Plasmids and cell culture
Monomeric RFP (mRFP) was ligated into pcDNA3.1(+) (Invitrogen, Carlsbad, CA) to create pcDNA3.1-mRFP. AURKA and derivatives were expressed from pcDNA3.1-mRFP vectors. Amino acid substitution mutations were introduced into wild-type human AURKA cDNA by site-direct mutagenesis, using the QuikChange XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). Primer sequences are available on request. Tetracycline- or doxycycline-inducible AURKA wild type and mutated derivatives were subcloned into pLUT-lentiviral vector (a gift from Alexey V. Ivanov, WVU, MBRCC), allowing expression as RFP-fused chimeras. 293T packaging cells were used to produce virus and infect the recipient cells. AURKA expression was induced by addition of doxycycline in the medium at a concentration of 1 μg/ml for 6–12 h. HEK293, HeLa, and hTERT-RPE1 cells were maintained in DMEM/F12 with 10% fetal bovine serum (FBS) plus penicillin/streptomycin. We transiently transfected HEK293 cells with expression constructs for AURKA using Lipofectamine Plus reagent (Invitrogen), according to the manufacturer's instructions.
For analysis of ciliary disassembly, hTERT-RPE1 cells were plated at 30% confluence in tissue culture plates containing glass coverslips and starved for 24–48 h in serum-free Opti-MEM to induce cilia formation, followed by treatments described in Results. For assessment of AURKA mutants in supporting ciliary disassembly, two independent siRNAs directed to the 3′ untranslated region (UTR) of AURKA were used to deplete endogenous AURKA (in parallel with a control siRNA) during incubation of cells in serum-free medium to induce cilia. Concurrently, we treated cells with doxycycline or vehicle at 60 h post siRNA transfection, allowing 12 h for production of AURKA or mutated derivatives. Cells were then incubated for 2 h in medium containing serum (to induce disassembly) or without serum and then scored.
Transient transfection of siRNAs was carried out using Lipofectamine RNAiMAX or Oligofectamine transfection reagents (Invitrogen). Cells were assayed 48–72 h after transfection. RNA oligonucleotide duplexes targeted to NEDD9 (Hs_NEDD9_2 [SI00657370], CGCTGCCGAAATGAAGTATAA, and Hs_NEDD9_1 [SI00657363]), AURKA (Hs_STK6_5 [SI03114111] and Hs_AURKA_1_HP, TCCCAGCGCATTCCTTTGCAA [SI00053452], and for 3′ UTR targeting, CCCUCAAUCUAGAACGCUA [D-003545-35-0005, siAUR1] and CGGUAGGCCUGAUUGGGUU [J-003545-27-0005, siAUR2]) and HDAC6 (GGGAGGUUCUUGUGAGAUC [J-003499-05] and GUUCACAGCCUAGAAUAUA [J-003499-08]) were purchased from Qiagen (Valencia, CA) or Thermo Scientific (Waltham, MA; HDAC6, 3′ UTR-targeting AURKA), as well as scrambled negative controls. After transfection of siRNAs, degree of depletion of target proteins was determined by Western blot.
Immunofluorescence microscopy and live cell imaging
Cells growing on coverslips were fixed with 4% paraformaldehyde (10 min) and then cold methanol (5 min), permeabilized with 1%Triton X-100 in phosphate-buffered saline (PBS), blocked in 1× PBS with 3–5% BSA, and incubated with antibodies using standard protocols. Alternatively, to maximize clear signals at centrosomes, cells were fixed in cold methanol (−20°C) for 2 min, blocked, and incubated with antibody. Primary antibodies included mouse anti-AURKA (BD Biosciences, San Jose, CA), rabbit anti-AURKA, rabbit polyclonal anti–phospho-AURKA/T288 (Cell Signaling, Beverly, MA), anti–acetylated α-tubulin monoclonal antibody (mAb; clone 6-11B-1; Sigma-Aldrich, St. Louis, MO, and clone K(Ac)40, Biomol International, Enzo Life Sciences, Plymouth, PA), monoclonal anti-calmodulin (Santa Cruz Biotechnology, Santa Cruz, CA, and Millipore, Billerica, MA), and mouse anti–γ-tubulin mAb (Sigma-Aldrich). Secondary antibodies labeled with Alexa 488, Alexa 568, and 4′,6-diamidino-2-phenylindole (DAPI) to stain DNA were from Molecular Probes/Invitrogen (Carlsbad, CA). Confocal microscopy was performed using a Nikon C1 Spectral confocal microscope (Nikon, Melville, NY) equipped with a numerical aperture (NA) 1.40, oil immersion, 63× Plan Apo objective (Nikon). Images were acquired at room temperature using EZ-C1 3.8 (Nikon) software and analyzed using MetaMorph (Molecular Devices, Union City, CA) and Photoshop, version CS2 (Adobe, San Jose, CA) software. Adjustments to brightness and contrast were minimal and were applied to the whole image.
