Sequences encoding human EHD proteins were PCR amplified from clones (ImageClone 5229002 for EHD1, 4908085 for EHD2, 5459130 for EHD3 (ATCC) and Ultimate ORF clone IOH6327 for EHD4 (Invitrogen)) using the primers listed in Additional File 7. The PCR products were cloned into pENTR/SD/D-TOPO vector following the manufacturer's instructions (Invitrogen) and sequences of correct clones were verified. The inserts were then transferred into pcDNA-pDEST47 (Invitrogen) vector using an LR Clonase reaction for CMV promoter-driven expression as C-terminal GFP-fusion proteins. GFP-tagged ΔEH deletion mutants were generated using reverse PCR primers excluding the C-terminal EH domain, the sizes of the mutant proteins with respect to number of amino acids were: EHD1 ΔEH (1–435), EHD2 ΔEH (1–439), EHD3 ΔEH (1–434) and EHD4 ΔEH (1–437); these mutants also contain an N-terminal Myc-tag. Myc-tagged wild type and ΔEH deletion mutants were also generated using the PCR with the inclusion of sequences for the Myc-tag in the forward primers followed by LR-mediated transfer into pcDNA-6.2/cLumio-DEST vector. While Myc-tagged proteins proved very useful in biochemical analyses, anti-Myc immunostaining was not fully comparable to corresponding EHD-GFP fluorescence patterns. To independently confirm the EHD-GFP localization patterns, DsRed-Monomer-tagged wild type EHD constructs were generated specifically for use in colocalization studies using forward primers with an Xho I site and reverse primers with a Hind III site, and cloned into Xho I-Hind III cut DsRed-Monomer-N1 vector (Clontech). All DsRed constructs used in this study were DsRed-Monomer and will be referred to as DsRed.
The EHD3 ImageClone was derived from a neuroblastoma and was found to lack a cytosine (C) at position 1552 of the ORF predicted by the Unigene sequence (ACCESSION: NM_014600), which we confirmed using the reverse transcription-PCR cloning and sequencing of EHD3 mRNA from 20 separate clones from four fibroblast and mammary epithelial cell lines. Therefore, the missing C in the ImageClone-derived EHD3 was replaced by site-directed mutagenesis. Primers used in the study are listed in Additional File 7.
The Rab5-GFP and Rab11-GFP constructs in EGFPN1 vector were provided by Dr. Victor Hsu (Brigham and Women's Hospital, Harvard Medical School, Boston, MA). The GFP-MTMR3 construct was provided by Dr. Michael Clague (University of Liverpool, Liverpool, UK). The Myc-SNX1 construct was provided by Dr. JoAnn Trejo (UNC School of Medicine, Chapel Hill, North Carolina).
HeLa cells (obtained from Dr. Victor Hsu) and human embryonic kidney (HEK) 293T cells were grown in Dulbecco's Modified Eagle Medium (DMEM) containing 5% fetal bovine serum (FBS, Hyclone Inc., Logan, UT), 20 mM HEPES, pH 7.35, 1 mM sodium pyruvate, 1 mM each of nonessential amino acids, 100 units/ml penicillin and 100 μg/ml streptomycin (all supplements from Invitrogen).
Primary (76N) and immortalized (16A5, MCF10A) human mammary epithelial cell strains were grown in DFCI-1 medium as described . Human Breast carcinoma cell line T47D, cervical carcinoma cell line SiHa and osteosarcoma cell line U2OS were cultured in alpha-minimal essential medium (Invitrogen) supplemented with 5% fetal calf serum (Hyclone Inc., Logan, UT).
Gene knock-down by small interfering RNA (siRNA)
Small interfering RNA (siRNA) oligonucleotides (synthesized by Dharmacon, Lafayette, CO) were transfected using Oligofectamine (Invitrogen) following the manufacturer's instructions. Demonstrable knock-down of protein expression was seen 48 h after transfection. siRNA sequences targeting EHD1 and EHD3 were as in Caplan et al. 2005 (EHD1, 5'-gaa aga gat gcc caa tgt c, bases 945–963; EHD3, 5'-act gga cat ctc tga tga g, bases 945–963) . siRNA sequences targeting EHD2 and EHD4 (EHD2, 5'-gtc tac atc ggc tcc ttc t, bases 754–772; EHD4, 5'-tgg agg acg ccg act tcg a, bases 158–176) were generated using the SFold algorithm . An irrelevant siRNA control (siCONTROL Non-Targeting siRNA, Cat. #D-001210-01-20) was purchased from Dharmacon (Lafayette, CO).
