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First published online 18 March 2008
doi: 10.1242/jcs.025015

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Trans-endocytosis of CD47 and SHPS-1 and its role in regulation of the CD47–SHPS-1 system

Laboratory of Biosignal Sciences, Institute for Molecular and Cellular Regulation, Gunma University, Gunma, Japan

View larger version (72K): [in this window] [in a new window]   Fig. 1. Trans-endocytosis of CD47 on CD47-expressing cells by neighboring SHPS-1-expressing cells. (A-I) CHO–SHPS-1 or CHO-Ras (CHO) cells were cocultured with CHO-CD47 cells for 1 hour. The cells were then fixed and subjected to immunostaining with a mAb to CD47 (green; A,D,G) and staining with Rhodamine-conjugated phalloidin (red; B,E,H) in the presence of 0.1% Triton X-100. Merged images are shown in C, F and I. Arrows indicate CD47-positive vesicle-like structures in SHPS-1-expressing cells, and the dotted lines in A, D and G indicate CHO–SHPS-1 (A,D) or CHO-Ras (G) cells. Bar, 20 µm. (J-L) CHO–SHPS-1 and CHO-CD47 cells were cocultured as in A-I, fixed, and stained sequentially with a mAb to CD47 before (green; J) or after (red; K) permeabilization with 0.1% Triton X-100 (see Materials and Methods). The merged image is shown in L. The dotted lines in J-L indicate a CHO–SHPS-1 cell. Bar, 20 µm. (M) CHO–SHPS-1 and CHO-CD47 cells were cocultured for 1 hour, fixed, and stained with a mAb to CD47 in the absence or presence of 0.1% Triton X-100. The percentage of cells with CD47-positive vesicles was determined for CHO–SHPS-1 cells adjacent to CD47-expressing cells by assay A (see Materials and Methods). The effect of treatment of CHO-CD47 cells with the miap301 mAb to CD47 (50 µg/ml) for 15 minutes before and during coculture was also determined. (N) CHO–SHPS-1 and CHO-CD47 cells were cocultured, fixed, and stained as in J-L. The numbers of surface, internalized, and total CD47-positive vesicles in CHO–SHPS-1 cells adjacent to CHO-CD47 cells were determined by assay B (see Materials and Methods). Data in M and N are means ± s.e.m. from three separate experiments. **P<0.01.

View larger version (124K): [in this window] [in a new window]   Fig. 2. Trans-endocytosed CD47 is sorted into multivesicular bodies. (A,B) CHO–SHPS-1 (A) or CHO-Ras (CHO) cells (B) were cocultured with CHO-CD47 cells for 1 hour. The cells were then fixed, and stained with a mAb to CD47 and immunogold-labeled secondary antibodies. Ultrathin sections were prepared after silver enhancement and stained with uranyl acetate and lead citrate as described in Materials and Methods. MVBs in CHO–SHPS-1 (A) and CHO-Ras cells (B) are shown. Bar, 100 nm. (C,D) CHO–SHPS-1 cells were cocultured with CHO-CD47 cells for 1 hour. The cells were then fixed and subjected to double-immunostaining with a mAb to CD47 (green) and pAbs to cathepsin D (red) in the presence of 0.1% Triton X-100. A higher magnification image of boxed region in C was shown in D. The dotted line in C indicates a CHO–SHPS-1 cell. Bar, 10 µm.

