Antibodies, plasmids, recombinant proteins, and mice
The following antibodies were used in this study: mouse monoclonals against AP3-δ (SA4) and SV2 from the Developmental Studies Hybridoma Bank at the University of Iowa (Iowa City, IA), synaptophysin (SY38) from Chemicon International/Millipore (Billerica, MA), and VAMP7-TI (a generous gift of Andrew Peden, Cambridge University, Cambridge, United Kingdom), as well as rabbit polyclonals against PI4KIIα peptide 51-71 PGHDRERQPLLDRARGAAAQ (Uniprot/Swiss-Prot entry Q99M64 [P4K2A_RAT] UniProtKB/Swiss-Prot) generated in this study and VAMP-2 from Synaptic Systems (Göttingen, Germany). Chicken anti-GFP was obtained from Aves Labs (Tigard, OR). Rat monoclonal anti-HA was from Roche (Indianapolis, IN). Glutathione S
-transferase full fusion protein of PI4KIIα was prepared as previously described (Craige et al., 2008
). pEGFP-C2 wild-type rat PI4KIIα and pEGFP-C2-PI4KIIαL60-61A mutant plasmids were previously described (Craige et al., 2008
). C-Terminally FLAG-tagged muted (EXT4795-M14) and N-terminal–tagged FLAG-tagged Dysbindin (EX-Mm12550-M12) were obtained from Genecopoeia (Rockville, MD). Both constructs were in a pReceiver vector backbone, and sequences were independently confirmed. EGFP-GAP43-ps was expressed from pCAG-mGFP Addgene (Cambridge, MA) plasmid 14757 (Matsuda and Cepko, 2007
(STOCK gr +/+ Ap3d1mh
/J, here referred to as Ap3d1mh/mh
) and its control grizzled
(STOCK gr +/+ Ap3d1+
/J, here referred to as Ap3d+/+
) and pallid
/J, here referred to as Pldnpa/pa
) breeding mouse pairs were obtained from Jackson Labs (Bar Harbor, ME) and bred in-house following Institutional Animal Care and Use Committee (IUCAC)–approved protocols. Muted mice and their controls (B6C3 Aw-J/A-Mutedmu
, and CHMU+/mu
; Zhang et al., 2002
) were obtained from Richard Swank (Roswell Park Cancer Institute, Buffalo, NY) and bred in-house. Sandy
mice in C57B6 background were previously described (Cox et al., 2009
). All mice were bred in-house following IUCAC-approved protocols.
Cerebrocortical neurons were cultured from postnatal day 4 (P4) mice (Ap3d1+/+, Ap3mh/mh, CHMU, muted, C57 and sandy) and maintained in neurobasal media containing B27, l-glutamine, and 100 μg/ml penicillin and streptomycin (Hyclone, Logan, UT) at 5% CO2 and 37°C. Dissociated neurons were plated on poly-l-lysine (Sigma-Aldrich, St. Louis, MO)–coated glass coverslips and cultured for 3–7 d in vitro (DIV). Dissociated neurons (2 × 106) were transfected with 3 μg of plasmid DNA using Amaxa nucleofection electroporation (Lonza, Cologne, Germany). Neurons were plated at a density of 6 × 104 cells per well in a 12-well plate.
HEK-293, SH-SY5Y, wild-type and pallidin-deficient mouse skin primary culture fibroblasts, and PC12 cells (American Type Culture Collection, Manassas, VA) were cultured in DMEM supplemented with either 10% fetal bovine serum (FBS) or, in the case of PC12 cells, a mix of 5% FBS plus 10% equine serum, respectively (Hyclone). Media were supplemented with 100 μg/ml penicillin and streptomycin (Hyclone). Cells were maintained at 37°C with 10% CO2. PC12 cells were transfected with 3 μg/μl DNA by nucleofection using Amaxa Cell Line Nucleofector Kit V (Lonza) and plated in PC12 culture media supplemented with 100 ng/ml NGF 2.5S (murine, natural) (Invitrogen, Carlsbad, CA) on Matrigel-coated, glass-bottom culture dishes (MaTek, Ashland, MA). PC12 cells were differentiated for 48–72 h at 37°C with 10% CO2.
