Materials and antibodies
Lipofectamine 2000, terminal deoxynucleotidyl transferase, Fura-2 acetoxymethyl ester, and Hoechst 33258 were purchased from Invitrogen; MK801, NBQX, and nimodipine were purchased from Sigma-Aldrich; SNX482 was purchased from Alomone Labs; DL-AP3 was purchased from Tocris Bioscience; bafilomycin A1 and lactacystin were purchased from EMD; chloroquine was purchased from MP Biomedicals; biotin–deoxy-UTP was purchased from TriLink BioTechnologies; and fluorescein-conjugated streptavidin (DTAF-streptavidin) was purchased from Jackson ImmunoResearch Laboratories, Inc. Antibodies to CaV1.2 (1:500; AB5156; Millipore), PIKfyve (1:500; 6C7; Abnova), Myc (1:500; 4A6; Millipore), HA (3F10; Roche), Flag (1:500; M2; Sigma-Aldrich), GAPDH (1:1,000; 6C5; Applied Biosystems), or DsRed (1:500; E64-1077; BD) was used for immunoblot analysis. Antibodies to CaV1.2 (1:100; AB5156), MAP2 (1:500; MAB378; Millipore), Myc (1:500; 4A6), HA (1:500; 3F10), PtdIns(3,5)P2 (1:100; Z-P035; Echelon), or GFP (1:500; 598; MBL) was used for immunocytochemistry.
Cell culture, stimulation, and transfection
HEK 293T and NIH3T3 cells were cultured in Dulbecco's modified minimum essential medium containing 10% FBS, 100 U penicillin and 100 µg streptomycin (P/S), and 2 mMl-glutamine (Q). Neuro2A cells were cultured in minimum essential medium containing 10% FBS and P/S. Primary neuronal cultures were prepared from embryonic day 17–19 Sprague-Dawley rats by dissociating dissected cortices with enzyme solution (Hanks’ balanced salt solution with 600 U papain and 0.32 mg/ml cystein). Neurons were plated on coverslips coated with 20 µg/ml polyornithine and 1.42 µg/ml laminin and maintained in culture in Basal Medium Eagle with 5% FBS, P/S, Q, and 0.6% glucose. For immunocytochemistry, neurons were stimulated with glutamate in Tyrode's solution without Mg (129 mM NaCl, 5 mM KCl, 3 mM CaCl2, 30 mM glucose, 0.1% BSA, and 25 mM Hepes, pH 7.4). Cell lines and cortical neurons were transfected with plasmids using Lipofectamine 2000 according to the manufacturer's instructions. The ratio of plasmids used for specific experiments is given as follows: for surface expression assay, α1C/β1b/α2δ = 2:1:1 () and α1C/β1b/α2δ/shRNA = 2:1:1:2 (); for colocalization assay, α1C/β1b/α2δ/LAMP = 2:1:1:1 () and α1C/β1b/α2δ/LAMP/shRNA = 2:1:1:1:1 (); for coimmunoprecipitation, PIKfyve/CaV1.2 CT = 1:1 (), PIKfyve/α1C/β1b = 2:2:1 (), PIKfyveΔ/CaV1.2 CT = 1:1 (), and PIKfyve/CaV1.2 ΔCT = 1:1 (); for relocalization of the GRAM domain, GRAM/shRNA/PIKfyve = 1:2:2 (); for CaV1.2 degradation assay, PIKfyve/shRNA = 1:1 () and α1C/β1b/α2δ = 2:1:1 (); and for cell death assay, shRNA/PIKfyve = 1:1 ().
