Cell culture and transfection
Mouse inner medullary collecting duct cells (IMCD3; from ATCC, Manassas, VA, USA) were maintained in DMEM/F12 medium (Cellgro, Manassas, VA, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA), non-essential amino acids (BioWhittaker, East Rutherford, NJ, USA) and penicillin (100 U/ml)/streptomycin (100 µg/ml) (GIBCO, Carlsbad, CA, USA) at 37°C in a humidified incubator under 5% CO2. To induce cilium formation, we cultured IMCD3 cells to confluence, followed by an additional 2 days in serum-deprived medium.
Stable and transiently transfected IMCD3 cells, containing either shRNAi sequences directed against Ahi1 or a scramble sequence, were analysed. The RNAi targeting sequences for mouse Ahi1 (RNAi 1: 5′-GATTTCTCACCCAATGGTAAA-3′; RNAi 2: 5′-TGAAATTCCTTCTGGACGTTT-3′; RNAi 3: 5′-GAAACTGTCACAGAGGTGATA-3′) and for a control scramble sequence (5′-GAAACAAGGGTGCCAGTGTCT-3′) were synthesized. The sense and antisense oligonucleotides were cloned into a pSUPER-neo/gfp plasmid according to the manufacturer's instructions (OligoEngine, Seattle, WA, USA). To establish the stable Ahi1-knockdown cells, we linearized the pSUPER-neo/gfp-shRNA plasmids with SacI, and transfected them into IMCD3 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). After 24 h, cells were cultured in G418 selecting medium (1 mg/ml G418; Sigma, St Louis, MO, USA) until cell colonies formed. For cells with Ahi1 RNAi knockdown, 10 EGFP-expressing/G418-resistant clones were assayed for Ahi1 protein levels, by western blotting, resulting in four lines (shAhi1-1, -2, -3, -4). Control scramble RNAi-expressing IMCD3 cells were established using the same procedure (scramble-1 and -2). Once the stable clones were established, cells were maintained in normal IMCD3 medium.
For transient transfection, IMCD3 cells were plated on glass coverslips in 24-well culture dishes for 24 h before transfection with Lipofectamine 2000. Cells were fixed for immunostaining 24 h after transfection.
Cell cycle synchronization
Localization of Ahi1 at time points throughout the cell cycle was determined through synchronization of the cells at various stages of the cell cycle. To enrich the population of cells at specific stages of the cell cycle, we cultured IMCD3 cells under the following conditions: for G0/G1 phase (serum-depriving medium for 24 h); for G1 phase (100 µM mimosine (Sigma) for 16 h); for G1/S phase (1 µg/ml aphidicolin (Sigma) for 16 h); for M phase (100 nM paclitaxel (Sigma) for 16 h). Low cell-density IMCD3 cultures, in normal medium without any treatments, were also assessed for cell cycle stage on the basis of DNA and cell morphological-based criteria.
Immunostaining and imaging
Overexpressed hemagglutinin (HA)-tagged Rab8a was transfected into the stably integrated IMCD3 cells using Lipofectamine 2000, with fixation of cells 48 h later. In addition, untransfected cell lines were also analysed throughout our studies.