We analyzed PI4KIIα distribution using two immunoperoxidase-labeling protocols. First, 11 male and 11 female C57BL6/J mice 3–6 mo old were deeply anesthetized with sodium pentobarbital (1.0 mg/kg) and perfused transcardially with saline, followed by neutral buffered Formalin. Their brains were then postfixed in the same fixative overnight, embedded in paraffin, and sectioned coronally at 6 μm. The sections were mounted and air dried. They were next dewaxed in xylenes, rehydrated in descending concentrations of ethanol, and immersed for 30 min in 5% hydrogen peroxide dissolved in absolute methanol to quench endogenous peroxidase activity. Following a 10-min water rinse, the tissue was subjected to antigen retrieval by boiling in 1 mM EDTA in 0.1 M Tris buffer, pH 8.0, for 10 min (Pileri et al., 1997
). After cooling and rinsing, the sections were reacted immunohistochemically with the PI4KIIα antibody (1:300) generated for this study using a standard avidin/biotin/peroxidase method with nickel sulfate amplification of the 3,3'-diaminobenzidine (DAB) reaction product (Talbot et al., 2004
). Results were analyzed on a BX61 Olympus (Center Valley, PA) research microscope equipped with an Olympus DP71 cooled charge coupled device (CCD) digital camera.
Tissue used in brain slice preparations was obtained from either male or female mice between 6 and 8 wk of age, unless otherwise indicated. Following deep anesthesia with Nembutal, animals were transcardially perfused with fixative (4% paraformaldehyde with 0.1% glutaraldehyde). Brains were postfixed in 4% paraformaldehyde, which was replaced with phosphate-buffered saline (PBS) within 12–18 h. With use of a vibrating microtome, brains were cut into 60-μm-thick sections and stored in antifreeze (0.1 M sodium phosphate monobasic, 0.1 M sodium phosphate dibasic heptahydrate, 30% ethylene glycol, 30% glycerol) at −20°C until immunohistochemical preparation.
The 60-μm-thick brain sections were rinsed in PBS and then incubated in sodium borohydride, followed by 100% cryoprotectant (phosphate buffer 0.05 M, pH 7.4, 25% sucrose, 10% glycerol) for 20 min at −80°C and returned to decreasing amounts of cryoprotectant. Sections were preincubated in PBS with 1% normal horse serum (NHS) and 1% BSA, followed by primary antibody incubation with 1:500 affinity-purified anti-PI4KIIα. Sections were rinsed in PBS and then incubated in a secondary goat anti–rabbit immunoglobulin G antibody solution (1:1000 dilution) (Jackson ImmunoResearch Labs, West Grove, PA) for 90 min at room temperature. Sections were rinsed with PBS and then incubated in a 1:1000 dilution of rabbit peroxidase antiperoxidase (Jackson ImmunoResearch Labs). Sections were rinsed in PBS with a final rinse in Tris buffer (50 mM, pH 7.6) before a 10-min incubation in 0.025% DAB, 1 mM imidazole, and 0.005% hydrogen peroxide in Tris buffer at room temperature. At this point sections were mounted and analyzed by light microcopy or they were further processed for electron microscopy after rinsing sections in phosphate buffer (0.1 M, pH 7.4). Sections were incubated in 1% osmium tetroxide for 10 min and then returned to phosphate buffer before being dehydrated in increasing concentrations of ethanol. At the 70% ethanol incubation, 1% uranyl acetate was added, and sections were incubated in the dark for 35 min. After dehydration, sections were treated with propylene oxide and embedded in epoxy resin overnight (Durcupan ACM; Fluka, Buschs, Switzerland). Next tissues were mounted onto slides and incubated at 60°C for 48 h. Tissue samples of the dentate gyrus were removed from the slides, mounted on resin blocks, cut into 60-nm-thick sections, collected on Pioloform-coated copper grids, and stained with lead citrate for 5 min.