For live cell imaging, transfected HEK293 cells expressing RFP-AURKA, RFP-fused AURKA mutants, or RFP were incubated for 18 h with 2 mM thymidine, washed twice in PBS, then returned to fresh medium and observed for 24 h. Videos of RFP-positive cells were captured with an Inverted Nikon TE300, equipped for phase and epifluorescence with a Ludl MAC2000 x-y stage control and z-axis motor (Nikon) and a Spot RT (Diagnostic Instruments, Sterling Heights, MI) monochrome camera (12-bit images) using a 20× Plan Fluor, NA 0.45, ELWD, WD 7.4 objective. Image processing was performed using MetaMorph software as described in the manufacturer's guidebook. MetaVue (Molecular Devices) software was used for processing and analysis of immunofluorescent images.
Protein expression, Western blotting, and immunoprecipitation
Recombinant hexahistidine wild-type and mutant AURKA were produced in a baculovirus expression system at the Recombinant Protein Facility at the Wistar Institute (Philadelphia, PA). For Western blotting and immunoprecipitation, mammalian cells were disrupted in CelLytic M lysis buffer (Sigma-Aldrich) supplemented with protease and phosphatase inhibitor cocktails (Roche, Basel, Switzerland). Whole cell lysates were used either directly for SDS–PAGE or for immunoprecipitation. Immunoprecipitation samples were incubated overnight with antibody at 4°C and subsequently incubated for 2 h with protein A/G–Sepharose (Pierce, Rockford, IL), washed, and resolved by SDS–PAGE.
For detection of binding with CaM, cell lysates in lysis buffer (PBS with 1% Triton X-100) or purified proteins were diluted in binding buffer (50 mM Tris-HCl, pH 7.6,120 mM NaCl, 2 mM CaCl2, 1% Brij) and incubated with Calmodulin-Sepharose 4B (GE Healthcare, Piscataway, NJ) or control Sepharose for 3–4 h at 4°C as indicated in the figure legends. After washing, beads were boiled in SDS sample buffer and separated by SDS–PAGE followed by Western blotting.
Western blotting was done using standard procedures. Primary antibodies included mouse anti-NEDD9 mAb (clone 2G9; Pugacheva and Golemis, 2005
), anti-AURKA (BD Biosciences, San Jose, CA), anti–phospho-AURKA-T288
(Cell Signaling), polyclonal anti-HDAC6 (Millipore), monoclonal anti-calmodulin (Millipore), and anti–β-actin mAb (AC15; Sigma-Aldrich). Polyclonal anti-AURKA agarose–immobilized conjugate (Bethyl, Montgomery, TX) was used for immunoprecipitations. Secondary anti-mouse and anti-rabbit horseradish peroxidase–conjugated antibodies (GE Healthcare) were used at a dilution of 1:10,000 for visualization of Western blots and blots developed by chemiluminescence using the West-Pico system (Pierce). Image analysis was done using ImageJ (National Institutes of Health, Bethesda, MD), with signal intensity normalized to β-actin or total level of detected proteins.
Histone H3 (Upstate, Charlottesville, VA) was used as substrate for AURKA phosphorylation, using standard methods. Parallel aliquots without [γ-32P]ATP were processed for SDS–PAGE/Coomassie blue staining (Invitrogen). To assess calcium and CaM-dependent Aurora-A activation, we performed the in vitro kinase assay using AURKA purified from baculovirus or according to the protocol described in the Protein expression, Western blotting, and immunoprecipitation section in the presence of 1 μM CaM (Calbiochem, La Jolla, CA) with 1 mM Ca2+ or with 1 mM EGTA.
Cell cycle synchronization
Cells were incubated for 18 h with 2 mM thymidine, washed twice in PBS, and then either assayed directly (for observation at the G1/S boundary) or returned to fresh medium and allowed to grow for 9–12 h to observe synchronized progression to mitosis. Alternatively, to obtain an M-phase cell population, cells were synchronized by 2 mM nocodazole for 16 h, and rounded cells were collected by shakeoff. Collected cells were suspended in fresh DMEM containing 10% FBS and analyzed at 0 and 30 min after release. For all synchronization procedures, the predicted cell cycle progression was confirmed by flow cytometry analysis using Guava (Millipore), with data analyzed using the Guava Cell Cycle Software for the EasyCyte Plus System (Millipore).
Statistical comparisons were made using a two-tailed Student's t test. Experimental values were reported as the means ± SE. Differences in mean values were considered significant at p < 0.05. For multiple group comparison, repeated-measures analysis of variance (ANOVA) was used to analyze the p values and the significance of the difference between the groups. A value of p < 0.0001 for each group with a significant difference is labeled by double or triple asterisks. The ANOVA test was followed by Tukey's test, with a significance level 0.01. All calculations of statistical significance were made using InStat software (GraphPad, San Diego, CA).