Cells and reagents
NIH3T3 (murine fibroblast cells) and NMuMG (mammary gland cells; CRL-1636) cell lines were purchased from the American Type Culture Collection. The antibodies used were anti–phospho-Erk1/2 (Cell Signaling Technology), anti–phospho-Smad2 (Cell Signaling Technology), anti-Smad4 (Santa Cruz Biotechnology, Inc; H552), anti-Smad2 (Cell Signaling Technology), anti-M6PR (Affinity BioReagents, Inc.), anti-EEA1 (BD Biosciences), anti-TGN38 (BD Biosciences), anti–mouse transferrin receptor (BD Biosciences; C2), anti–mouse LAMP1 (BD Biosciences; ID4B), and anti-GAPDH4 (Fitzgerald Industries International). An anti-CHMP5 polyclonal antibody was generated by immunizing rabbits with a 17-aa synthetic peptide corresponding to the COOH-terminal sequence of mouse CHMP5 and affinity purified with immobilized antigen. Antibodies specific to LBPA, Rab8, and MHC II (I-Aβ) were gifts from T. Kobayashi (Institute of Physical and Chemical Research, Wako, Saitama, Japan) and I. Mellman (Yale University School of Medicine, New Haven, CT). Plasmids encoding HA-TBI and HA-TBII were a gift from J.L. Wrana (University of Toronto, Toronto, Canada).
Cloning of Chmp5
CHMP5 was copurified with IκBα from rabbit lung tissue extract. After separation on SDS-PAGE and silver staining, a 32-kD band was excised from the gel and trypsinized, and five peptides were microsequenced. One mouse EST (available from GenBank/EMBL/DDBJ under accession no. W51061) matched these peptides and was used as a probe to screen a mouse liver cDNA library. One clone containing the Chmp5 open reading frame was obtained and an additional 5′ sequence was cloned by 5′ rapid amplification of cDNA ends.
Generation of Chmp5−/− mice and Chmp5−/− ES cells
Four Chmp5 genomic clones were isolated from a 129/SvJ mouse genomic DNA library (Stratagene). The targeting vector consists of a 5.3-kb 5′ homology region and a 4.7-kb 3′ homology region. A loxP-PGKneo-loxP cassette was inserted between the two regions and a PGK-TK cassette was placed upstream of the 5′ homology region, resulting in a vector designed to delete exons 3–7 of Chmp5. Linearized targeting vector was electroporated into TC1 ES cells. Clones resistant to G418 and gancyclovir were selected, and homologous recombination was confirmed by Southern blotting. Two targeted clones were injected into C57BL/6 blastocysts and both produced germline chimeras. Chimeras were mated with C57BL/6 females, and heterozygous male offspring were bred with female splicer mice to delete the floxed neo cassette. Their offspring were screened for the targeted allele without the neo cassette and in the absence of the cre transgene. Positive mice were interbred and maintained on a mixed 129 × C57BL/6 background.
To establish Chmp5−/−
ES cell lines, Chmp5+/−
mice were mated, blastocysts were collected, and ES cell lines were isolated from the inner cell mass and cultured as previously described (Robertson, 1987
). The genotypes of ES cell lines were determined by Southern blotting and immunoblotting with anti-CHMP5 antibody.
Whole-mount in situ hybridization and TUNEL assay
Whole-mount TUNEL assay and in situ hybridization using digoxigenin UTP-labeled riboprobes were performed as previously described (Sasaki and Hogan, 1993
). Embryos were placed on 60-mm Petri dish containing 1% agarose that was filled with PBS and oriented as indicated. The embryos were imaged on a dissecting microscope (model Stemi 2000-C; Carl Zeiss MicroImaging, Inc.) using 16×/16, NA 2.5, objectives (AxioCam; Carl Zeiss MicroImaging, Inc.). Image data was acquired and stored as TIFF files using AxioCam software.
Isolation of cells from mouse embryos
E8.5 embryos from breeding of Chmp5+/− mice were dissected free of maternal tissues and had their Reichert's membrane removed, after which they were washed with PBS and incubated with 0.1% collagenase (Sigma-Aldrich) and Trypsin-EDTA (Invitrogen) for 30 min at 37°C. The cell suspension was plated in 24-well plates precoated with 0.2% gelatin (Sigma-Aldrich) and cultured in DME supplemented with 15% FBS (Sigma-Aldrich). Chmp5−/− cells were identified by immunostaining with CHMP5 antibody or PCR from extraembryonic tissues.