GFP-PIKfyve (mouse) and GFP-HA-KV
1.2 constructs were provided by P. Cullen (University of Bristol, Bristol, England, UK; Rutherford et al., 2006
) and L.Y. Jan (University of California, San Francisco, San Francisco, CA; Gu et al., 2003
), respectively. The cDNA for PIKfyve was amplified by PCR from the GFP-PIKfyve construct and cloned into the XbaI sites of pCS4-Myc and pCS4-HA. The pCS4-GFP, pCS4-Cherry, and pCS4-HA-GFP plasmids were constructed by the insertion of the EGFP and Cherry coding sequence into the XbaI site in pCS4 and pCS4-HA, respectively. The Rab5, LAMP1, and MTM1 cDNAs were amplified by PCR from the human ORFeome cDNA library (Thermo Fisher Scientific) and subcloned into the BamHI sites of pcDNA3-GFP, pCS4-GFP, pCS4-Cherry, and pCS4-Myc to generate three different vectors encoding each protein. The GluR2 cDNA was amplified from the human ORFeome cDNA library and subcloned into the XbaI sites of pCS4-HA-GFP. The dihydropyridine-resistant CaV
1.2 α1C, β1b, α2δ, and pEYFP-HA-CaV
1.2 constructs were described previously (Dolmetsch et al., 2001
; Gomez-Ospina et al., 2006
; Green et al., 2007
). The pGW1-Flag-CaV
1.2, pGW-Flag-α2δ, GST–CaV
1.2 CT, and pDEST27-GST-YFP were generated using Gateway technology (Invitrogen). The cDNA for CaV
1.2 CT was amplified by PCR and cloned into the BamHI site of pcDNA3-Flag. Short hairpin oligonucleotides were designed and inserted into the RNAi-Ready pSIREN-DNR-DsRed-Express vector (Takara Bio Inc.) by ligation into the BamHI and EcoRI sites. For specific experiments, DsRed was eliminated from the RNAi-Ready pSIREN-DNR-DsRed-Express vector using the NcoI restriction enzyme. Site-directed mutagenesis was performed using QuikChange (Agilent Technologies) to generate RNAi-resistant kinase-negative PIKfyve (PIKfyve KEres; Sbrissa et al., 2000
). PIKfyve KEres consisted of changing the K1820
to E in the kinase domain. The cDNA from the GRAM domain of myotubularin (residues 8–112) was amplified by PCR from the MTM1 construct and cloned into pcDNA3-GFP.
The primers used were as follows: PIKfyve forward, 5′-ATCTAGAATGGCCACAGATGACAAG-3′; and reverse, 5′-GTCTAGATCAGCAATTCAGATCCAA-3′; PIKfyve FYVE forward, 5′-AGGATCCATGGCCACAGATGACAAG-3′; and reverse, 5′-GGGATCCCTAACTTAAGGCTATTTT-3′; PIKfyve DEP forward, 5′-GGGATCCTATGCTCATTCTACAGAC-3′; and reverse, 5′-AGGATCCTTATGAGAGCTGCTGTCC-3′; PIKfyve Cpn/TCP-1 forward, 5′-GAGATCTATAAGTGATGCCTTCATC-3′; and reverse, 5′-AAGATCTTTATGAGCATCTCATCCC-3′; PIKfyve linker forward, 5′-GAGATCTACTCGAGATTATTTTCCA-3′; and reverse, 