Antibodies and Western blotting
Rabbit polyclonal antibodies were raised against synthetic peptides coupled through an N-terminal cysteine to Keyhole Limpet Hemocyanin (KLH) (CVSKDARRKKEPELF for EHD1, CSKRRHKGSAE for EHD2, CNLKRMQDQLQAQ for EHD3, and CSHRKSLPKAD for EHD4) using a commercial vendor (Animal Pharm Services, Inc., Healdsburg, CA). Primary immunization with 150 μg of KLH-peptide in Freund's Complete Adjuvant was followed by three booster injections of 100 μg in Freund's Incomplete Adjuvant. The monoclonal antibody 9E10 (anti-Myc)  was purified from serum-free culture supernatants using Protein G Sepharose 4 Fast Flow beads (Amersham Biosciences, Piscataway, NJ). Rabbit anti-green fluorescent protein (anti-GFP, sc-8334) was from Santa Cruz Biotechnology (Santa Cruz, CA).
For Western blotting, cell lysates were prepared in Laemmli SDS-PAGE sample buffer, and protein concentration was determined using the Bio-Rad Dc Protein Assay (Bio-Rad Laboratories, Hercules, CA) with bovine serum albumin as standard. Aliquots of 100 μg protein lysate were separated by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes (PerkinElmer, Boston, MA) and immunoblotted with 1:2000 dilutions of the indicated antisera, followed by 1:10,000 dilution of horseradish peroxidase (HRP)-conjugated protein A (Cappel/Organon Teknika Corp., West Chester, PA), as described . Signals were detected using Western Lightning Chemiluminescence Reagent Plus (PerkinElmer, Boston, MA) and Kodak X-Omat Blue XB-1 film (PerkinElmer, Boston, MA). Figures were prepared by direct scanning of films with a Hewlett Packard Scanjet 7400c scanner and Photoshop 6.0 software.
Preparation of mouse tissue lysates
Mice (18 week-old male and female C57BL/6J) were sacrificed and organs were dissected (brain, heart, lung liver, spleen, kidney, intestine, and testes (male) or mammary gland (female)), washed thoroughly in PBS, chopped into fine pieces, and rocked at 4°C in tissue lysis buffer (50 mM Tris-HCl (pH7.5), 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 1 mM DTT, 0.1% Tween-20, 10% glycerol, 2 mM Na3VO4, 20 mM NaF, 1 mM PMSF) overnight. 100 μg of aliquots of lysate protein were separated using 8% SDS-PAGE and Western blotted as described above.
HEK 293T cells were transfected with 2.5 μg each of Myc-EHD and EHD-GFP DNA constructs using a modified version of the calcium phosphate method , grown for 26–30 h, lysed with cold lysis buffer (1% Triton X-100, 150 mM NaCl, 50 mM Tris-HCl, pH 7.6) supplemented with 0.1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and 10 mM sodium fluoride , and rocked at 4°C overnight or 1–2 hours for Myc-EHD and Myc-EHD ΔEH immunoprecipitations, respectively. Immunoprecipitations were carried out as described previously [48, 49] using 1 mg aliquots of protein lysate, 3 μg of anti-Myc antibody and 20 μl of Protein G Sepharose beads. Beads were washed five times, and bound proteins were eluted in Laemmli sample buffer with 2-mercaptoethanol and resolved by 8% SDS-PAGE followed by immunoblotting. Myc-tagged proteins were detected with mouse-anti-Myc 9E10 at 500 ng/ml and rabbit anti-mouse IgG (H+L)-HRP conjugate (Zymed, San Fransisco, CA) at 1:25,000. GFP-tagged proteins were detected with rabbit anti-GFP (Santa Cruz Biotechnology, Santa Cruz, CA) at 200 ng/ml followed by Protein A-HRP (Zymed, San Fransisco, CA) at 1:25,000. Chemiluminescence detection was as described above.
