Chromaffin Cell Preparation
Bovine adrenal chromaffin cells were isolated as described previously (Meunier et al., 2005
) and cultured in DMEM supplemented with 10% fetal bovine serum. The cells were maintained at 37°C in 5% CO2
incubator for at least 24 h before use in experiments.
PC12 cells and human embryonic kidney (HEK) cells growing on (poly-lysine–treated coverslips were transfected using Lipofectamine LTX (Invitrogen, Carlsbad, CA) according to manufacturer's instructions. Cells were processed for immunocytochemistry or time-lapse confocal microscopy imaging 15–24 h after transfection.
Cells plated on poly-d
-lysine–coated coverslips for 15–24 h, were briefly washed with buffer A: 145 mM NaCl, 5 mM KCl, 1.2 mM Na2
, 10 mM glucose, and 20 mM HEPES-NaOH, pH 7.4, and fixed with 4% paraformaldehyde (PFA) for 30 min. In some experiments, transfected cells were pretreated with stated concentrations of wortmannin (Sigma-Aldrich, New Castle, NSW, Australia) for 25–30 min before fixation. For digitonin permeabilization, cells were incubated with 20 μM digitonin in KGEP buffer: 139 mM K-glutamate, 5 mM glucose, 5 mM EGTA, and 20 mM piperazine-N
′-bis(2-ethanesulfonic acid) (PIPES)-NaOH, pH 6.7, containing 2 mM free Mg2+
and 2 mM ATP in the continuing presence of 2xFYVE-glutathione transferase (GST) (17 μM; a kind gift from G. Schiavo, Cancer Research UK) for 10 min. The cells were briefly washed in KGEP buffer, fixed with 4% PFA, and processed for immunocytochemistry. Coverslips were blocked using blocking buffer (3% normal horse serum, 0.5% bovine serum albumin (BSA), and 0.05% Triton X-100) before incubation with the primary antibodies (anti-GST polyclonal antibody; Sigma-Aldrich), anti-synaptotagmin 1 (M48; a kind gift from G. Schiavo; Matthew et al., 1981
), anti-early endosomal antigen 1 (BD Biosciences, San Jose, CA) and anti-P18 (a kind gift from S. Tooze, Cancer Research UK, London, United Kingdom). After washing with phosphate-buffered saline (PBS), coverslips were incubated with Alexa 488- and Alexa 546-conjugated secondary antibodies (Invitrogen), washed with PBS, and mounted (Prolong Gold; Invitrogen). Images were examined by confocal microscopy (LSM 510 Meta; Carl Zeiss, Jena, Germany). Quantification of the degree of colocalization was carried out using the color range tool in the LSM510 software. Briefly, immunopositive puncta were selected (pixel intensity >100 arbitrary units) solely in the green channel as regions of interests (by switching off the red channel). Twelve to 132 regions of interests were selected per cell for at least nine cells. By adjusting the red channel (pixel intensity >100 arbitrary units), the organelles showing a high degree of colocalization in the merged image were scored over those that did not colocalize and are expressed as a percentage.
Subcellular Fractionation of Chromaffin Cells
Chromaffin granules were prepared as described previously (Bon et al., 1990
; Vitale et al., 1996
; Meunier et al., 2005
; Osborne et al., 2007
). Briefly, bovine adrenal medulla were taken and finely chopped in 0.32 M sucrose (10 mM Tris, 1 mM EGTA, pH 7.4, and protease inhibitor [Roche Products, Dee Why, NSW, Australia]). Medullas were homogenized and centrifuged at 800 × g
for 15 min. After filtering of the supernatant, filtrates were centrifuged at 12,000 × g
for 20 min. Pellets were resuspended in 0.32 M sucrose, layered on a 1.6 M sucrose gradient, and centrifuged 1 h at 127,000 × g
. Pink chromaffin granules pellet fraction collected and analyzed for its concentration by Bradford assay.
Chromaffin Granules Kinase Assay and Lipid Analysis
Frozen purified chromaffin granules were slowly thawed on ice. In each assay, 300 μg of chromaffin granules were used. Chromaffin granules were diluted in KGEP buffer containing 2 mM MgCl2
, protease inhibitor with or without bovine adrenal medulla cytosol (500 μg) (Panaretou and Tooze, 2002
), 2 mM ATP, and indicated concentrations of CaCl2
, pH 6.8. After 30 min at 37°C, the reactions were stopped by mixing with 1 M HCl. Lipids were extracted by addition of chloroform (CHCl3
):methanol (MeOH) (1:1, vol/vol) and 2 M KCl to the acidified samples. The lower organic phase was collected and dried under a low stream of N2
. Dried lipids were resuspended in chloroform and spotted on oxalate-treated thin layer chromatography (TLC) plates. For control experiment, synthetic phosphoinositides PtdIns(3)P, phosphatidylinositol-4-phosphate [PtdIns(4)P], phosphatidylinositol-5-phosphate [PtdIns(5)P], PtdIns(3,5)P2
, phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2
], and phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3
] (diC16, acid form) (Cell Signals, Columbus, OH) were dried and resuspended in chloroform and spotted on oxalate-treated TLC plates. Lipids were separated on TLC plates in a pre-equilibrated tank containing CHCl3
OH (30%) (9:7:1:1, vol/vol). TLC plates were dried overnight before processing for overlay assay.