View larger version (34K): [in this window] [in a new window]   Fig. 3. Participation of clathrin, epsin, and dynamin in trans-endocytosis of CD47. (A-C) HEK293T cells were transfected with the vector siRNAchc-1, which encoded both mouse SHPS-1 and a siRNA specific for human CHC mRNA. Twenty-four hours after transfection, the cells were isolated, plated on new culture dishes, and incubated for 48 hour before the addition of CHO-CD47 cells. After coculture for 1 hour, the cells were fixed and subjected to two-color immunostaining with mAbs to SHPS-1 (green; A) and to CHC (red; B). The merged image is shown in C. Asterisks indicate cells in which CHC was depleted and exogenous SHPS-1 was expressed. Bar, 20 µm. (D) HEK293T cells transfected with siRNAchc-1 or siRNAchc-2 were treated as in A-C and then fixed and stained as in Fig. 1J-L. The cells were also stained with pAbs to SHPS-1 and AMCA-conjugated goat pAbs to rabbit IgG in order to confirm the expression of SHPS-1. As a control, HEK293T cells were transfected with the corresponding SHPS-1 expression vector without the insert for CHC siRNA. The numbers of surface, internalized, and total CD47-positive vesicles in transfected HEK293T cells located adjacent to CHO-CD47 cells were determined by assay B as described in Materials and Methods, with the exception that data were collected from 20 cells in each experiment. (E) CHO-Ras cells were transfected with an expression vector for SHPS-1 and that for the indicated DN mutants of epsin or dynamin 1 (or with the corresponding empty vector). Twenty-four hours after transfection, the cells were cocultured with CHO-CD47 cells for 1 hour. All cells were then fixed and stained as in Fig. 1J-L. They were also stained with mAbs to the Myc or HA epitope tags in order to confirm expression of the epsin or dynamin 1 mutants. The numbers of surface, internalized, and total CD47-positive vesicles in SHPS-1-expressing CHO-Ras cells positioned adjacent to CHO-CD47 cells were determined by assay B. Data in D and E are means ± s.e.m. from three separate experiments. *P<0.05, **P<0.01 versus corresponding control value.

View larger version (37K): [in this window] [in a new window]   Fig. 4. Role of a juxtamembrane region of SHPS-1 in trans-endocytosis of CD47. (A) Schematic representation of mouse SHPS-1 mutants. Black, gray and white boxes indicate the extracellular (Ex), transmembrane (TM) and cytoplasmic (Cyto) regions of SHPS-1, respectively. WT, wild type. Y and F indicate the positions of tyrosine and phenylalanine, respectively. (B) Lysates (15 µg of protein) of CHO-Ras cell lines expressing wild-type or mutant forms of SHPS-1 were subjected to immunoblot analysis with the p84 mAb to SHPS-1. (C) CHO-Ras cells expressing wild-type or mutant forms of SHPS-1 were cocultured with CHO-CD47 cells, fixed, and stained as in Fig. 1J-L. The numbers of surface, internalized, and total CD47-positive vesicles in SHPS-1-expressing cells located adjacent to CHO-CD47 cells were determined by assay B. The extent of CD47 trans-endocytosis in each cell line was determined as the percentage of the total number of CD47-positive vesicles (Ntotal) that were present at the cell surface (Nsur) or intracellularly (Nin). Data are means ± s.e.m. from three separate experiments. **P<0.01 versus the corresponding value for cells expressing wild-type SHPS-1.

View larger version (59K): [in this window] [in a new window]   Fig. 5. Regulation of trans-endocytosis of CD47 by Rac, Cdc42 and Rab5. (A) CHO-Ras cells were cotransfected with an expression vector for SHPS-1 and with a vector for either Rac(T17N) or NWASP-CRIB (or with the corresponding empty vector). Twenty-four hours after transfection, the cells were cocultured with CHO-CD47 cells for 1 hour. CHO–SHPS-1 cells were also treated with 0.5 µM cytochalasin D (CytoD) or with dimethyl sulfoxide (DMSO) vehicle for 30 minutes before coculture with CHO-CD47 cells. All cells were then fixed and stained as in Fig. 1J-L. The numbers of surface, internalized, and total CD47-positive vesicles in SHPS-1-expressing cells adjacent to CD47-expressing cells were determined by assay B. Cells were also stained with a mAb to the Myc epitope in order to confirm the expression of Myc-tagged Rac(T17N) or NWASP-CRIB. Data are means ± s.e.m. from three separate experiments. *P<0.05, **P<0.01 versus the corresponding control value. (B-D) CHO–SHPS-1 cells were transfected with an expression vector for Rab5(Q79L; C) or for Rab5(S34N; D). Twenty-four hours after transfection, the cells were cocultured with CHO-CD47 cells for 1 hour, fixed, and subjected to immunostaining with mAbs to CD47 (red; B-D) and to the HA epitope tag of the Rab5 mutants (green; C,D). In a control experiment (B), CHO–SHPS-1 cells were transfected with a vector for GFP and then analyzed as for the cells expressing the Rab mutants, with the exception that GFP was detected on the basis of its intrinsic fluorescence (green). Bar, 20 µm.