Transmission electron microscopy
E8.0–8.25 embryos were dissected and washed with PBS, and then fixed with 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, for 1 h at room temperature. Further procedures were performed by standard protocols.
For immunofluorescence analysis, cells were grown on 0.2% gelatin-coated coverslips, washed with PBS, fixed with 4% paraformaldehyde for 30 min at room temperature, and permeabilized with permeabilization buffer (0.05% saponin, 1% FBS, 10 mM Hepes, and 10 mM glycine in PBS, pH 7.5) for 30 min at room temperature. Cells were incubated with the indicated primary antibodies for 30 min at room temperature and then incubated with either goat anti–rabbit or anti–mouse antibodies conjugated with FITC or Texas red under identical conditions. Subsequently, cells were washed three times with PBS, mounted in GEL/MOUNT (Biomeda), and examined under either a Plan Apochromat 63× oil objective or a Plan Neofluar 40× oil objective on a fluorescence microscope (Axioplan 2; all Carl Zeiss MicroImaging, Inc.) equipped with a charge-coupled device camera (Orca ER; Hamamatsu). Image data was acquired and stored as TIFF files using OpenLab software (Improvision, Inc.).
Generation of CHMP5 ShRNAs
shRNAs with murine CHMP5 target sequence Sh2 (5′-CCTGGCCCAACAGTCCTTT-3′) and murine CHMP5 control sequence Sh1 (5′-AAGCGAAACCCAAGGCTCC-3′), or human CHMP5 target sequence Sh3 (5′-AAGGACACCAAGACCACGGTT-3′) were produced by chemically synthesized DNA oligonucleotides and cloned into pSUPER.retro vector following the manufacturer's instruction (OligoEngine). To test ShRNAs, Flag-tagged Chmp5 cDNA and CHMP5 ShRNAs were cotransfected into COS1 cells. 48 h after transfection, cells were lysed and immunoblotted with antibodies specific for Flag and GAPDH4.
Endosome–lysosome transport assay
NIH3T3 cells were cultured in medium containing protease inhibitors to inhibit protein degradation in lysosomes throughout the experiment. Cells were incubated with biotin-conjugated HRP for 3 h to label lysosomes before being transfected with CHMP5 shRNA. 48 h after transfection, cells were incubated with streptavidin for 10 min, washed, chased for the indicated time points, and lysed with biotin-containing lysis buffer. Lysates were applied to antistreptavidin-coated plates, incubated for 1 h, and washed; HRP enzymatic activity was measured by colorimetric assay and is expressed as arbitrary units.
Subcellular fractionation and protease protection assay
HEK293 cells were transfected with COOH-terminal HA-TβRII in the presence or absence of human CHMP5 shRNA (Sh3). 48 h after transfection, cells were treated with 10 ng/ml TGFβ for 5 h or left untreated and fractionated using Percoll gradients as previously described (Marsh et al., 1987
). In brief, cells were washed and resuspended in homogenization buffer (10 mM triethanolamine, 10 mM acetic acid, 1 mM EDTA, and 0.25 M sucrose, pH 7.4) and disrupted with 20 strokes in a dounce homogenizer. Microscopic analysis assured that the cell breakage was nearly complete. The homogenate was centrifuged at 1,000 g
for 5 min at 4°C to remove nuclei and unbroken cells. The postnuclear supernatant was mixed with Percoll (Sigma-Aldrich) in homogenization buffer to give a final concentration of 27%, and the mixture was underlaid with a 27.6% nycodenz solution (Sigma-Aldrich). The gradients were centrifuged in a SW41Ti rotor at 17,500 rpm for 1 h at 4°C, and 14 fractions were collected from the top of gradient.
Protease protection assays have traditionally been used to determine the sidedness of proteins relative to a sealed membrane compartments (Blobel and Sabatini, 1970
; Sabatini and Blobel, 1970
). These experiments typically involve the complete digestion of exposed domains of proteins on the outside of a sealed compartment and the protection of those domains or proteins that reside on the inside of the compartment (Wu et al., 2003
; Sik et al., 2004
). To apply the protease protection assay to our system, we modified the original protocol of the protease protection assay. For protease protection assay, 25-μl aliquots of each fraction were treated with 2–5 μg proteinase K for 30 min at room temperature or left untreated. The reaction was stopped by adding SDS loading buffer, and proteinase K was inactivated by boiling at 90°C. The samples were subjected to 10% SDS-PAGE, followed by immunoblotting with the indicated antibodies.