Antibodies and reagents
Antibodies were raised in rabbits against the amino acids 427–719 of human Hook2, affinity purified as previously described (Walenta et al., 2001
), and their specificity was further tested (Szebenyi et al., 2007
). Anti-Hook2 antibodies were also raised in rats against the peptides 65 (CLGRLASLNLRPTDKH) and 66 (DTPDSLSPETYGNFDS) present in the human Hook2 C-term and N-term domains, respectively. The rat anti-Hook2 antibodies were used uniquely for WB and IP analyses and gave the same results as the rabbit anti-Hook2 antibodies. Antibodies were also raised in rabbits against mouse Hook1 and Hook3 and affinity purified as previously described (Walenta et al., 2001
). Mouse monoclonal anti-cenexin antibody (clone CD1B4) was a gift from K. Gull (Lange and Gull, 1995
). Rabbit anti-cenexin antibody was from Atlas Antibodies (St. Louis, MO). Mouse monoclonal anti-giantin antibody was a gift from H. P. Hauri (Linstedt and Hauri, 1993
). Mouse monoclonal anti–α-tubulin (clone B-5-1-2), anti–γ-tubulin (clone GTU-88), and anti–acetylated α-tubulin (clone 6-11B-1) antibodies were from Sigma (Lyon, France). Mouse monoclonal anti-Rab11 (clone 47) and anti-EEA1 (clone 14) antibodies were from BD Biosciences PharMingen (Le Pont de Claix, France). Mouse monoclonal anti–c-Myc (clone 9E10) and anti-GFP (clone B-2) and rabbit polyclonal anti–glyceraldehyde-3-phosphate dehydrogenase (FL-335) antibodies were from Santa Cruz Biotechnology/Clinisciences (Montrouge, France). Mouse anti–Lamp1/CD107a (clone H4A3) antibody was from Biolegend/Ozyme (Saint Quentin en Yvelines, France). Sheep anti–human TGN46 and anti–mouse TGN38 antibodies were from AbDSerotec (Düsseldorf, Germany). Affinity-purified rabbit anti-CEP170 and anti-PCM1 antibodies were from Bethyl Laboratories (Montgomery, TX). Mouse anti-prohibitin antibody (II-14-10) was from Abcam (Cambridge, United Kingdom). Secondary antibodies were from Jackson ImmunoResearch (Immunotech, Marseille, France) or molecular probes (Invitrogen, Cergy Pontoise, France). Tris, bovine serum albumin (BSA), methanol, PFA, saponin, sucrose, and 4′,6-diamidino-2-phenylindole were from Sigma. Other reagents were as follows: glycine and coverslips (VWR, Fontenay, France), SDS and Tx100 (Euromedex, Mundolsheim, France), 1,4-diazabicyclo[2.2.2]octane (DABCO) and Mowiol (Calbiochem/Merck, Meudon, France), Tissue Matrix Optimal Cutting Temperature (OCT) and Superfrost Plus slides (Labonord, Templemars, France), phosphate-buffered saline (PBS; Eurobio, Les Ulis, France), glutaraldehyde EM grade 25%, osmium, and silver lacquer (Electron Microscopy Sciences, Ayguesvives, France), and protein A–Sepharose CL-4B and protein G–Sepharose 4 fast flow (GE Healthcare, La Penne sur Huveaune, France).