Cells were either fixed with ice-cold methanol at −20°C for 15 min or with 4% paraformaldehyde, and were then permeabilized in 0.04% Triton X-100/PBS (PBSTx) for 10 min at room temperature. Fixed cells were incubated in 10% normal donkey serum (for Odf2 antibodies) or 10% normal goat serum (all other antibodies) in PBSTx for 1 h at room temperature. Primary antibodies were diluted in 1% donkey serum (for Odf2 antibodies) or 1% normal goat serum (all other antibodies) in PBSTx, and were then incubated with cells at 4°C overnight. The following antibodies were used for immuno-labelling: rabbit polyclonal anti-Ahi1 (1:1000; (34
)), mouse monoclonal anti-γ-tubulin (1:1000; Sigma), mouse monoclonal anti-acetylated α-tubulin (1:1000; Sigma), mouse monoclonal anti-GM130 (1:1000; BD Biosciences, San Jose, CA, USA), rabbit polyclonal anti-Golph4 (1:500; Abcam, Cambridge, MA, USA), mouse monoclonal anti-Rab8a (1:50; Novus Biologicals, Littleton, CO, USA), rabbit polyclonal anti-ninein (1:100; Millipore, Billerica, MA, USA), goat polyclonal anti-Odf2 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse monoclonal anti-HA (1:1000; Cell Signalling Technology, Danvers, MA, USA), and rabbit polyclonal anti-HA (1:100; Clontech, Mountain View, CA, USA). Cells were then incubated with fluorescently conjugated secondary antibodies (1:500, Invitrogen (Molecular Probes) or Jackson Immunoresearch Laboratories, West Grove, PA, USA) for 1 h at room temperature, followed by Hoechst DNA staining (1 µg/ml). After staining, coverslips were mounted on slides with Fluoromount-G antifade solution (Southern Biotechnology, Birmingham, AL, USA).
For actin staining, IMCD3 (shAhi1-1, shAhi1-2, scramble and wild-type) cell lines were grown for 2 days on glass coverslips and then fixed in 4% paraformaldehyde for 15 min. Cells were permeabilized in PBSTx and incubated in rhodamine-conjugated phalloidin (Sigma, 100 µM) for 2 h. The cells were washed and then incubated in Hoechst dye for 5 min. Coverslips were washed again and mounted for imaging.
All immunofluorescent images were visualized with a Zeiss AxioImager-Z1 microscope, and were collected with a Zeiss AxioCam MRm monochrome camera with AxioVision Rel 4.6 software. The objective lenses were a Zeiss EX Plan-NeoFluar 40X/0.75, ∞/0.17 lens and a Zeiss Plan-Apochromat 63X/1.4 oil DIC, ∞/0.17 lens. The immersion medium for the 63X lens was Zeiss Immersol 518F immersion oil. All images were taken at room temperature. Fluorochromes were Alexa Fluor 488 (Invitrogen); Alexa Fluor 546 (Invitrogen); Cy5 (Jackson Immunoresearch). Contrast and brightness of images were adjusted through linear level adjustments, as needed, to optimize the intensity ranges of the images using Adobe Photoshop CS2 (version 9.0.2; Adobe Systems Inc., San Jose, CA, USA).
Generation of mice with a targeted deletion of Ahi1
The mouse genomic region coding Ahi1
was identified through DNA database searches. A BAC (RPCI23-225H6) or genomic DNA, from C57BL/6J mice, which contained the complete genomic structure of Ahi1
, was used as a template for generating the 5′- and 3′-arms of the targeting construct. Our targeting strategy involved deleting coding exons 2–5 (genomic exons 3–6), producing a downstream frameshift and premature stop codon (Supplementary Material, Fig. S4A
). The targeting construct was a modified pSA-BGAL-PGK-NEO plasmid (courtesy Sheila Thomas, Beth Israel Deaconess Medical Center, Boston, MA, USA) containing a phosphoglycerate kinase (PGK) promoter-neomycin resistance (Neo
) gene flanked by short polylinker sites, and a PGK-promoter driving a diphtheria toxin cassette. A 5 kb fragment of intronic region surrounding and containing exon 2 (containing the ATG start-site) of Ahi1
was PCR amplified (using primers containing AscI
sites) and subcloned into the AscI
