Yeast Two-Hybrid Screen
The yeast strain AH109 was transformed with a GAL4 DNA-binding domain fusion vector, pGBKT7 (Clontech, Mountain View, CA), containing residues 1340-1756 of murine pericentrin. After determining that the GAL4 DBD/pericentrin fusion failed to autotransactivate the reporter genes, a 50-ml culture was grown overnight and then mated with the yeast strain Y187, which had been pretransformed with a human testes cDNA library (Clontech). Diploid clones were plated out onto to synthetic defined medium lacking leucine, tryptophan, histidine, and adenine to select for positive interactants.
cDNA Clones, Cloning Techniques, and Expression Constructs
cDNAs encoding CHD3, CHD4, and pericentrin were obtained from the following sources: CHD3 C-terminal sequence was from molecular analysis of genomes and their expression (IMAGE) clone 642405 (IMAGE consortium); CHD4 human testis cDNA library described above; and murine pericentrin as described previously (Purohit et al., 1999
). Sequences encoding pericentrin, CHD3, and CHD4 were amplified by polymerase chain reaction (PCR) by using Pfu
Turbo DNA polymerase (Stratagene, La Jolla, CA), cloned into a donor vector of the Creator system (Clontech), and sequenced to verify the fidelity of the amplifying enzyme (Applied Biosystems, Foster City, CA). Coding sequences were transferred, by Cre-mediated recombination, into a range of expression vectors that included pLP-GBK-T7 (Clontech), pLP-CMV-myc (Clontech), and an existing FLAG-tagged vector that was converted for use with the system. The recombination protocol was as follows: 200 ng of each vector (donor and acceptor) were incubated at 37°C for 1 h in 1× Cre recombinase buffer with 1 U of Cre recombinase (New England Biolabs, Ipswich, MA), the enzyme was then heat inactivated at 70°C for 5 min, and the reaction was allowed to slowly cool to room temperature. Chemically competent DH5α were transformed with 2 μl of heat-inactivated recombination reaction.
Cell Culture and Transfection
COS and HeLa cells were cultured in DMEM supplemented with l-glutamine and 10% fetal calf serum, whereas retinal pigment epithelial (RPE)-1 cells (Clontech) were cultured in DMEM-F-12 supplemented with l-glutamine, sodium bicarbonate, and 10% fetal calf serum (Invitrogen). Cells were transfected either with Lipofectamine (Invitrogen, Carlsbad, CA) or by calcium phosphate precipitation. COS were transiently transfected with 5 μg of DNA by using Lipofectamine Plus reagent (Invitrogen) according to the protocol provided by the manufacturer. For immunofluorescent studies, 4 × 106 HeLa or RPE cells were electroporated with 20 μg of plasmid DNA in 500 μl of electroporation buffer (50 mM HEPES, pH 7, and 100 mM NaCl in phosphate-buffered saline (PBS) by using a Gene Pulser II electroporator (Bio-Rad, Hercules, CA) with a capacitance of 975 μF and a voltage of 290 V. Cells were plated out onto coverslips coated with 1 μg/μl fibronectin (Sigma-Aldrich, St. Louis, MO) and 5 μg/μl collagen, and they were fixed at various times with −20°C methanol.
The following antibodies were used either for immunofluorescent staining or Western blotting purposes: anti-pericentrin (M8) rabbit polyclonal (Doxsey et al., 1994
), anti-pericentrin B (gift from Trisha Davis as used in Mikule et al., 2007
) anti-RbAp46 (Abcam, Cambridge, MA), anti-CHD3 (Orbigen, San Diego, CA), anti-CHD3 (gift from W. Wang) anti-CHD4 (Orbigen), anti-CHD3/4 and anti-MTA2 (gifts from P. Wade). Secondary antibodies for immunofluorescent staining were obtained from the following sources: anti-mouse Alexa 488, anti-mouse AMCA and anti-rabbit Alexa 488 (all from Invitrogen); anti-mouse Cy3, anti-rabbit Cy3, and anti-human Cy5 (all from Jackson ImmunoResearch Laboratories, West Grove, PA). Horseradish peroxidase-linked anti-mouse and anti-rabbit secondary antibodies for Western blotting purposes were obtained from GE Healthcare (Little Chalfont, Buckinghamshire, United Kingdom).