siRNA, shRNAs, plasmids, and yeast-two hybrid assay
Hook2 siRNA H1 (ID 20301: 5′ggagacucugauuuuauau3′), H2 (ID 20392: 5′ggaccacauccagagaauc3′), H3 (ID 20208: 5′ggucagcaaucugaagaug3′), and control siRNA against a sequence of luciferase (5′cguacgcggaauacuucga3′) were from Ambion/Applied Biosystems (Courtaboeuf, France). A mix of the three siRNAs targeting Hook2 was used throughout the study unless otherwise indicated. Control shRNA targeting luciferase sequence as well as Hook2 shRNA–expressing plasmids that encode H1 or H2 siRNA targeting sequence and that also encode GFP were generated from the pRNAi vector. ON-TARGETplus SMARTpools of 4 Hook2 siRNAs (L-020408-02) that do not overlap H1, H2, or H3 positions, four PCM1 siRNAs (L-005165-00), four Hook1 siRNAs (L-016845-01), and four Hook3 siRNAs (L-013558), as well as ON-TARGETplus nontargeting pool control siRNAs (D-001810-10), were from Dharmacon/Thermo Scientific (Lafayette, CO). Hook2 full-length cDNA clone (IRAVp968E0334D) expressing untagged mouse Hook2 was from ImaGenes (Berlin, Germany), and the control empty vector pcDNA3.1 was from Invitrogen. The plasmid pEGFPC::Rab8a (wild type) was from A. Echard (Institut Pasteur, Paris, France), pEGFPC::Rab10 was from D. M. Scidmore (Cornell University College of Veterinary Medicine, Ithaca, NY), and pEGFPN::Arl13b was from G. Pazour (University of Massachusetts Medical School, Worcester, MA). Yeast two-hybrid screens were performed as described (Thalappilly et al., 2008
). All yeast media were prepared as described (Walhout and Vidal, 2001
; Thalappilly et al., 2008
). C-term Hook2 (Bam
H1 to end) inserted in the Gateway System (pZEO; Invitrogen) was used as bait to screen a human colon cDNA Library. The positive control on all phenotypes was pCL1 (full-length GAL4) with pPC86 (AD), and the negative control on all phenotypes was pPC97-CYH2 (DB) with pPC86 (AD). Seventy-five positive clones were sequenced.
Cell culture, transfections, and generation of clones
ARPE19 and HK-2 cells (American Type Culture Collection [ATCC], Manassas, VA) were cultured in DMEM/F12 (1:1) with l-glutamine containing 10% fetal bovine serum (Perbio Thermo Scientific, Brebières, France) and insulin/transferrin/selenium-A. For HK-2, the complete medium was supplemented with 10 μg/l epidermal growth factor, 36 μg/l hydrocortisone, and 4 μg/l 3′,3′,5-triiodo-l-thyronine sodium salt. The 4T1 cell (ATCC) complete medium was RPMI 1640 containing 2 mM l-glutamine, 10% fetal bovine serum, 1.5 g/l sodium bicarbonate, 4.5 g/l glucose, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, and 1 mM sodium pyruvate. HEK 293F cells were cultured in DMEM with l-glutamine and 10% fetal bovine serum. For ciliogenesis, ARPE19, HK-2, and HEK 293F cells were cultured for 7 d in their complete medium without any serum starvation. ARPE19 cells were transfected with Fugene HD transfection reagent using the manufacturer's instructions (Roche, Indianapolis, IN). Transfected cells were selected to generate stable cell lines using genetin/G418 sulfate. All cell culture reagents were from Life Technologies/Invitrogen (Cergy-Pontoise, France), unless otherwise indicated.
Immunofluorescence staining and confocal microscopy
Cultured cells on glass coverslips were fixed either with 3% PFA for 20 min, rinsed, and permeabilized with 0.5% Tx100 for 10 min (except for Lamp-1 and EEA1 stainings) or with methanol for 4 min at −20°C. The cells were rinsed in PBS, quenched with 0.2% BSA and 0.05% saponin in PBS for ½ h, and stained with the indicated antibodies. P0 CD1 mice were fixed in 4% PFA for 1 h, washed three times in PBS, incubated in 20% sucrose overnight at 4°C, embedded in OCT, and frozen on dry ice. The 12 μM embedded head cryosections were dried on Superfrost Plus slides and permeabilized for 10 min with PBS containing 2% BSA, 0.05% saponin, and 0.5% Tx100. The cryosections were quenched with 2% BSA and 0.05% saponin in PBS for ½ h and immunostained. Coverslips and slides were mounted in DABCO/Mowiol and observed with a Zeiss Meta confocal microscope (Zeiss, Le Pecq, France) with a UV laser and 100× and 63× objectives for cells and 40× and 10× objectives for tissues. Confocal image analyses were performed using LSM5 Image Browser (Zeiss), ImageJ (National Institutes of Health, Bethesda, MD), and Photoshop (Adobe, San Jose, CA) softwares.
