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First published online 12 February 2008
doi: 10.1242/jcs.016964

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The R-Ras interaction partner ORP3 regulates cell adhesion

¹ Department of Molecular Medicine, National Public Health Institute, Biomedicum, FI-00251 Helsinki, Finland
² Folkhälsan Institute of Genetics, Folkhälsan Research Centre Biomedicum, Biomedicum, University of Helsinki, Helsinki, Finland
³ Wihuri Research Institute, Helsinki, Finland
⁴ Department of Forensic Medicine, University of Helsinki, Finland
⁵ VTT Medical Biotechnology, Turku, Finland
⁶ Department of Pathology, Haartman Institute, University of Helsinki, Finland

View larger version (101K): [in this window] [in a new window]   Fig. 1. Localization of endogenous ORP3 in HEK293 cells. Immunofluorescence microscopy analysis of HEK293 cells using anti-ORP3 antibodies. F-actin was stained with OG488P. The arrows in A and D indicate ORP3 staining at the filopodial plasma membrane extensions. The low-magnification views (G-I) visualize the loss of ORP3-positive filopodia in cultures of increased cell density. Specificity of the staining pattern is demonstrated by preincubation of the ORP3 antibody with the immunizing peptide, leading to loss of the immunoreactivity (J-L). Scale bars: 20 µm.

View larger version (70K): [in this window] [in a new window]   Fig. 2. ORP3 overexpression induces cell-surface protrusions in HEK293 cells. HEK293 cells were transfected with N-terminally Xpress-tagged ORP3 plasmid constructs. At 20 hours post transfection, the cells were fixed and stained using anti-XpressTM antibodies. F-actin was visualized with OG488P. (A-C) Wild-type ORP3 (protrusions indicated by arrows), (D-F) ORP3mFFAT, (G-I) ORP3mPH2, (J-L) FR1(398-886), (M-O) FR6(1-555). Scale bars: 20 µm.

View larger version (34K): [in this window] [in a new window]   Fig. 3. ORP3 interacts with R-Ras. (A) HEK293 cells doubly transfected with ORP3/R-Ras(wt), ORP3/R-Ras(38V) or ORP3/R-Ras(43N) cDNA constructs were lysed in detergent-containing buffer, followed by removal of insoluble material by centrifugation. The insoluble pellets (P) and the soluble fractions (S) are depicted in the top panel. The S fractions were subjected to immunoprecipitation (IP) with anti-R-Ras or irrelevant control (Control IgG) rabbit antibodies. ORP3 and R-Ras in the immunocomplexes were visualized by western blotting (WB) using anti-XpressTM and anti-R-Ras antibodies, respectively. (B) HEK293 cells doubly transfected with the ORP3 cDNA and with R-Ras(wt), the constitutively active R-Ras(38V), or with the dominant inhibitory R-Ras(43N) and stained with anti-ORP3 and anti-R-Ras antibodies. (C) Untransfected HEK293 cells double-stained for endogenous ORP3 and R-Ras. The inset shows a magnified view of a protrusion end. The bottom panels display a similar specimen treated with the secondary antibody conjugates only, anti-rabbit Alexa Fluor 568 (A568R) and anti-goat-FITC (AGFITC), to demonstrate specificity of the staining patterns. Scale bars: 20 µm.

View larger version (46K): [in this window] [in a new window]   Fig. 4. Effects of ORP3 gene silencing on the actin cytoskeleton and HEK293 cell morphology. (A) Western blot analysis of ORP3 protein in HEK293 cells (NT) and cells treated with siORP3 or siC1 siRNAs twice over a 3-day period. (B) HEK293 cells transfected with the indicated siRNAs, or cotransfected with siORP3 and a resistant mutant ORP3 cDNA (ORP3mut), were trypsinized, replated on fibronectin-coated coverslips and allowed to spread for 20, 40, 60 and 120 minutes (as indicated) in the presence of normal culture medium. The F-actin was stained using OG488P. The insets in the siORP3 ORP3mut specimens, stained with anti-ORP3, verify expression of the ORP3 mutant cDNA in the imaged cells. (C) siORP3-transfected cells at the 120 minute time point. The arrow indicates a cell that expresses ORP3 protein at a higher level than the neighbouring cells. (D) R-Ras(38V)-transfected cell at the 120 minute time point of a spreading experiment. Scale bars: 20 µm.