Electron microscopy was performed with a Zeiss EM-10C electron microscope with a CCD camera (DualView 300W; Gatan, Pleasanton, CA). Images were acquired with Gatan Digital Micrograph Software, version 3.10.1 (Gatan). Analysis was focused on the interface between the granule cells and hilar neuropil, where PI4KIIα was concentrated across mice strains. In the electron microscope, only tissue areas from the surface of the blocks with optimal antibody penetration were examined. These sections were scanned at 10,000×, and random fields of view containing at least one asymmetric synapse were photographed at 75,000×. One hundred ninety micrographs from three blocks of tissue in three animals covering 773 μm2
were examined for the control and 172 micrographs from three blocks of tissue in three animals covering 700 μm2
were examined. From this material, the total number of PI4KIIα-immunoreactive or immunonegative terminals forming asymmetric axospinous synapses was counted to estimate the relative percentage of positively labeled terminals. In addition, in wild-type (Ap3d1+/+
) mice, all positive immunoperoxidase-labeled neuronal elements in the field were counted and categorized as axons, spines, dendrites, or glia, based on ultrastructural criteria defined by Peters et al. (1991
). Light microscopy of brain sections was performed with a Leica DMRB microscope with a 10×/0.3 differential interference contrast (DIC) objective (Leica Microsystems, Bannockburn, IL), and images were captured with a CCD camera and the Leica IM50 software (Leica DC500).
Immunofluorescence labeling for confocal microscopy
Confocal microscopy was performed with an Axiovert 100M microscope (Carl Zeiss, Jena, Germany) coupled to Ar and He–Ne lasers. Images were acquired using Plan Apochromat 10×/0.5 dry, 20×/0.5 dry, and 40×/1.3 and 63×/1.4 DIC oil objectives. Emission filters used for fluorescence imaging were BP 505-530 and LP 560. Images were acquired with ZEN and LSM 510 software (Carl Zeiss). Hippocampal-formation 60-μm-thick brain sections were first rinsed with PBS and then incubated in 1% sodium borohydride in PBS for 20 min at room temperature, followed by extensive washing with PBS. Samples were preincubated in a solution of PBS with 5% NHS and 1% BSA and 0.3% Triton X-100 for 60 min at room temperature. Samples were incubated overnight at 4°C in primary antibody solutions of PBS with 1% NHS and 1% BSA and anti-PI4KIIα with anti-synaptophysin (SY38) (dilutions of 1:500 and 1:10,000, respectively). After rinsing in PBS, sections were incubated for 60 min in a secondary antibody PBS solution with 1% NHS and 1% BSA and 1:500 dilutions of the following Alexa-conjugated secondary antibodies: anti–mouse 555 (for anti-synaptophysin) and anti–rabbit 488 (for anti-PI4KIIα) (Invitrogen Molecular Probes, Carlsbad, CA). Following PBS rinses, sections were incubated in cupric sulfate (3.854 wt/vol ammonium acetate, 1.596 wt/vol cupric sulfate in distilled water, pH 5) for 30 min. Sections were washed with PBS and mounted on slides with Vectashield (Vector Laboratories, Burlingame, CA).
HEK293 cells, PC12 cells, and cortical neurons were washed in ice-cold PBS and fixed in 4% paraformaldehyde for 20 min on ice. Cells were then incubated in blocking solution (2% BSA plus 1% fish skin gelatin plus 15% horse serum plus 0.02% saponin in PBS) for 30 min at room temperature. Next cells were incubated with antibody for 30 min at 37ºC, rinsed, and then incubated with secondary antibody diluted in block for 30 min at 37ºC. Cells were then rinsed and mounted with Gelvatol and sealed. Cortical neurons were selected from a field of neurons using the following criteria. Neurons were chosen where a single neuron could be discerned from neighboring neurons, where the morphology met the criteria for the proper development in vitro (Goslin and Banker, 1989
), and only chosen based on the staining of the control vesicle marker illuminating the entire neuron. Micrographs were analyzed by creating a region of interest (ROI) outlining only the axon or only the cell body and then determining total number of fluorescent pixels in that specific ROI for the VAMP2 channel and the PI4KIIα channel.
Live imaging of PC12 cells expressing PI4KIIα-GFP, PI4KIIαL60-61A-GFP, or EGFP-GAP43-ps was performed on an A1R Laser Scanning Confocal Microscope (Nikon, Melville, NY) equipped with a hybrid scanner, Perfect Focus, and an environmental chamber for regulation of temperature to 37°C and 10% CO2. Confocal images with a Z-step of 1μm were captured for 10 min (no delay) with an Apo total internal reflection fluorescence 60×/1.49 oil differential interference contrast (DIC) objective on NIS-Elements AR 3.1 (Nikon) software. Neurite tips of PC12 cells were photobleached for 2 s at 30% laser power. Images were captured for 15 s (no delay) before photobleaching and for 15 min every 5 min after photobleaching, followed by 2 h every 15 min. PC12 cell imaging was performed in Hank's balanced salt solution minus phenol red and NaHCO2 (Sigma-Aldrich) and supplemented with 10% Donor Equine Serum (Hyclone), 5% fetal bovine serum (Hyclone), and 100 ng/ml NGF 2.5S. Imaris 6.3.1 (Bitplane, St. Paul, MN) and ImageJ 1.41 (National Institutes of Health, Bethesda, MD) software were used for image analysis. For FRAP experiments an ROI representing neurite tips was selected, and fluorescence intensity was measured using ImageJ and normalized to a second ROI in the cell body to compensate for photobleaching due to imaging. Voxel fluorescence intensity was measured in neurites and cell bodies of PC12 cells using Imaris software.
