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First published online 10 June 2008
doi: 10.1242/jcs.021717

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Association of tetraspanin CD9 with transmembrane TGF confers alterations in cell-surface presentation of TGF and cytoskeletal organization

Department of Cell and Tissue Biology, Program in Cell Biology, University of California–San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143, USA

View larger version (65K): [in this window] [in a new window]   Fig. 1. CD9 alters the localization and presentation of TGF. MDCK cells stably expressing CD9 or TGF, or coexpressing CD9 and TGF were analyzed. (A) Cells were fixed, permeabilized, stained and analyzed by confocal immunofluorescence microscopy (green, CD9; red, TGF). (B) Cell-surface immunostaining of TGF of MDCK cells that express TGF, or CD9 and TGF together. Top panels show cell-surface staining of non-permeabilized cells on ice, whereas the lower panels shows total immunofluorescence for TGF of permeabilized and fixed cells. The middle panels show phase-contrast microscopy of the stained cells in the top panels. (C) The cell-surface proteins labeled by biotinylation were subjected to immunoprecipitation for TGF, thus showing the level of cell-surface TGF in cells expressing CD9, TGF, or CD9 and TGF together (top panel). Middle and lower panels show immunoblotting for the expression levels of total TGF and CD9, respectively. Arrows in the middle panel mark the three differentially processed TGF forms (Bringman et al., 1987; Shi et al., 2000) (see also Fig. 2A). The arrow in the upper panel marks biotinylated cell-surface TGF, corresponding to unglycosylated transmembrane TGF without its pre-sequence (Bringman et al., 1987); this band corresponds to the lower one in the middle panel. (D) CD9 does not alter the distribution of TGF that lacks the TGF core sequence. MDCK cells stably expressing CD9, TGFE or both were fixed, permeabilized, stained and analyzed by confocal immunofluorescence microscopy (green, CD9; red, TGFE).

View larger version (16K): [in this window] [in a new window]   Fig. 2. The tetraspanin protein CD9 alters the stability of the processed forms of transmembrane TGF. CHO cells expressing TGF or coexpressing CD9 and TGF were 35S-Met–35S-Cys pulse-labeled for 12 minutes, chased for times indicated, lysed and subjected to immunoprecipitation with anti-TGF antibodies. (A) SDS-PAGE analyses of immunoprecipitated 35S-labeled TGF. (B) Quantification of the transmembrane TGF bands as a function of the chase time.

View larger version (34K): [in this window] [in a new window]   Fig. 3. Expression of CD9 redistributes TGF to the apical site in polarized epithelial cells. MDCK cells that express CD9, TGF or both proteins were grown in Transwell chambers to allow them to fully establish their polarized phenotype. (A) Cells were fixed and stained for CD9 or TGF, and immunofluorescence was visualized by confocal microscopy (xz section). (B) Expression of CD9 does not alter the distribution of transmembrane TGF that lacks the TGF core sequence in polarized epithelial cells. MDCK cells stably expressing CD9, TGFE or both were grown in Transwell chambers to establish a polarized phenotype. Cells were fixed, stained for CD9 or TGFE, and immunofluorescence was visualized by confocal microscopy (xz section). (C) Cells were subjected to biotinylation of cell-surface proteins in the apical or basolateral compartments, and cell-surface TGF and CD9 were visualized by immunoblotting. Arrow indicates the position of TGF form III. (D) Same experiment as in C, but visualizing the biotinylated E-cadherin or p58, two proteins that are largely expressed in the basolateral compartment, and the apical marker gp135.

View larger version (61K): [in this window] [in a new window]   Fig. 4. Effect of CD9 on TGF cell-surface expression in 3D cell cultures. MDCK cells expressing CD9, TGF or both proteins were embedded in Engelbreth-Holm-Swarm (EHS) tumor basement membrane extract and grown until cysts had formed. The cells were fixed and stained for CD9 or TGF, and immunofluorescence images were captured using confocal microscopy. Scale bars: 20 µm.

View larger version (49K): [in this window] [in a new window]   Fig. 5. (A) Increase in EGFR internalization in MDCK cells expressing CD9, TGF or CD9 and TGF. Cells were cultured in Transwell chambers, thus allowing full polarization, fixed and immunostained for EGFR. Immunofluorescence was visualized using confocal microscopy (xy section). (B) Cells were grown in regular culture medium, lysed, immunoprecipitated with anti-EGFR antibody and immunoblotted with antibodies against EGFR phosphorylated at tyrosine, EGFR or tubulin to detect phosphorylated EGFR (P-EGFR), total EGFR or tubulin.

View larger version (46K): [in this window] [in a new window]   Fig. 6. Coexpression of CD9 and TGF confers changes in actin stress fibers and activities of Rho family of small GTPases that can be rescued by blocking the EGFR activation. (A) MDCK cells expressing CD9, TGF or CD9 and TGF were stained with Alexa-Fluor-594–phalloidin (red) or by immunofluorescence for paxillin (green), and analyzed by confocal microscopy. The last two images (top and bottom) show MDCK cells expressing CD9 and TGF in the presence of a blocking EGFR antibody. (B) The same cells as shown in A were evaluated for the level of activated, GTP-bound forms of the small GTPases RhoA, Cdc42 or Rac1. (C) RhoA and Rac1 GTPases activities were analyzed in the presence of the EGFR inhibitor AG1478. (D) CD9 expression does not change the distribution of actin stress fibers or focal adhesions when expressed with transmembrane TGF that lacks the TGF core domain. MDCK cells that express TGFE, or TGFE and CD9 together were stained using phalloidin (red) or paxillin (green) and analyzed by confocal immunofluorescence microscopy.

View larger version (20K): [in this window] [in a new window]   Fig. 7. Coexpression of CD9 and TGF alters cell migration and adhesion. (A) Migration of MDCK cells expressing CD9, TGF or both was analyzed in a Boyden chamber assay. Cell migration towards medium with 10% serum was quantified after 24 hours by counting those cells that had migrated through the membrane. Numbers of migrating cells were plotted in percent; 100% migration was defined as the number of migrated wild-type cells. (B) Cell adhesion was examined by Crystal Violet staining of the adherent cells after 20 or 40 minutes after seeding onto fibronectin-coated wells.

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