3, which are largely dissociated from each other in the GC/TGN perinuclear area, do co-localize (yellow) in most of the discrete puncta scattered in the rest of the cytoplasm (A,B). Also note that expression of CgA does not induce any co-appearance of CgB (C). Scale bars: 8 µm. D shows western blots (25 µg/lane) of different cell lines expressing (PC12-15; PC12-64) and not expressing (CV-1) endogenous CgA, some of which were also analysed after stable transfection with human CgA (hCgA PC12-64; hCgA CV-1). The same blot was immunostained with the polyclonal anti-CgA antiserum GE-19 (a); with the monoclonal anti-hCgA antibody LK2H10 (b); and with the polyclonal anti-CgB antiserum PE-11 (c; similar results were obtained with the monoclonal CIRO). Notice that GE-19 recognizes both rCgA (86-100 kDa) and, to a minor extent, also hCgA (70-63 kDa), whereas LK2H10 recognizes hCgA only. In PC12-64 cells expression of the transfected CgA is accompanied by a decrease of the endogenous granin. Mr of protein standards is indicated to the right in kDa.

View larger version (72K): [in a new window]   Fig. 4. Expression, distribution and release pattern of CgA in PC12-15 and in transfected CV-1 cells. A,B show groups of PC12-15 cells immunolabelled for CgA before (A) and after (B) three hours of treatment with cycloheximide (10 µg/ml): the protein synthesis blocker had no appreciable effect on the levels and distribution of the granin. C,D illustrate the same experiment in CV-1 cells transfected with CgA. Before the treatment the cells showed accumulation of the granin in the GC/TGC area and in multiple discrete puncta scattered in the cytoplasm (C). After exposure to cycloheximide the signal was reduced to a faint trace (D). The images shown are 3D reconstructions of focal plane series comprising the whole cell thickness. Exposure conditions were the same for controls and cycloheximide-treated cells. Scale bars: 7 µm. (E) results of pulse-chase experiments showing the release pattern of huCgA from transfected CV-1 cells during a chase time of 3 hours. The percentage of released CgA measured following immunoprecipitation of the protein from the cell media and lysates at the different time-points was calculated over the total CgA (released + intracellularly retained).