Immunoprecipitations and Western Blotting
Before lysis, cells were briefly washed with PBS, placed on ice, and excess medium was removed by aspiration. Cells were lysed in either a low-stringency buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM Na2HPO4, 1 mM EDTA, pH 8, and 1% Triton X-100) or a high-stringency buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% sodium deoxycholate, 1% Triton X-100, and 0.1% SDS). Lysates were cleared by centrifugation at 14, 000 rpm for 10 min at 4°C and then added to antibodies that had been incubated with 25 μl of protein A/G beads (Santa Cruz Biotechnology, Santa Cruz, CA) for 30 min on ice. Proteins were immunoprecipitated overnight at 4°C with gentle mixing, the beads washed were five times with low-stringency lysis buffer, and then they were resuspended in 50 μl of 2X SDS sample buffer. Proteins were denatured at 90°C for 3 min, fractionated on SDS polyacrylamide gels, and transferred to Immobilon membrane (GE Healthcare). Membranes were blocked with 01.% Tween 20, 5% nonfat dried milk in PBS for 1 h at room temperature, and incubated with primary antibodies diluted either in the same buffer or 5% bovine serum albumin (BSA), PBS, and 0.1% Tween 20 for 2 h at ambient temperature or overnight at 4°C. Blots were washed four times with 0.1% Tween 20, 5% nonfat dried milk in PBS, incubated with secondary antibodies diluted in the same buffer for 1 h at ambient temperature, and washed three times with 0.2% Tween 20 in PBS. Revelation of blots was carried out using enhanced chemiluminescent development (Kirkegaard and Perry Laboratories, Gaithersburg, MD).
Cells were grown on 12-mm acid-washed circular coverslips and fixed with either −20°C methanol or 3.7% paraformaldehyde for 10 min at room temperature. Fixed cells were rehydrated by sequentially washing in PBS and then with a buffer consisting of PBS, 1% BSA, and 0.5% Triton X-100 (PBSA). Antibodies were diluted in PBSA, pipetted onto the surface of the coverslip, and incubated at room temperature for 1 h. The cells were washed with PBSA, secondary antibodies diluted in the same buffer were added, and then cells were incubated for 30 min at room temperature. DNA was stained using 1 μg/ml 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich).
Small-interfering RNA (siRNA)
The following duplexes were synthesized for the purpose of gene silencing: CHD3, AAGCGUGACAGUGAGGAGGAA and AAGGCCAUCGAUCGGU-UUAAU; CHD4, AAGGAUGAUGAUGAUGAUGAU and AACAGUUACC-AAGAAGACUUA; MTA2, AACCGGUAUAUUCAGCAGAAA; and lamin, AACUGGACUUCCAGAAGAACA (Dharmacon RNA Technologies, Lafayette, CO); pericentrin and green fluorescent protein (GFP) siRNA have been described previously (Mikule et al., 2007
). Cells were transiently transfected with siRNA at a final concentration of 200 nM by using oligofectamine (Invitrogen) according to the protocol provided.
Immunofluorescence Microscopy and Live Cell Imaging
Images of cells were taken using a Lecia microscope equipped a 100× objective lens and a CoolSNAP camera (Princeton Instruments). Deconvolution and fluorescence intensity measurements were carried out using MetaMorph software (Molecular Devices, Downingtown, PA) according to previously published protocols (Khodjakov and Rieder, 1999
; Piehl et al., 2004
). Briefly, a 1-μm-diameter disk was placed over the centrosome, and the average fluorescence intensity was measured within this area. In cases where it was difficult to discern the location of the centrosome, regions were drawn onto an image of γ-tubulin staining and then copied and pasted onto the images to be measured. Live cell imaging was performed in 35-mm glass-bottomed dishes overlaying medium with mineral oil (Sigma-Aldrich). A heated chamber (Harvard Apparatus, Holliston, MA) perfused with CO2
was used to maintain a temperature of 37°C, and constant pH. Images were taken every 5 min with an Olympus microscope fitted with a 20× objective lens.