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doi: 10.1242/10.1242/jcs.00140

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Identification of a novel sorting determinant for the regulated pathway in the secretory protein chromogranin A

¹ Department of Medicine, University of California at San Diego, La Jolla, CA 92093, USA
² Center for Molecular Genetics, University of California at San Diego, La Jolla, CA 92093, USA
³ Veterans Affairs San Diego Healthcare System, San Diego, CA 92161, USA

View larger version (37K): [in a new window]   Fig. 3. Schematic representation of chimeric photoproteins (CgA fragment-GFP fusion proteins) designed to study CgA trafficking to the dense core secretory granules of PC12 cells. Construction of plasmids encoding GFP fusion proteins was performed as described in Materials and Methods. Plasmid numbering refers to the position of the 3' end of the CgA fragment subcloned, within the original human CgA cDNA (GenBank NM_001275). For example, in pCMV-CgA805-EGFP, the 3' end of the subcloned CgA fragment (at amino acid +224) is located at 805 bp in the original cDNA. Amino acid residue positions are indicated by numbers alongside the chimera. Proteins are drawn proportional to scale.

View larger version (69K): [in a new window]   Fig. 1. Subcellular distribution of chimeric photoproteins in PC12 cells. 48 hours after transfection with expression plasmids encoding GFP, SgP-GFP, SgP-CgA-GFP or VAMP2-pHluorin, aldehyde-fixed cells were examined by deconvolution microscopy. Optical sections along the Z axis were acquired with increments of 0.2 µm using 60x or 100x oil immersion objectives (1.4 NA). GFP was excited at ex 490±10 nm and imaged at em 528±238 nm. Nuclei were visualized with Hoechst 33342 (ex 350 nm/em 461 nm). Data were processed to generate 3D/volume (a1, b1, c1, d1) or section (0.2 µm; a2, b2, c2, d2) views. Bars, 5 µm. Postnuclear supernatants prepared from [3H]-L-norepinephrine-labeled cells transiently expressing GFP (a3), SgP-GFP (b3), SgP-CgA-GFP (c3) or VAMP2-pHluorin (d3), were centrifuged to equilibrium on sucrose density gradients (0.6-2.2 M). Fractions were collected from the bottom of the gradient and assayed for [3H]-L-norepinephrine, sucrose concentration and detection of GFP fluorescence.

View larger version (30K): [in a new window]   Fig. 2. Biochemical detection of SgP-CgA-GFP in sucrose density gradient fractions and in the secreted medium from PC12 cells. (A) Subcellular localization of SgP-CgA-GFP in PC12 cells. Postnuclear supernatants prepared from [3H]-L-norepinephrine-labeled PC12 cells transiently expressing SgP-CgA-GFP were centrifuged to equilibrium on sucrose density gradients. Fractions were assayed for sucrose concentration, GFP fluorescence and scintillation counting. (B) Immunochemical detection of the SgP-CgA-GFP chimera in sucrose density gradient fractions. Gradient fractions were concentrated using SepPak C-18 cartridges and processed for immunoblot using an anti-GFP antibody. (C) Immunochemical detection of stimulated release of SgP-CgA-GFP or proANF-GFP. Regulated secretion from SgP-CgA-GFP- or proANF-GFP-expressing PC12 cells was triggered by 2 mM BaCl2. Extracellular media were concentrated using C-18 SepPak cartridges and processed for immunoblot using an anti-GFP antibody. Immunoreactivity was visualized by chemiluminescence.

View larger version (33K): [in a new window]   Fig. 4. Immunochemical detection of the expression of GFP fusion proteins in PC12 cells. Total PC12 cell lysates were prepared 72 hours after transfection with expression plasmids encoding the GFP fusion proteins indicated. Expression of GFP fusion proteins was evaluated by SDS-PAGE followed by immunoblotting using an anti-GFP antibody. Immunoreactivity was visualized by chemiluminescence. Estimation of size (Mm, calculated molecular mass, based upon primary structure of the chimera; Ma, apparent molecular mass by SDS-PAGE): SgP-CgA1-16-GFP (28.6 kDa; 27 kDa); SgP-CgA1-37-GFP (30.9 kDa; 29.2 kDa); SgP-CgA1-39-GFP (31.2 kDa; 29.5 kDa); SgP-CgA1-77-GFP (35.6 kDa; 35 kDa); SgP-CgA1-115-GFP (39.9 kDa; 40 kDa); SgP-CgA1-153-GFP (43.8 kDa; 48.5 kDa); SgP-CgA1-168-GFP (45.6 kDa; 52.5 kDa); SgP-CgA1-209-GFP (49.9 kDa; 62 kDa); SgP-CgA1-224-GFP (51.6 kDa; 71 kDa); SgP-CgA-GFP (75.8 kDa; 105 kDa); SgP-CgAC17E-GFP (75.8 kDa; 105 kDa); SgP-CgA233-439-GFP (50.3 kDa; 56 kDa).