For overlay assay, TLC plates were blocked in 2.5% BSA (>98% fatty acid free; Sigma-Aldrich) in PBS for 2 h at room temperature. 2xFYVE-GST (250 ng/ml) was added to the blocking buffer for an additional 2 h. Plates were extensively washed with PBS and incubated with anti-GST (1 in 5000) in 1% BSA for 1 h. After washes with PBS, the plates were incubated with the HRP-labeled secondary antibodies for 1 h in 1% BSA, washed in PBS, and signals were detected by SuperSignal West Pico (Pierce Chemical, Rockford, IL).
Image Analysis of 2xFYVE-Enhanced Green Fluorescent Protein (EGFP) Vesicular Staining during Wortmannin Treatment
To analyze changes in fluorescence intensity of 2xFYVE-positive vesicles and cytosol before and after wortmannin treatment (15–20 min), three or more fluorescent vesicular structures and cytosolic regions were selected (avoiding puncta disappearing out of the optical section in the time lapse), and a region of interest outlined to measure their fluorescence intensity over time by using the LSM 510 software. The loss in 2xFYVE-positive organelles staining after wortmannin treatment was calculated based on the percentage change in the ratio of the organelles' fluorescence over cytosolic fluorescence.
Time-Lapse Confocal Microscopy Imaging
Confocal microscope (LSM 510 Meta; Carl Zeiss) laser power was set <4% for 488 nM argon laser and <20% for 543 nM HeNe laser; pinhole was 173 μm and optical section generally was kept at 1 μm by using a 63× water immersion objective (numerical aperture = 0.93). The pinhole was chosen to give rise to 1.7-μm confocal z-sections. The frequency of acquisition was two frames per minute over the incubation period indicated in the figure. Nicotine (100 μM) was applied by injection using a Hamilton syringe as indicated in the figures.
Four-Dimensional (4D) Confocal Analysis of 2xFYVE-EGFP Staining during Nicotine Treatment
Labeled organelles that remained in the same optical section throughout the duration of imaging were selected for intensity analysis. The changes in intensity of 2xFYVE-EGFP fluorescence from regions of interest in the cytosol and identified organelles were followed over time before and after nicotine treatment as described in the previous section but using maximum projection of z-stacks. The values were recorded and expressed as percentage increase of the normalized initial fluorescence intensity of 2xFYVE-EGFP.
Calcium Sensor Fluo-4/Acetoxymethyl Ester (AM) Experiment
PC12 cells were transfected with 2xFYVE-cherry for 15–24 h before use in experiment. Cells were briefly washed with Hanks' balanced salt solution (HBSS): 137 mM NaCl, 5.4 mM KCl, 0.25 mM Na2HPO4, 0.44 mM KH2PO4, 1.3 mM CaCl2, 1 mM MgSO4, and 4.2 mM NaHCO3, pH 7.4, before being loaded with 2 μM Fluo-4/AM (Invitrogen) plus 1 μM Pluronic acid for 45 min at room temperature in HBSS. Dye-loaded cells were washed with HBSS for an additional 30 min at room temperature, and changes in fluorescence intensity were monitored by confocal microscopy.
Time-Lapse Confocal Microscopy Imaging of Digitonin-permeabilized Cells
Transfection of chromaffin cells were performed with an Amaxa Rat Neuron Nucleofactor kit (Quantum Scientific, Murarrie, QLD, Australia) according to the manufacturer's instructions. After 48–72 h, cells were washed with the indicated KGEP buffer and visualized by confocal microscopy. For time-lapse movies, cells were captured at 3 s/frame over 7-min periods. Cells were digitonin (2.5 μM)-permabilized in KGEP buffer containing Mg-ATP in the presence or absence of free Ca2+. The image analysis was performed as described above. Values were expressed as the ratios of the peak fluorescence intensity over the initial fluorescence intensity.