Transfection, immunofluorescence and confocal microscopy
HeLa cells were grown on 12-mm diameter glass coverslips (Fisher Scientific, Pittsburgh, PA) for 1 day, transfected using the calcium phosphate co-precipitation method with the desired plasmids for 24 h, rinsed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde in PBS at 4°C o Cvernight. The cells were rinsed with PBS, followed by the addition of permeabilization and blocking buffer (PBS containing 5% FBS and 0.05% saponin from Quillaja bark (Sigma, St. Louis, MO) at 25°C for 30 min. For anti-Myc staining, cells were incubated with mouse-anti-Myc (0.5 μg/ml) at 25°C for 1 h. After rinsing with wash buffer (0.05% saponin in PBS), cells were incubated with Alexa Fluor 594-conjugated goat anti-mouse IgG (Molecular Probes, Eugene, OR, A11005) at 25°C for 1 h. The cells were washed extensively with wash buffer and mounted on glass slides using Vectasheild with DAPI for nuclear staining (Vector Laboratories, Burlingame, CA). Fluorescently-stained cells were scanned using a Nikon Eclipse 80i confocal microscope equipped with a Nikon D-eclipse C1 scanning head (Nikon) and analyzed using the EZ-C12.10 software.
Live cell imaging
Live cell video microscopy was performed on transfected HeLa cells grown on Poly-D-Lysine-coated 35-mm coverslip bottom dishes (BD Biocoat Cell Environments). During imaging, cells were immersed in CO2-independent medium (Life Technologies, Grand Island, NY). The cells were imaged every 5 seconds for 7 minutes using a 100× oil immersion objective on an inverted microscope (Model TE2000-U; Nikon) equipped with a charge-coupled device camera controlled by Metamorph software (Universal Imaging Corp.). Image control and post-capture image analysis were performed using MetaMorph software.
Wild type or Myc-EHD ΔEH-transfected HeLa cells were loaded with Alexa Fluor 488-labeled transferrin (Molecular Probes, Eugene, OR, T-13342) at 10 μg/ml in internalization media (DMEM, 10 mM HEPES pH 7.4, 0.1% BSA) at 37°C for 30 min, rinsed with ice-cold PBS followed by transferrin chase by incubation at 37°C in regular serum-containing media . At the indicated time points, cells were washed twice and fixed with 4% paraformaldehyde, stained with anti-Myc antibody and mounted as above, followed by image acquisition. For colocalization of transferrin with Rab5 and Rab11 in siRNA-transfected cells, cells were transfected with Rab5 and Rab11-GFP 24 h after siRNA transfection using the FuGene 6 reagent (Roche Diagnostics, Indianapolis, IN). After 24 hours, the cells were loaded with transferrin as described above, fixed in 4% paraformaldehyde and mounted on glass slides using Vectasheild with DAPI for nuclear staining (Vector Laboratories, Burlingame, CA). Confocal analyses were performed with Zeiss inverted LSM510 confocal microscopy system.
C. elegans strains, culture conditions, and rme-1(b1045) rescue experiments
C. elegans worms were cultured at 22°C under standard growth conditions . Strains used in this study were: Bristol strain N2 (wild type) and rme-1(b1045) with a mutation in the EHD ortholog . To test whether human EHD proteins could rescue the vacuolated intestine phenotype of rme-1 worms, full length human EHD cDNAs were expressed downstream of a worm intestine-specific Vha-6 promoter  in pENtr vector containing the SL2-gfp operon cassette  (details of plasmids available upon request). The rescue constructs (50 ng/μl) were co-injected with a GFP marker (myo::gfp at 100 ng/μl) into the gonads of hermaphrodite rme-1(b1045) worms using standard methodology . The intestinal vacuoles were counted in three independent lines of transgenic adult worms (grown 3–4 days) expressing GFP in intestinal cells. At least 25 worms were counted for each independent line. The basolateral endocytosis assay of the intestinal vacuoles was performed in adult hermaphrodites by microinjection of 1 mg/mL Texas-Red BSA into the pseudocoelom as described .