Synaptosomes were prepared according to Nagy and Delgado-Escueta (1984
) from 4-wk-old mice. Briefly, mice were anesthetized by CO2
and brains quickly transferred to ice cold PBS. Tissue was homogenized by 16 strokes of a Potter-Elvehjem homogenizer at 800 rpm in 0.32 M sucrose, 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 0.5 mM EDTA supplemented with Complete antiprotease inhibitor (Roche). Homogenates were spun at 1000 × g
for 10 min, and S1 supernatants were further sedimented at 12,000 × g
for 20 min. This P2 pellet was resuspended in 8.5% Percoll (Sigma-Aldrich) and then loaded on a discontinuous gradient comprised of 10 and 16% Percoll. Gradients were spun at 15,000 × g
for 20 min.
PC12 cell microvesicles were prepared as described (Clift-O'Grady et al., 1998
). Briefly, cells were lifted from culture dishes with PBS plus 5 mM EDTA on ice. Cells were spun at 800 rpm for 5 min and resuspended in bud buffer (38 mM potassium aspartate, 38 mM potassium glutamate, 38 mM potassium gluconate, 20 mM 4-morpholinepropanesulfonic acid, pH 7.2, 5 mM reduced glutathione, 5 mM sodium carbonate, 2.5 mM magnesium sulfate) and spun again for 5 min at 800 rpm. Cells were then passed through the cell homogenizer (Isobiotec, Heidelberg, Germany) for 16 passes. Cell homogenates were spun for 5 min at 1000 × g
and supernatant resolved 10–45% sucrose gradient. Gradient were spun in a SW55 rotor for 1 h at 116,000 × g
(Lichtenstein et al., 1998
To assess low-affinity interactions between PI4KIIα, AP-3, and BLOC-1 subunits, we performed cross-linking in intact cells with DSP (Craige et al., 2008
; Salazar et al., 2009
; Zlatic et al., 2010
). Briefly, SHSY5Y cells stably transfected either with FLAG-dysbindin or FLAG-muted were placed on ice, rinsed twice with PBS, and incubated either with 10 mM DSP (Pierce, Rockford, IL) or as a vehicle control DMSO, diluted in PBS for 2 h on ice. Tris, pH 7.4, was added to the cells for 15 min to quench the DSP reaction. The cells were then rinsed twice with PBS and lysed in buffer A (150 mM NaCl, 10 mM HEPES, 1 mM ethylene glycol tetraacetic acid, and 0.1 mM MgCl2
, pH 7.4) with 0.5% Triton X-100 by incubation for 30 min on ice. Cells were scraped from the dish, and cell homogenates were centrifuged at 16,100 × g
for 10 min. The clarified supernatant was recovered, and at least 500 μg was applied to 30 μl of Dynal magnetic beads (Invitrogen) coated with antibody and incubated for 2 h at 4°C. In some cases, immunoprecipitations were done in the presence of the antigenic peptide as a control. The beads were then washed four to six times with buffer A with 0.1% Triton X-100. Proteins were eluted from the beads either with sample buffer or by 2 h of incubation with 200 μM antigenic peptide (either PI4KIIα peptide 51-71 or 3× FLAG peptide) on ice. Samples were resolved by SDS–PAGE and contents analyzed by immunoblot.
Experimental conditions were compared with the nonparametric Wilcoxon–Mann–Whitney rank sum test or one-way analysis of variance, Dunnett's multiple comparison using Synergy KaleidaGraph, version 4.03 (Reading, PA) or StatPlus Mac Built5.6.0pre/Universal (AnalystSoft, Vancouver, Canada). Data are presented as boxplots displaying the four quartiles of the data, with the “box” comprising the two middle quartiles, separated by the median. The upper and lower quartiles are represented by the single lines extending from the box.