5′-AAGATCTTTAGATGAATTCCTCCTC-3′; PIKfyve PIP5K forward, 5′-AGGATCCCGTTCCCTTTCTCACTCA-3′; and reverse, 5′-GGGATCCTCAGCAATTCAGATCCAA-3′; PIKfyve KEres sense, 5′-AGATTCATTCTGGAGCAAATGCCTCGTTTG-3′; and antisense, 5′-CAAACGAGGCATTTGCTCCAGAATGAATCT-3′; CaV1.2 CT-1 forward, 5′-GCCACCATGGTCGGCAAGCCCTCGCAGAGG-3′; and reverse, 5′-CTAGTTGCCAAACAGGCCTCCAGCCCTCCTG-3′; CaV1.2 CT-2 forward, 5′-GCCACCATGCACGTCAGCTACTACCAGAGTG-3′; and reverse, 5′-CTACTGTACCCGGACAGCAGGGGACAAGGG-3′; CaV1.2 CT-3 forward, 5′-GCCACCATGGAGGCAGCATGGAAACTCAGC-3′; and reverse, 5′-CTACTTTAGACATTCCAGATGGAAGGAGGC-3′; CaV1.2 CT-4 forward, 5′-GCCACCATGCGACAAAAGGATCAAGGGGGAG-3′; and reverse, 5′-CTAGCCCCCACTACAGGCTGTGGTCTCCTC-3′; CaV1.2 CT-5 forward, 5′-GCCACCATGAGCAGCATGGCCCGGAGAGCC-3′; and reverse, 5′-CTACAGGTTGCTGACATAGGACCTG-3′; GFP forward, 5′-AATCTAGAATGGTGAGCAAGGGCGAG-3′; and reverse, 5′-GGACTAGTTTACTTGTACAGCTCGTC-3′; Cherry forward, 5′-CTCTAGAGCCACCATGGTGAGCAAGGGCGAG-3′; and reverse, 5′-GACTAGTTTACTTGTACAGCTCGTCCATGCCGCC-3′; Rab5 forward, 5′-AGGATCCATGGCTAGTCGAGGCGC-3′; and reverse, 5′-GGGATCCTTAGTTACTACAACACTGATTCCTGG-3′; LAMP1 forward, 5′-AGGATCCGCCACCATGGCGGCCCCCGGCAG-3′; and reverse, 5′-GGGATCCGATAGTCTGGTAGCCTGCGTGACTCC-3′; MTM1 forward, 5′-AGGATCCGCCACCATGGCTTCTGCATCAACTTC-3′; and reverse, 5′-AGGATCCTCAGAAGTGAGTTTGCACATGGGG-3′; 2× GRAM forward, 5′-GGGATCCGCCACCATGAAATATAATTCACACTCC-3′; and reverse, 5′-AGAATTCAGTAATATCTAGACCATAGGAATT-3′; 2× GRAM forward, 5′-AGAATTCATGAAATATAATTCACACTCCTTG-3′; and reverse, 5′-AGGATCCAGTAATATCTAGACCATAGGAATTTTCTCC-3′; and GluR2 forward, 5′-GTCTAGAGCCACCATGCAAAAGATTATGCATAT-3′; and reverse, 5′-GTCTAGAAATTTTAACACTTTCGATGCCATA-3′.
The oligonucleotides used were as follows: mouse PIKfyve shRNA sense, 5′-GATCCGGACAGGGTTGGATCTGAATTCAAGAGATTCAGATCCAACCCTGTCCTTTTTTACGCGTG-3′; and antisense, 5′-AATTCACGCGTAAAAAAGGACAGGGTTGGATCTGAATCTCTTGAATTCAGATCCAACCCTGTCCG-3′; rat PIKfyve shRNA sense, 5′-GATCCGGACAGGGTTGGATCTCAATTCAAGAGATTGAGATCCAACCCTGTCCTTTTTTACGCGTG-3′; and antisense, 5′-AATTCACGCGTAAAAAAGGACAGGGTTGGATCTCAATCTCTTGAATTGAGATCCAACCCTGTCCG-3′; and shScrambled shRNA sense, 5′-GATCCTGTAGGTCGAGAGCGTAGATTCAAGAGATCTACGCTCTCGACCTACATTTTTTACGCGTG-3′; and antisense, 5′-AATTCACGCGTAAAAAATGTAGGTCGAGAGCGTAGATCTCTTGAATCTACGCTCTCGACCTACAG-3′. RNAi-resistant PIKfyve cDNA was made by introducing silent mutations using conventional PCR. The PCR primers used were as follows: forward, 5′-TCTAGAATGGCCACAGATGACAAGAGTTCCCCGACACTGGACTCTGCTAATGATTTG-3′; and reverse, 5′-TCTAGATTAGCAATTTAAGTCTAGTCCCGTCCAGTGGTCTGGCACCATCAAGAAATACTT-3′.
