Nuclear localization of neutral sphingomyelinase 1: biochemical and immunocytochemical analyses
Yukiko Mizutani1,
Keiko Tamiya-Koizumi1,
Noriko Nakamura2,
Miya Kobayashi3,
Yoshio Hirabayashi4 and
Shonen Yoshida1,*
1 Laboratory of Cancer Cell Biology, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Showa-ku, Nagoya, 466-8550, Japan
2 Molecular Cell Biology, Nagoya University School of Medicine, Showa-ku, Nagoya 466-8550, Japan
3 Department of Anatomy, Nagoya University School of Medicine, Showa-ku, Nagoya 466-8550, Japan
4 Neuronal Circuit Mechanisms Research Group, Brain Science Institute, The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-0198, Japan
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Fig. 1. Specific detection of nSMase 1 by western blotting using a rabbit polyclonal antibody. (A) Detection of GST-tagged nSMase 1 expressed in E. coli with immune rabbit serum. Lysate of E. coli expressing GST-tagged recombinant nSMase 1 was electrophoresed on 8% SDS-polyacrylamide gel, transferred to a membrane, and probed with immune rabbit serum (dilution 1:1000, lane 2), as described in Materials and Methods. An immunoreactive protein band was detected at 76.1 kDa, the predicted value for the GST-tagged nSMase 1. As a control, preimmune rabbit serum was used (lane 1). (B) Western blot analysis of the whole cell lysates and a nuclear extract using an affinity-purified anti-rnSMase 1 antibody. Whole cell lysates of AH7974 (lane 3) and 3Y1 (lane 5) were analyzed by western blotting using an affinity-purified antibody. Histidine-tagged nSMase 1 (49.4 kDa, lane 1) and GST-conjugated nSMase 1 (76.1 kDa, lane 2) were also stained as positive controls. Nuclear extract of AH7974 was also analyzed (lane 4). The arrow indicates the immunoreactive band detected at 47.6 kDa, corresponding to the predicted value for the full-length rat nSMase 1.
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Fig. 2. Detection of nSMase 1 in subcellular fractions of AH7974 cells. (A) Scheme for isolating subcellular fractions of AH7974 cells (for details, see Materials and Methods). Pellets obtained by centrifugation’s at 8000 g and 105,000 g were designated as mitochondrial and microsomal fractions, respectively. They may contain some of other organelle but were not purified further. Nucleus and plasma membrane were highly purified by this protocol, as shown in the electron microscopic photographs (B). (C) Subcellular fractions of AH7974 (50 µg protein each) were subjected to western blotting with the anti-rnSMase 1 antibody. An immunoreactive protein band was detected at 47.6 kDa. The microsomal marker Grp78 was also detected with an anti-KDEL antibody in the same way. Extracts of whole cells and organelle showed the bulk activities of neutral SMases (nmol/mg protein/hour) as follows: whole cell, 17.7; nucleus, 5.80; mitochondria, 20.4; microsome, 26.3; plasma membrane, 22.2.
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Fig. 3. Detection of nSMase 1 in subnuclear fractions of AH7974 cells. (A) Neutral SMase activity in subnuclear fractions, such as nuclear envelope, chromatin, nuclear matrix, and denuded nucleus were measured. 10 µl aliquots of each sample (4 ml) were assayed for neutral SMase activity. Activities were relative to that in the intact nucleus, which was taken as 100%. (B) Western blotting of subnuclear fractions were carried out using the anti-rnSMase 1 antibody with aliquots of subnuclear fractions corresponding to 75 µg DNA, as indicated. Immunoreactive protein bands were detected at 47.6 kDa.
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Fig. 4. Identification of nuclear neutral SMase as nSMase 1. (A) Immuno-precipitation of the neutral SMase activity in extracts from nuclei and plasma membranes with anti-rnSMase 1 antibody. Extracts were prepared as described in Materials and Methods. Aliquots (20 µl) containing activity of neutral SMase (2.5 nmol/hour) were mixed with the anti-rnSMase 1 antibody, and antigen-antibody complexes were adsorbed onto protein A-Sepharose beads. After the incubation, the mixture was centrifuged and supernatants were assayed for neutral SMase activity, indicated as% of the control. B. Nuclear extract (10 ml) from AH7974 cells was applied to a Sephacryl S-300 column (1.2 x 85 cm) and 7 ml fractions were collected. Activity of neutral SMase was assayed with 10 µl aliquots of each fraction, and the peak fractions (10 µl each) were subjected to Western blotting using anti-rnSMase 1 antibody. As a molecular size marker, histidine-tagged nSMase 1 (49.4 kDa) was shown in the first lane. Open circles, optical density at 280 nm; closed circles, neutral SMase activity.
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Fig. 5. Immunocytochemistry and immuno-electron microscopy of AH7974 cells. AH7974 cells were stained with anti-rnSMase 1 antibody and the localization of enzyme was observed by immuno-fluorescence microscopy (A-G) and immuno-electron microscopy (H,I) as described in Materials and Methods. (A) Phase contrast of AH7974 cells. (B) Cells in A stained for nSMase 1 with anti-rnSMase 1 antibody (green fluorescence of FITC). (C) Cells in A stained for cytoplasm with anti-KDEL antibody (red fluorescence of CyTM3). (D) Cells in A stained of nuclear DNA with DAPI. (E) Phase contrast of AH7974 cells. (F) Control staining of cells as in E with the antibody pre-treated with purified rat nSMase 1 at 4°C for 3 hours. (G) Merge of two pictures of confocal laser microscopy stained with anti-rnSMase 1 (green) and anti-KDEL (red). (H) Immuno-electron microscopy of an AH7974 cell with anti-rnSMase 1 antibody. Localization of the enzyme was shown by gold colloid particles (10 nm) conjugated with second antibody. (I) Control staining of immuno-electron microscopy without first antibody. Bar, 200 nm. Abbreviations: Nuc, nucleus; Cyt, cytoplasm; arrows indicate clusters of gold particles, and open arrowheads indicate nuclear envelope.
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Fig. 6. Comparison between intracellular localization of endogenous nSMase 1 and overexpressed GFP-nSMase 1 in normal rat fibroblast 3Y1 cells. (A) Phase-contrast of 3Y1. (B) Cells in A stained for nSMase 1 with anti-rnSMase 1 antibody (green). (C) Cells in A stained for cytoplasm with anti-KDEL antibody (red). (D) Cells in A stained for nuclear DNA with DAPI. (E) Phase contrast of 3Y1. (F) Control staining of cells in E with the antibody pre-treated with purified rat nSMase 1. (G) Phase contrast of 3Y1 cells in H. (H) Fluorescence of 3Y1 cells transformed with plasmid expressing GFP-nSMase 1 (green).
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