View larger version (95K): [in this window] [in a new window]   Fig. 6. Time-lapse imaging of CD47 trans-endocytosis. (A-J) CHO-Ras cells, which were transfected with an expression vector for either SHPS-1–CFP or CD47-YFP, were cocultured, and CFP and YFP images were acquired every minute for 75 minutes. Images obtained at the indicated times between 0 and 60 minutes are shown. Filled white (A,B) and open (C,D) arrowheads indicate lamellipodium-like and filopodium-like protrusions, respectively, formed by a CD47-YFP-expressing cell. White arrows (E-H) indicate vesicle-like structures labeled with both CFP and YFP in a cell expressing SHPS-1–CFP. Endocytosis of vesicles at the contact site was followed by retraction of the filopodium-like protrusion by the CD47-exressing cell and the near disruption of cell-cell contact (yellow arrowheads; I,J). Also see Movie 1 in supplementary material. (K-N) CHO-Ras cells were treated as in A-J with the exception that an expression vector for dynamin 1(K44A) was introduced together with that for SHPS-1–CFP. CFP and YFP images were acquired as in A-J. Images obtained at 0, 10, 20 and 60 minutes are shown. Also see Movie 2 in supplementary material. Bars, 20 µm.

View larger version (39K): [in this window] [in a new window]   Fig. 7. Transient aggregation of CHO-CD47 and CHO–SHPS-1 cells. (A) CHO–SHPS-1 cells were mixed with CHO-CD47 cells, incubated for the indicated times, fixed, and examined by phase-contrast microscopy. Bar, 20 µm. (B,C) CHO–SHPS-1, CHO–SHPS-1–C404, or CHO-Ras cells were mixed with CHO-CD47 cells and incubated for the indicated times (B). Alternatively, CHO–SHPS-1 cells were pretreated with 0.5 µM cytochalasin D or DMSO for 30 minutes, mixed with CHO-CD47 cells or control CHO-Ras cells, and incubated for the indicated times (C). Cells were then fixed and the extent of cell aggregation was quantified as the ratio of the number of cells at the indicated time (Nt) to that at the initiation of incubation (No) as described in Materials and Methods. Data are means ± s.e.m. from three separate experiments. *P<0.05, **P<0.01 versus the corresponding value for CHO–SHPS-1 cells (B) or DMSO-pretreated cells (C).

View larger version (71K): [in this window] [in a new window]   Fig. 8. Trans-endocytosis of CD47 from cultured hippocampal neurons to neighboring glial cells or to HEK293T cells expressing SHPS-1. (A-C) Mouse hippocampal neurons were cocultured for 1 hour with HEK–SHPS-1 cells that had been further transfected with an expression vector for GFP to allow visualization of cell shape. The cells were then fixed, stained in the presence of 0.1% Triton X-100 with a mAb to CD47 (red; A), and examined for GFP fluorescence (green; B). A merged image is shown in C. Arrows indicate CD47-positive vesicles in a HEK–SHPS-1 cell. Bar, 20 µm. (D-I) Mouse hippocampal neurons (and glial cells) were cocultured for 3 hour with HEK293T cells that had been transfected with an expression vector for CD47-YFP. The cells were then fixed and stained with mAbs to MAP2 (red; E) or to GFAP (red; H). Expression of CD47-YFP was confirmed by detection of YFP fluorescence (green; D, G). Merged images are shown in F and I. Arrows indicate CD47-positive vesicles in neighboring GFAP-positive cells. Bar, 20 µm. (J-L) Glial cells in mouse hippocampal neuron cultures were fixed and stained with mAbs to SHPS-1 (green; J) and to GFAP (red; K). A merged image is shown in L. Bar, 20 µm. (M-O) Hippocampal neurons (and glial cells) were isolated, cultured for 24 hours, and then transfected with an expression vector for CD47-YFP. Twelve hours after transfection, the cells were fixed and stained with a mAb to GFAP (red; N). Expression of CD47-YFP was confirmed by detection of YFP fluorescence (green; M). A merged image is shown in O. Enlarged images of the boxed regions are shown in the insets. Arrows indicate CD47-positive vesicles in a GFAP-positive astrocyte. Bar, 20 µm.

View larger version (14K): [in this window] [in a new window]   Fig. 9. Model for trans-endocytosis of the CD47–SHPS-1 complex. See text for details.

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