Cortical neurons plated on 12- or 15-mm coverslips and grown in 12- or 24-well plates were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. The coverslips were washed in PBS once and incubated with a blocking solution (5% BSA in PBS) with (for regular immunocytochemistry) or without (for surface expression assay) 0.4% Triton X-100 for 30 min. The samples were incubated with the indicated primary antibodies for 1 h at room temperature or overnight at 4°C and then washed three times in PBS at room temperature before incubation with secondary antibodies (Alexa Fluor 350 anti–mouse IgG [1:1,000], Alexa Fluor 488 anti–mouse/rabbit IgG [1:1,000], and/or Alexa Fluor 594 anti–mouse/rabbit IgG [1:1,000]) for 30 min at room temperature in the blocking solution. For surface expression assays, the samples were incubated with anti-HA antibodies (3F10) for 1 h at room temperature or overnight at 4°C and then incubated with secondary antibodies (Alexa Fluor 594 anti–rat IgG [1:1000]) for 30 min at room temperature in the blocking solution. Cells were imaged using an epifluorescence microscope (TE2000U; Nikon) equipped with 10× S Fluor 0.5 NA (Nikon), 40× S Fluor 1.30 NA (Nikon), and 60× Plan-Apochromat 1.40 NA (Nikon) objective lenses and a cooled charge-coupled device camera (ORCA-ER; Hamamatsu Photonics) controlled by a computer (Macintosh; Apple) running Open Laboratory software (PerkinElmer).
To measure the surface expression levels, the single regions were defined to encompass the entire cell body and calculate the ratio of HA to YFP fluorescence. To measure the relocalization of the GFP–2× GRAM in neurons, fluorescent images were obtained and analyzed using Open Laboratory software. For each cell, five circular regions of interest (ROIs) were defined around the periphery of the cell nucleus, and a single ROI was defined to encompass the entire cell body. The mean fluorescence intensity calculated from the ROIs around the nucleus was divided by the mean intensity of the entire cell body to generate a perinuclear ratio value (P). The percent change in P in stimulated cells was calculated using the following equation: %P = (PS
, where PS
is the ratio after stimulation and PR
is the ratio in resting cells. Stimulation of cells with glutamate resulted in an increase in puncta that could be detected using the anti-PtdIns(3,5)P2
antibodies. To quantify the number of puncta, the raw fluorescence images were intensity sliced to generate binary images that included only pixels that were part of a puncta. The area covered by the puncta was divided by the total area of the cell to generate a value for the percentage of pixels in a cell that formed part of a puncta. Colocalization of Flag-Myc-CaV
1.2 and LAMP-GFP was evaluated by using line profiles drawn along neuronal dendrites for each type of staining using Igor Pro software (WaveMetrics). The coefficient of correlation between the Flag-Myc-CaV
1.2 and LAMP-GFP line profiles was calculated according to the following equation:
Cells were washed with PBS and then lysed in extraction buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EGTA, 5 µM NaF, 1 µM Na3VO4, 0.5% Triton X-100, and 1 mM dithiothreitol) containing a protease inhibitor cocktail tablet (Roche). The cell lysates were analyzed by SDS-PAGE, transferred to polyvinylidene fluoride, probed with primary antibodies, and detected with HRP-conjugated secondary antibodies and luminol reagent (SuperSignal West Dura Extended Duration Substrate; Thermo Fisher Scientific).
Transfected HEK 293T cells were washed with PBS and then lysed in extraction buffer containing protease inhibitors (see Immunoblot analysis). The cell lysates were centrifuged at 13,200 rpm for 10 min, and the resulting supernatant was subjected to immunoprecipitation with antibodies to 10 µg/ml PIKfyve (6C7), 2.5 µg/ml Myc (4A6), or anti-Flag M2 agarose beads (Sigma-Aldrich) for 3 h at 4°C. The immunoprecipitates were subjected to immunoblot analysis with antibodies to CaV1.2, Myc, Flag, and HA.