sites to form the 5′-arm of the targeting vector. A 7.5 kb fragment of intronic region surrounding and containing exons 7–9 (containing coding exons 6–8) of Ahi1
was PCR amplified (using primers containing NheI
sites) and subcloned into the NheI
sites to form the 3′-arm of the targeting vector. The vector was linearized with AscI
and electroporated into C57BL/6-derived Bruce-4 embryonic stem cells resulting in positive recombinant heterozygous clones. Targeting of Ahi1
was determined by Southern blotting of EcoRV
digested genomic DNA with a PCR-amplified 1.1 kb probe derived from a genomic region upstream of the 5′-arm of the targeting vector (Supplementary Material, Fig. S4A
). Southern blotting confirmed homologous recombination (Supplementary Material, Fig. S4B
) and two independent cell lines were injected into blastocysts. Correctly targeted embryonic stem cells were injected into recipient C57BL/6 -Tyrc-2J
blastocysts (Jackson Laboratories, Bar Harbor, ME, USA). Resulting chimeric males were crossed to wild-type C57BL/6J females to obtain germline transmission of the mutant allele. Genotyping was performed by PCR using primers specific to a region in exon 4 and the ensuing intronic region for detecting the wild-type allele (Ahi1, For-WT: 5′-CAGTTCATCCCAAGTGCTTGCTGGATGATG-3′; Rev-WT: 5′-CCACGAGGGGCAGCAGAGAGGATTTCTAGT-3′; 228 bp product), and primers specific for lacZ (Ahi1, For-KO: 5′-GTTGCAGTGCACGGCAGATACACTTGCTGA-3′; Rev-KO: 5′-GCCACTGGTGTGGGCCATAATTCAATTCGC-3′; 389 bp product) for detecting the mutant allele. Heterozygous Ahi1
mice were maintained by breeding to wild-type C57BL/6J mice. Ahi1+/−
mice were bred to produce Ahi1+/+
mice for the analysis. Mice were maintained on a normal 12 h light–dark cycle (06:00 to 18:00) with unlimited access to food and water. Western blot analysis and immunohistochemistry on brain tissue further confirmed that Ahi1−/−
mice had no detectable Ahi1 protein (Supplementary Material, Fig. S4C
; data not shown). Mice from both ES cell lines had identical phenotypes (data not shown). All mouse procedures were performed under approval from the Institutional Animal Care and Use Committees of both Rensselaer Polytechnic Institute and the Wadsworth Center (NY State Department of Health), in accordance with The National Institutes of Health Guide for the Care and Use of Laboratory Animals
Mouse embryonic fibroblast isolation from Ahi1+/+ and Ahi1−/− embryos
Mouse embryonic fibroblasts (MEFs) were prepared (69
) from mouse embryos (E14.5–E16.5) derived from intercrossing Ahi1+/−
mice. In brief, embryos were removed from the uterus and placed in sterile PBS containing 10% fetal bovine serum. Skin tissue was minced in 0.05% trypsin/EDTA followed by a 10 min incubation at 37°C. The tissue was then gently dissociated to release the cells. The cell suspension was collected and then cultured in DMEM with 10% fetal bovine serum and 1% penicillin/streptomycin. For immunostaining, MEFs were cultured on gelatin-coated glass coverslips to ~90% confluency and then starved for 3 days in media containing 0.1% fetal bovine serum. The cells were fixed and processed for immunostaining as described previously.
Scanning electron microscopy
IMCD3 cells (shAhi1, scramble and wild-type) were grown on coverslips in six-well dishes. They were fixed with cold 6.5% glutaraldehyde in PBS (pH 7.2) for 2 h, washed in PBS and then in 0.1 m cacodylate buffer (pH 7.2) and post-fixed with 2% osmium in cacodylate buffer for 1 h. After washing in double-distilled water, the fixed cells on coverslips were dehydrated in a graded ethanol series and were stored in 100% ethanol overnight at 4°C. The coverslips were then critical-point dried in a Samdri-795 critical point dryer (Tousimis, Rockville, MD, USA) and coated with gold using a SPI sputter coater (West Chester, PA, USA). Cells were observed and photographed on a LEO 1550 VP field emission scanning electron microscope (Carl Zeiss SMT, Peabody, MA, USA).