View larger version (95K): [in a new window]   Fig. 5. Subcellular distribution of the CgA fragment-GFP chimeric photoproteins in PC12 cells. 48 hours after transfection with expression plasmids encoding the GFP fusion proteins indicated, aldehyde-fixed cells were examined by deconvolution microscopy. GFP was excited at ex 490±10 nm and imaged at em 528±238 nm. Nuclei were visualized with (blue) Hoechst 33342 (ex 350 nm/em 461 nm). Optical sections along the Z axis were acquired with increments of 0.2 µm using 60x or 100x oil immersion objectives (1.4 NA). Data were processed to generate 3D/volume views of the GFP chimera distributions. Bars, 5 µm.

View larger version (46K): [in a new window]   Fig. 6. The Golgi apparatus. Analysis for colocalization between CgA fragment-GFP chimeric photoproteins and ß1,4-galactosyltransferase cyan fluorescent protein (GALT-CFP) chimera, a Golgi apparatus marker. (A) 48 hours after transfection with expression plasmids encoding SgP-GFP (a), CgA1-77-GFP (b), CgA1-115-GFP (c), CgA1-224-GFP (d), SgP-CgA-GFP (e), CgAC17E-GFP (f) fusion proteins, together with the expression plasmids encoding for the targeting sequence of ß1,4-galactosyltransferase fused to CFP (GALT-CFP), aldehyde-fixed cells were examined by deconvolution microscopy. GFP was excited at ex 490±10 nm and imaged at em 528±38 nm; CFP was excited at ex 436±10 nm and imaged at em 465±30 nm. Optical sections along the Z axis were acquired with increments of 0.2 µm using a 100x oil immersion objective (1.4 NA). Data were processed to generate combined 3D/volume views of the GFP and CFP chimera distribution. Representative 0.2 µm XY optical sections acquired in the middle region of the cells, and corresponding three-color mask images generated by Nearcount Image Analyzer are shown. The extent of cyan (CFP) and green (GFP) fluorescent signal 3D colocalization in cells within the boxed areas (crop region) was assessed using Nearcount Image Analyzer software. (B) Nearcount image quantification of GFP (green) chimeras and CFP-Golgi (blue) 3D colocalization. `Overlap' is defined is defined as the total number of blue (CFP) pixels within the crop region that are above a threshold value and have an above-threshold green (GFP) pixel at the same location (same pixel). `Nearness' is defined as the total number of blue (CFP) pixels within the crop region that are above a threshhold value and have an above-threshhold green (GFP) pixel within a 3D 2x2x2 pixel window centered on a green (GFP) pixel. Areas of overlap or nearness are displayed in red (`mask'). Bars, 5 µm.

View larger version (64K): [in a new window]   Fig. 7. Secretory granules. Analysis for colocalization between CgA fragment-GFP chimeric photoproteins and dopamine ß-hydroxylase (DßH), a dense-core secretory (chromaffin) granule marker. (A) 48 hours after transfection with expression plasmids encoding SgP-GFP (a), SgP-CgA1-77-GFP (b), SgP-CgA1-115-GFP (c), SgP-CgA1-224-GFP (d), SgP-CgA-GFP (e), SgP-CgAC17E-GFP (f), SgP-CgA233-439-GFP (g) or proANF-GFP (h), cells were aldehyde-fixed and processed for immunocytochemistry. Cells were incubated with a mouse anti-rat DßH followed by Alexa-Fluor-594 (red)-conjugated goat anti-mouse. GFP was excited at ex 490±10 nm and imaged at em 528±38 nm; the red Alexa Fluor 594 conjugate was excited at ex 555±28 nm and imaged at em 580±20 nm. Nuclei were visualized with Hoechst 33342 (ex 350 nm/em 461 nm). Optical sections along the Z axis were acquired with increments of 0.2 µm with a Delta Vision imaging system using a 100x oil immersion objective. Volume views (left panel) of the GFP chimeras and Alexa-Fluor-594-conjugated antibody distributions are shown. The extent of intracellular 3D colocalization of red (Alexa 594) and green (GFP) fluorescent signals was assessed using Nearcount Image Analyzer software. Representative 0.2 µm XY optical sections acquired in the middle region of the cells, and corresponding three-color mask images generated by Nearcount Image Analyzer are shown. (B) Nearcount image quantification of GFP (green) chimeras and DßH (red) 3D colocalization. The area of analysis (crop region) was defined as a rectangle that included the whole cell. `Overlap' is defined as the total number of red (Alexa 594) pixels within the crop region that are above a threshold value and have an above-threshold green (GFP) pixel at the same location (same pixel). `Nearness' is defined as the total number of red (Alexa 594) pixels within the crop region that are above a threshold value and have an above-threshold green (GFP) pixel within a 3D 2x2x2 pixels window centered on a green (GFP) pixel (i.e. ±2 pixels). At the total magnification used here, a chromaffin granule spans 9-15 pixels (as determined by CgA-GFP or proANF-GFP). Areas of overlap or nearness are displayed in yellow (`mask'). Bar, 5 µm.

View larger version (69K): [in a new window]   Fig. 8. Immunochemical analysis of CgA domain-GFP fusion protein release upon stimulation of regulated secretion. PC12 cells were transiently transfected with expression plasmids encoding the GFP fusion proteins indicated. 48 hours after transfection, regulated secretion was triggered by 2 mM BaCl2 for 15 minutes. Extracellular media were collected and concentrated using C-18 SepPak cartridges, and processed for immunoblot using an anti-GFP antibody. Immunoreactivity was visualized by chemiluminescence.