Reagents and plasmids
Nocodazole was from Calbiochem, and thapsigargin was from Alexis Biochemicals. STIM1 antibodies were from ProSci (C-terminus; Product 4119) and Sigma (N-terminus; Product S6072). The Orai1 antibody was from Sigma. The eYFP, α-tubulin, and β-actin antibodies were from AbCam. The siRNA targeted to human Orai1 had the sequence cccuucggccugaucuuuaucgucu and the control siRNA was SiControl from Dharmacon. cDNAs encoding eYFP-tagged human STIM1 (eYFP-STIM1) and eYFP-tagged human STIM2 (eYFP-STIM2) were obtained from Dr. Tobias Meyer, Stanford University, and mutations of eYFP-STIM1 were generated using the QuikChange Site-Directed Mutagenesis kit (Stragagene). Untagged human Orai1 was purchased from Origene and CFP-tagged human Orai1 was constructed as previously described.37
Cell culture and transfections
HEK293 and HeLa cells (both from ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum and 2 mM glutamine in a humidified CO2 incubator at 37° C. Transient cDNA transfections were carried out on cells plated to 90% confluency using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions, and transfected cells were used in experiments 48–72 hours later. siRNA transfections were similarly carried out using the HiPerFect reagent (Stratagene), and transfected cells were used 48 hours later.
Most immunoprecipitation analyses of STIM1 phosphorylation were carried out using HeLa cells because we found that non-mitotic HeLa cells remained well attached to the culture dish during collection of mitotic cells, allowing for collection of relatively pure mitotic samples. In contrast, it was more difficult to obtain mitotic HEK293 cell samples for biochemical analyses without significant contamination of non-mitotic cells. Most of our functional analyses employed HEK293 cells to take advantage of more efficient expression of co-transfected proteins.
Intracellular Ca2+ measurements
measurements were carried out as previously described.38
Briefly, cells were loaded with the Ca2+
sensitive dye fura-5F and were maintained in HEPES-buffered saline solution (HBSS; in mM: 120 NaCl, 5.4 KCl, 0.8 MgCl2
, 11 glucose, and 20 HEPES, pH 7.4) at room temperature throughout the course of the experiments. Fluorescence values at excitation wavelengths of 340 and 380 nm were measured consecutively at 6 second intervals for 20–30 individual cells on a single coverslip using a microscope-based digital imaging system (Intracellular Imaging), and data representing relative intracellular Ca2+
changes are plotted as 340/380 fluorescence ratios. Cells expressing eYFP-STIM1 or mutants thereof were selected at the beginning of the experiments based on eYFP fluorescence when excited at 477 nm.
Confocal microscopy was carried out using a Zeiss LSM 510 laser scanning system and a 63X oil-immersion lens (NA 1.4). For the mitotic image shown in , a stack of 128 images was deconvolved using Huygens software based on a theoretical point-spread function. For fixation, cells plated on LabTek chamber slides (Nalge Nunc) were rinsed in phosphate buffered saline (PBS; in mM: 137 NaCl, 2.68 KCl, 1.47 KH2PO4, 14.9 Na2HPO4, pH 7.4), fixed for 1 minute in 0.25% gluteraldehyde, lysed for 1 minute in Karsenti’s extraction buffer (in mM: 80 Pipes, 1.0 MgSO4, 5.0 EGTA, 0.5% Triton X- 100), post-fixed in gluteraldehyde for 10 minutes, and quenched in 1 mg/ml Na- borohybride for 10 minutes. Fixed cells were incubated with primary antibodies diluted in PBS containing 0.1% Tween-20 and 3.0 mM NaN3 (PBS-TA) for 1 hour at 37° C, washed in PBS-TA, and incubated with secondary antibody (Alexa Fluor 633 goat anti-mouse; Invitrogen). Following further washes, cells were mounted using ProLong Gold with DAPI reagent (Invitrogen). For live cells, cells were maintained in HBSS at room temperature throughout the course of the experiments.
Whole-cell currents were measured in cells bathed in HBSS at room temperature using the patch-clamp technique. The intracellular pipette solution contained in mM 145 cesium methanesulfonate, 20 BAPTA, 10 HEPES, and 8 MgCl2 (pH 7.2 with CsOH). The pipette solution also contained 25 μM inositol 1,4,5-trisphosphate (IP3; Sigma) to rapidly deplete intracellular Ca2+ stores upon break-in. Voltage ramps (+100 to −100 mV) of 250 ms were recorded every 2 seconds from a holding potential of 0 mV, and currents were acquired using pCLAMP 10 software (Axon Instruments). All currents were normalized based on cell capacitance, and leak currents were taken immediately after break-in before Icrac activation. Solutions were applied using a gravity-based multibarrel focal perfusion system (Perfusion Pencil, Automate Scientific).