Proteomics and mass spectrometry
The protein complex associated with the C terminus of CaV1.2 was purified and analyzed using mass spectrometry three independent times. One immunoprecipitated sample was analyzed in duplicate to generate a total of five mass spectrometry data samples. For each experiment, three 10-cm plates of Neuro2A cells were transfected with GST-YFP or with GST–CaV1.2 CT using Lipofectamine 2000 according to the manufacturer's instructions (24 µg DNA/plate in 1.5 ml of Opti-MEM-I and 60 µl of Lipofectamine in 1.5 ml of Opti-MEM-I). 2 d after transfection, the cells were lysed in lysis buffer (50 mM Tris-HCl, 1% Triton X-100, 150 mM NaCl, and 10 mM EDTA) for 30 min at 4°C with shaking. The lysis buffer was supplemented with an inhibitor cocktail tablet (Roche) and calpain I and II inhibitors. The lysates were centrifuged for 20 min at 14,000 rpm and incubated with 100 µl of glutathione Sepharose beads (GE Healthcare) for 2 h at 4°C. The beads were washed five times in lysis buffer and treated with trypsin before analysis. The peptides were separated using capillary electrophoresis and analyzed by tandem mass spectrometry in the Stanford University Mass Spectrometry facility. Finally, all of the peptide sequences were analyzed using MASCOT software (Matrix Science).
Cortical neurons were loaded with 1 µM Fura-2 for 30 min at 37°C in Tyrode's solution (129 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 30 mM glucose, 0.1% BSA, and 25 mM Hepes, pH 7.4), washed with Tyrode's solution, and placed in a perfusion chamber on the stage of an inverted fluorescence microscope (TE2000U; Nikon). The cells were treated with 50 µM glutamate in Tyrode's solution without Mg (129 mM NaCl, 5 mM KCl, 3 mM CaCl2, 30 mM glucose, 0.1% BSA, and 25 mM Hepe, pH 7.4) for 10 min before imaging in normal Tyrode's solution and then stimulated with high KCl Tyrode's solution (67 mM NaCl, 67 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 30 mM glucose, 0.1% BSA, and 25 mM Hepes, pH 7.4). Imaging was performed at room temperature on an epifluorescence microscope (Eclipse TE2000U; Nikon) equipped with an excitation wheel and an automated stage. Open Laboratory software was used to collect and quantify time-lapse excitation ratio images. Fluorescence images were analyzed using Igor Pro software.
Cell death assay
Cortical neurons were stimulated with 50 µM glutamate for the indicated times and were then incubated for 10 h in the absence of glutamate. Neurons were then fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. The coverslips were incubated in a solution (10 µM Hoechst 33258 and 0.4% Triton X-100 in PBS) for 10 min to stain the nuclei. Fluorescence images were obtained by epifluorescence microscopy, and pyknotic nuclei were counted. For TUNEL assay, neurons were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature and incubated with 0.1% Triton X-100 in PBS for 10 min. The samples were then incubated with TUNEL reaction solution (15 U terminal deoxynucleotidyl transferase, 40 µM biotin–deoxy-UTP, 0.1 M potassium cacodylate, 2 mM CoCl2, and 0.2 mM DTT) for 1 h at 37°C and washed three times in PBS at room temperature before incubation with DTAF-streptavidin (1:800) for 45 min at 37°C. The samples were then incubated in a solution (10 µM Hoechst 33258 in PBS) for 10 min to stain the nuclei.
Online supplemental material
Fig. S1 shows that glutamate stimulation promotes neuronal cell death. Fig. S2 shows that glutamate stimulation promotes CaV
1.2 relocalization and degradation. Fig. S3 shows localization of LAMP, PIKfyve, and the GRAM domain. Fig. S4 shows that knockdown of PIKfyve by PIKfyve shRNAs promotes vacuole formation. Fig. S5 shows that PIKfyve is important for specific channel internalization and degradation. Table S1 lists CaV
1.2-interacting proteins. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200903028/DC1