View larger version (29K): [in this window] [in a new window]   Fig. 5. ORP3 regulates HEK293 cell adhesion, spreading and β1 integrin activity. (A) Effect of ORP3 overexpression and silencing on cell spreading. HEK293 cells were transfected with ORP3 cDNA, with the empty vector (Mock), or with ORP3-specific (siORP3) or control (siC2) siRNAs. NT, non-transfected cells. Trypsinized cells were plated on fibronectin-coated coverslips for 1 hour, fixed and stained with OG488P. The area covered by the cells was determined from confocal microscope images. The figure shows mean area (± s.e.m.) of cells analysed from three different coverslips in the same experiment. The total numbers of cells analyzed are indicated. The expression levels of ORP3 and β-actin were assayed by western blotting of 20 µg total cell protein (2 µg for the ORP3-transfected specimen). (B) Effect of ORP3 silencing on cell adhesion on substratum. HEK293 cells treated with siC2 or siORP3 were trypsinized, allowed to adhere on collagen substratum for 1 hour and the number of adherent cells was determined. (C) ORP3 modulates β1 integrin activity. The amount of active integrin in cells transfected with ORP3 cDNA (ORP3), empty plasmid (Mock), or ORP3 siRNA was determined by FACS analysis using P4G11 antibody, which recognizes active β1 integrin. The amount of active integrin was calculated relative to total β1 integrin (P5D2 antibody) expressed on the cell surface. (D) Rescue of the ORP3-silencing phenotype. HEK293 cells were transfected with empty plasmid and ORP3 siRNA (siORP3+mock) or ORP3 mutant cDNA resistant to the siRNA and ORP3 siRNA (siORP3+ORP3mut), followed by analysis of the amount of active integrin on the cell surface. Western blot panels verify successful ORP3 overexpression and silencing. The results represent mean ± s.e.m.; *P<0.05; **P<0.01; ***P<0.001. (E) R-Ras regulates integrins downstream of ORP3. HEK293 cells transiently transfected with empty plasmid and ORP3 siRNA (siORP3+mock) or ORP3 siRNA and R-Ras constructs, were studied for integrin activity as above, or were allowed to spread on fibronectin-coated coverslips for 120 minutes, fixed and stained as indicated. Scale bars, 10 µm.

View larger version (70K): [in this window] [in a new window]   Fig. 6. Silencing of ORP3 results in impaired cell-cell adhesion. HEK293 cells were transfected with control siC2 (A-D) or siORP3 (E-H). After 3 days, the cells were fixed and stained using anti-β-catenin (β-CAT) and anti-pan-cadherin (pan-CAD) antibodies. (A,E) β-catenin staining. (B-D,F-H) High-magnification images showing both β-catenin and pan-cadherin staining. Scale bars: 20 µm.

View larger version (40K): [in this window] [in a new window]   Fig. 7. Effects of ORP3 overexpression in human primary macrophages. Human primary macrophages were transduced with AdGFP (A-C) or AdORP3 (D-F) recombinant adenoviruses. After 72 hours, the cells were fixed and stained for F-actin (Actin) and ORP3 (ORP3). The GFP expressed from both adenoviruses is shown in green (GFP). Scale bars: 20 µm. (G) Differentiated monocyte macrophages from three subjects were infected with AdORP3 or AdGFP for three days. Four million 3 µm polystyrene latex beads were loaded in each well containing 200,000 cells. After 1 hour, the cells were fixed, stained with AF568P, photographed and the beads were counted. The data represent the number of beads per cell, mean (± s.e.m.); the number of cells analyzed: Control (n=2169), AdORP3 (n=385), AdGFP (n=885). The P values indicate statistical significance.

View larger version (37K): [in this window] [in a new window]   Fig. 8. ORP3 is phosphorylated depending on the cell-adhesion status. (A) HEK293 cells were cultured in normal medium for 20 hours. Confluent adherent (AD) cells were lyzed directly on the plate or trypsinized briefly and cells were allowed to adhere and spread on the fibronectin-coated plate in normal medium for 1 hour (replated cells, RP). Adherent and replated cells were treated with 100 nM phorbol myristate acetate (PMA) or 10 µM all-trans retinoic acid (ATRA) for 60 minutes before lysis. (B) A time course in which trypsinized cells were replated and allowed to adhere and spread for 20-120 minutes (indicated at the top). (C) Cells treated as specified above were lyzed and treated without and with -phosphatase (indicated at the bottom). (D) Confluent adherent cells treated with 50 nM okadaic acid for the indicated times. PMA-treated adherent cell sample was used as a positive control for the band shift to the upper position. Quantified intensities of the two ORP3 bands (arrows) are indicated at the bottom. (E) Trypsinized cells (Trypsin) washed and lyzed directly in SDS-PAGE loading buffer or (F) incubated for 1 hour on a dish treated with denatured BSA to inhibit adhesion (DenBSA). Adherent and replated cells are shown for a comparison. (G) Comparison of adherent and replated cells with those at 40-60% confluency (AD Subconfluent). (H) Adherent cells were treated with 5 mM EGTA (AD EGTA) or E-cadherin-blocking antibodies (AD DECMA-1) for 30 minutes.

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