Immunoprecipitation and Western blotting
Cells were lysed in RIPA buffer (in mM: 50 Tris HCl, 150 NaCl, 1 EDTA, 1 phenylmethylsulfonyl fluoride, 1% v/v NP-40, and 0.25% w/v sodium deoxycholate) supplemented with 1X Complete Mini Protease Inhibitor (Roche Applied Sciences) and 1X Halt Phosphatase Inhibitor Cocktail (Thermo Scientific). Protein concentrations were determined using the DC protein assay kit (BioRad). For immunoprecipitations, lysates containing equal protein amounts were pre-cleared for 1 hour with Protein A/G beads (Thermo Scientific), incubated with primary antibody overnight, and incubated with Protein A/G beads for an additional 4 hours, all at 4° C. Beads were then washed 3 times with RIPA buffer, and proteins were eluted in Laemmli sample buffer containing 5% β-mercaptoethanol for 5 minutes at 95° C. For Western blotting, protein lysates diluted in Laemmli sample buffer containing 5% β-mercaptoethanol or eluates from immunoprecipitations were electrophoresed in 6% or 10% gels and transferred to PVDF membranes. All antibody dilutions and washes were carried out in Tris-buffered saline (TBS; in mM: 137 NaCl, 19 Tris HCl, 2.7 KCl, pH 7.4) containing 0.1% Tween-20 (TBS-T). Membranes were blocked in 3% BSA in TBS-T for 1 hour at room temperature, incubated with primary antibodies overnight at 4° C, and incubated with secondary antibodies (horseradish peroxidase-linked anti-mouse or anti-rabbit; GE Healthcare) for 45 minutes at room temperature. Blots were developed with ECL Plus (GE Healthcare) and exposed to film. Stripping was carried out using Restore reagent (Thermo Scientific). For quantification of band intensities, films were scanned and analyzed by densitometry using Photoshop software.
In Vitro Cdk1 Kinase Assay
Crude membrane fractions were prepared by scraping HEK293 cells into hypotonic buffer (in mM: 10 Tris-HCL, 10 NaCl, 1.5 MgCl2, 1 phenylmethylsulfonyl fluoride, pH 7.5) containing 1X Complete EDTA-free Protease Inhibitor (Roche) followed by homogenization in a Dounce homogenizer. Intact cells and nuclei were removed by centrifugation at 1,000 g for 5 min, and the supernatant was then centrifuged at 25,000 g for 30 min. Membrane pellets were resuspended in Cdk1 kinase buffer (in mM: 25 Tris-HCl, 10 MgCl2, 5 β-glycerophosphate, 0.1 Na3VO4, 2 dithiothreitol, 0.2 ATP, pH 7.5) by sonication. Recombinant Cdk1/cyclin B (Cell Signaling Technology) was added at a concentration of 200 ng per 100 μl reaction volume, and reactions were incubated at 37 °C overnight. Samples were then processed for Western blotting.
In-gel digestion with either trypsin or GluC, nanoLC-ESI-MS/MS, automated database searching, and manual spectral interpretation were performed essentially as previously described.39
In addition to traditional collision induced dissociation, electron transfer dissociation (ETD) was also employed for MS/MS experiments. ETD settings included the use of fluoranthine as the electron donor with negative ion source settings that included a 150 eV ionization energy and a 100 msec accumulation time. To enrich for phosphopeptides, metal oxide affinity chromatography was performed using TiO2
tips (Glygen Corp.) using essentially the manufacturer’s recommended protocol.
Proliferation Rate and Cell Cycle Analysis
For analysis of proliferation rates, equal numbers of eYFP-STIM1 or 482STOP and CFP-Orai1 co-transfected cells were plated, and on each day for three days cells were trypsinized and counted using a hemocytometer. The same populations of cells were then analyzed using a LSR II flow cytometer and FACSDiva software (BD Biosciences) to determine the proportion of CFP and eYFP double positive cells. eYFP fluorescence was determined by excitation with a 488 nm laser and a 530/30 nm emission filter, and CFP by excitation with a 405 nm laser and a 525/50 nm emission filter. Gates for eYFP and CFP positive cells were established using untransfected cells. A total of 10,000 viable cells were analyzed per sample, and the proportion of double positive cells was multiplied by the total number of cells obtained by counting to determine the total number of double positive cells per sample. The total number of double positive cells on day 3 was divided by that on day 1 to obtain the proliferation rate. For cell cycle analysis, trypsinized cells were fixed in 70% ethanol at 4 °C overnight, pelleted, and re-suspended in propidium iodide (PI) solution (20 μg/ml PI, 10 Units/ml Rnase in PBS) for 20 min. Cells were then analyzed by flow cytometry to initially identify CFP and YFP positive cells as described, followed by cell cycle analysis. DNA content was determined using PI fluorescence with an excitation at 488 nm and 575/26 nm emission on a double discrimination dot plot based on PI area versus width (to exclude cell aggregates). Data were then modeled using Modfit LT software (Verity Software House) to obtain proportions of cells at specific cell cycle stages.
Cells were arrested in mitosis by treating logarithmic phase populations of asynchronous cells with 1.67 μM nocodazole for 12–16 hours. This resulted in approximately 70–80% mitotic cells based on spherical morphology.23
For live-cell experiments, unless otherwise indicated, mitotic cells were then detached by gentle aspiration and plated onto poly-L-lysine (0.01%; Sigma) coated coverslips for 30 minutes. All solutions in these experiments contained 1.67 μM nocodazole to maintain mitotic arrest. For experiments with interphase cells, unless otherwise indicated, flat, well-spread cells were selected from populations of asynchronous cells. For biochemical analyses, nocodazole-arrested mitotic cells were detached in PBS, collected by centrifugation, and lysed in RIPA buffer.
Statistical analyses were carried out using GraphPad Prism Software.