Western blot analysis and co-immunoprecipitation
Cells were lysed or mouse brains were homogenized in RIPA buffer [50 mm Tris (pH 8), 150 mm NaCl, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 1 mm DTT, 1 mm phenylmethylsulfonyl fluoride and a 1X protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA)], incubated on ice for 30 min and centrifuged at 10 000g for 30 min at 4°C. The supernatant was collected, and the protein concentration was determined with the Advanced Protein Assay Reagent kit (Cytoskeleton Inc, Denver, CO, USA). Five micrograms of protein lysate was resolved on an SDS/PAGE gel and transferred onto PVDF membrane (Millipore). After blocking in 5% skim milk/TBSTx [100 mm Tris (pH 7.4), 150 mm NaCl and 0.01% Triton-X100] for 1 h at room temperature, the membrane was incubated with primary antibody [loading controls consisted of using chicken anti-βIII tubulin antibodies (Millipore)] diluted in blocking solution at 4°C overnight. Primary antibodies were detected with either the SuperSignal West Femto Maximum Sensitivity Substrate Chemiluminescence kit (Pierce, Rockford, IL, USA) or with fluorescent secondary antibodies (Alexa Fluor 488, Alexa Fluor 546 or Cy5). Signals were analysed with a Typhoon Trio+ scanner and ImageQuant analysis software (GE Healthcare, Piscataway, NJ, USA). In order to quantify and compare the signal intensities of each sample, the detected signals were unsaturated and in the linear range of detection.
For co-immunoprecipitation in IMCD3 cell lysates, Protein-A magnetic beads (Invitrogen) were used according to the manufacturer's manual. In brief, the lysate was incubated with Ahi1 antibodies (1 µg) at 4°C overnight, followed by a 15 min incubation with pre-washed Protein-A beads at room temperature. Following five washes with 0.1 m NaPO4 buffer (pH 8.0), the precipitated protein was eluted by boiling of the beads for 10 min in 1× SDS/PAGE sample buffer. The supernatant was collected after centrifugation and resolved on an SDS/PAGE gel for western blotting. Controls included a resin only condition to demonstrate that the co-immunoprecipitated proteins are not non-specifically binding to the resin.
For co-immunoprecipitation in HEK293 cells, cells were plated in 6-well plates and transfected with (i) Ahi1-EGFP (34
) and HA-tagged Rab8a, (ii) myc-tagged Ahi1 and HA-tagged Rab8a or (iii) myc-tagged Ahi1 and HA-tagged Rab11 using Lipofectamine 2000. Twenty-four hours after transfection, cell lysates were collected as described previously. Ahi1 antibodies (1 µg) were incubated with 100 µl of cell lysate for 1 h at room temperature, and then mixed with 30 µl of Protein-A Dynabeads and incubated at room temperature for 15 min. After three washes with PBS, proteins precipitated by antibodies were eluted by boiling the beads in 20 µl of 1× SDS/PAGE protein sample buffer and were resolved on a 12% SDS/PAGE gel for western blot analysis.
Cholera toxin and transferrin uptake assays
Cholera toxin B subunit and transferrin labelling experiments were carried out as previously described (46
). Briefly, IMCD3 cells were rinsed with serum-free medium three times before incubation with 1 µg/ml of Alexa Fluor 546 conjugated cholera toxin B (Invitrogen) in serum-free medium for 30 min on ice, washed, and then were incubated at 37°C for 30 or 60 min before fixing. For quantitative analysis, GM130 was used to mark the Golgi, and the cells with co-localization of cholera toxin B and GM130 were counted and compared with cells that did not have this co-localization. Analysis was conducted from five random image fields under a 40X objective from three independent experiments in Ahi1-knockdown, scramble and wild-type cells.
For the transferrin uptake assay, IMCD3 cells were washed three times with serum-free medium and incubated in the same medium for 2 h at 37°C. Alexa Fluor 546-conjugated-human transferrin (100 µg/ml; Invitrogen) was added and incubated for 30 min on ice, followed by three washes in serum-free medium. Cells were then incubated in serum-free medium for various times (10, 30 or 60 min) at 37° C. Cell surface-bound transferrin was removed by an acid wash (0.05% acetate, 0.5 m NaCl, pH 3) for 45 s. Cells were then rinsed with PBS three times before fixing. For quantification, the number of cells with transferrin in the perinuclear region were counted and compared with number of cells where the transferrin labelling was back on the plasma membrane. Analysis was conducted from six random image fields for Ahi1-knockdown cells, scramble and wild-type cells.