Chromaffin cells were fixed in 8% paraformaldehyde/0.1% glutaraldehyde in PBS and then processed for frozen sectioning. Processing, sectioning, and labeling for PtdIns(3)P with the 2xFYVE-GST was carried out as described previously (Gillooly et al., 2000
). Wild-type yeast or yeast strains lacking Vps34 were labeled in parallel to determine the specificity of the labeling for PtdIns(3)P, as in a previous study (Gillooly et al., 2000
Knockdown of PI3K-C2α by Small Interfering RNA (siRNA) in PC12 Cells
Two independent PI3K-C2α double-stranded siRNA (805, 807) clones (Ambion, Austin, TX) were used. The sequence of the forward oligonucleotide for clone 805 is 5′-CCUGCUGUUCAAAGAAGCCtt-3′. The complementary sequence for clone 805 is 5′-GGCUUCUUUGAACAGCAGGtc-3′. The sequence of the forward oligonucleotide for clone 807 is 5′-GGAGGUUCUACAGAAUAAUtt-3′. The complementary sequence for clone 807 is 5′-AUUAUUCUGUAGAACCUCCtc-3′. The negative control Silencer siRNA (provided by the manufacturer) composed of a 19-base pair scrambled sequences with 3′dT overhangs. Dried oligonucleotides were resuspended in nuclease-free water and stored at −20°C ready for use. PC12 cells were transfected twice 24 and 48 h after plating of cells with siRNA805 (20 nM), siRNA807 (20 nM), or negative control siRNA (20 nM) using Lipofectamine-LTX (Invitrogen). Forty-eight hours after the second transfection, 2xFYVE-EGFP was coexpressed in PC12 cells and examined after 15–24 h. Knockdown efficiency was analyzed by Western blot using antibodies against PI3K-C2α and β-actin (Sigma-Aldrich) as loading control.
Subcloning of the 2xFYVE in PmCherry Vector
PmCherry C1 vector (kindly provided by R. Tsien, University of California, San Diego, CA) and 2xFYVE-EGFP were both digested using Nhe1/BSrG1. Pmcherry insert was ligated in with 2xFYVE-C2 vector by using DNA ligase (Roche Products).
Chromaffin Granule Recruitment Assays
Purified chromaffin granules were prepared from bovine adrenal medulla as described previously (Bon et al., 1990
; Vitale et al., 1996
; Meunier et al., 2005
; Osborne et al., 2007
). We incubated 125 μg of granules per assay with 0.5 μg of 2xFYVE-GST for 30 min at 37°C in KGEP buffer containing 2 mM free Mg2+
and 2 mM ATP and the indicated concentrations of free calcium calculated using the WEBMAXC program (Patton et al., 2004
). Reactions were stopped by diluting with 750 μl of ice-cold KGEP buffer and chromaffin granules isolated by centrifugation for 45 min at 100,000 × gav
(TLA100.4). Pellets were resuspended in Laemmli sample buffer containing β-mercaptoethanol and heated for 3 min at 95°C before separation by SDS-polyacrylamide gel electrophoresis (PAGE) and analysis by Western blotting with anti-GST antibodies (Sigma-Aldrich).
Analysis of Phosphoinositide Kinase Activity
HEK293 cells were transiently transfected with a cDNA construct encoding EE-tagged PI3K-C2α-pcDNA3.1 by using calcium phosphate. After 2 d, cultures were lysed with 10 mM Tris-HCl, pH 7.6, 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 100 μM Na3VO4, 1% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride (lysis buffer) and clarified by centrifugation at 4°C (14,000× g for 20 min). Recombinant enzyme was isolated by immunoprecipitation using anti-PI3K-C2α antisera and protein A-Sepharose. Immune complexes were washed with lysis buffer and twice with 139 mM potassium glutamate, 5 mM EGTA, and 20 mM PIPES, pH 6.8 (KGEP buffer). Recombinant enzyme was then aliquoted and incubated at 4°C with KGEP buffer, pH 7.4, containing 2 mM ATP, 2 mM MgCl2, 10 μg of PtdIns, and varying concentrations of CaCl2. After addition of radiolabeled [γ32P]ATP (2 μCi) samples were incubated for 30 min at room temperature. Reactions were terminated by addition of 200 μl of 1 M HCl and phosphoinositides were extracted with 400 μl of chloroform:methanol (1:1, vol/vol). Reaction products were fractionated by TLC by using oxylate pretreated Silica Gel 60 plates and a 4 M NH4OH:chloroform:methanol (11:50:39) solvent system. Radiolabeled PtdIns(3)P was quantified using a PhosphorImager (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom), and data are expressed as phosphorimager units.
Data analysis was carried out using Student's t test. Experiments were performed at least three times. Values are expressed as mean ± SEM, and data are considered significant at *p < 0.05, **p < 0.01.