siRNA-treated cells cultured on glass coverslips, washed in PBS, fixed in 2.5% glutaraldehyde in PBS for 1h, washed in PBS, and postfixed in 1% osmium in PBS. The cells were dehydrated in a 25–100% graded series of ethanol and washed twice in alcohol/hexamethyldisilazane (1:1) for 2 h. The coverslips were pasted on SEM disk targets with silver lacquer and gold metalized in an Edwards S150B sputter coater (Boc Edwards, Guildford, United Kingdom). Cells were observed using a Leica S440 scanning electron microscope (Leica Microsystèmes SAS, Nanterre, France) under 30 kV.
Stable clones were cultured for 7 d on glass coverslips. The cells were incubated for 5 min with complete medium containing 2.5% glutaraldehyde, washed once in PBS, and fixed for 1 h in cacodylate sodium buffer 0.1 M, pH 7.2, containing 2.5% glutaraldehyde, 0.1% tannic acid, and 0.01% CaCl2
. The samples were washed three times in cacodylate buffer, postfixed for 1 h in cacodylate buffer containing 1% OsO4, washed three times, dehydrated in a graded series of ethanol, impregnated in propylene oxide, and embedded in Epon resin. Ultrathin 80-nm sections were obtained using a Leica Ultracut UCT microtome (Leica, Wetzlar, Germany) equipped with a diamond knife (Drukker, Cuijk, Netherlands). Sections were collected on copper grids and stained with uranyl acetate and lead citrate (Reynolds, 1963
). Centrosomal sections were selected, and observations were performed on an EM 912 electron microscope (Zeiss) at 100-kV acceleration equipped with a BioScan camera (Model 792; Gatan, Warrendale, PA). Images were acquired with the DigitalMicrograph software (Gatan).
WB and IP
For WB analyses, cells were resuspended either directly in Laemmli buffer or in lysis buffer A containing 50 mM Tris, pH 7.5, 150 mM NaCl, 50 mM NaF, 1 mM EDTA, 1% NP40, 1 mM orthovanadate, and protease inhibitor cocktail containing 1 μg/ml antipain, 1 μg/ml pepstatin, 15 μg/ml benzamidine, and 1 μg/ml leupeptin. The cell lysates were centrifuged, and the supernatants were completed with Laemmli buffer. The proteins were heat denaturated and processed by SDS–PAGE, transferred to nitrocellulose, stained with Ponceau red, and immunoblotted with the indicated antibodies. The proteins were detected with the Western Lightning Chemiluminescence Reagent Plus (PerkinElmer, Courtaboeuf, France). For co-IP of Hook2 and PCM1, HEK cells were lysed in buffer A and incubated overnight with control rabbit anti–c-Myc, rabbit preimmune serum, anti-Hook2, or anti-PCM1 antibodies. The immune complexes were precipitated with protein A/G agarose bead mix, and the immunoprecipitates were analyzed by WB with the indicated antibodies. For co-IP of Hook2 and PCM1 with GFP::Rab8a, HEK cells were transfected with GFP::Rab8a or GFP::Rab10, incubated for 48 h, lysed in buffer A, and incubated overnight with anti-GFP coupled to G protein/agarose beads. The immunoprecipitates were analyzed by WB as indicated.
Quantifications were performed using Excel software (Microsoft, Redmond, WA). The error bars represent SDs with n experiments (n is the number of distinct experiments, unless otherwise indicated). Statistical difference p was calculated using Student's t test.