The Tetraspanins CD9 and CD81 Regulate CD9P1-Induced Effects on Cell Migration

CD9P-1 is a cell surface protein with immunoglobulin domains and an unknown function that specifically associates with tetraspanins CD9 and CD81. Overexpression of CD9P-1 in HEK-293 cells induces dramatic changes in cell spreading and migration on various matrices. Experiments using time-lapse videomicroscopy revealed that CD9P-1 expression has led to higher cell motility on collagen I but lower motility on fibronectin through a β1-integrins dependent mechanism. On collagen I, the increase in cell motility induced by CD9P-1 expression was found to involve integrin α2β1 and CD9P-1 was observed to associate with this collagen receptor. The generation of CD9P-1 mutants demonstrated that the transmembrane and the cytoplasmic domains are necessary for inducing effects on cell motility. On the other hand, expression of tetraspanins CD9 or CD81 was shown to reverse the effects of CD9P-1 on cell motility on collagen I or fibronectin with a concomitant association with CD9P-1. Thus, the ratio of expression levels between CD9P-1 and its tetraspanin partners can regulate cell motility

<i>In situ</i> labeling of CD9P-1 and CD9.

<p>HEK-293, HEK/CD9P-1 and HEK/CD9P-1 transfected (tr) with CD9 were plated on collagen I and cultured in labtek chambers. After 24h, cells were fixed and <i>in situ</i> labeling was performed using CD9P-1 mAb (1F11, IgG1) and CD9 mAb TS9b (CD9, IgG2b). Secondary antibodies, anti-IgG1 coupled to Alexa 568 and anti-IgG2 coupled to Alexa 488, were used for differential staining. Chromatin was also stained using 300 nM DAPI. Images were acquired using optical microscopy with epifluorescence at the magnification 630×. Red: CD9P-1; green: CD9; blue: nucleus.</p

Quantification of the amount of CD9P-1 associated with tetraspanins.

<p>The amount of CD9P-1 associated with tetraspanins or that remaining outside the tetraspanin complexes was quantified in HEK-293 or HEK/CD9P-1 cells after transfection with CD9P-1 or/and CD9. Cells were lysed using the mild detergent Brij97. CD9P-1 associated with tetraspanins was investigated by depletion of tetraspanins from the lysate. Three rounds of subsequent immunoprecipitation were performed with mAbs directed against CD9 and CD81. After depletion of tetraspanin complexes, the amount of CD9P-1 still remaining in the lysate and therefore not associated with tetraspanins was measured by three additional rounds of immunoprecipitation using a mAb directed against CD9P-1 (1F11) antibody. All immunoprecipitates were separated by electrophoresis under reducing conditions and proteins were stained using colloidal Coomassie blue. Gels were scanned using the Odyssey device (upper panel). Signals obtained from CD9P-1 were measured and quantitated. When HEK-293 cells were transiently transfected with CD9P-1 (mfi = 300 <i>versus</i> 50 from endogenous), more than 70% of CD9P-1 was associated with tetraspanin partners CD9 (mfi = 150) and CD81 (mfi = 300). Furthermore, HEK-293 cells were transfected with both plasmids encoding CD9P-1 and CD9. Under these conditions when CD9 expression level was higher (mfi = 700), we observed that 100% of CD9P-1 was associated with tetraspanins. In HEK/CD9P-1 cells that stably expressed CD9P-1 with a high level (mfi = 1000), the amount of CD9P-1 associated with tetraspanins was estimated to be 30%. When HEK/CD9P-1 cells were transfected with CD9 (mfi = 700), a higher amount, corresponding to 50% of CD9P-1, was associated with tetraspanins CD9 and CD81 (lower panel). A representative experiment is shown. IP: immunoprecipitation; Tr: transfection.</p

CD9P-1 overexpression affects HEK-293 cell spreading.

<p>HEK-293 or HEK/CD9P-1 cells were enzymatically dissociated and plated on various matrices for 24h. (A) Laminin-5. (B) Matrigel. (C) Collagen I. (D) Fibronectin. Cells were photographed with phase contrast (200×). Close up sections are shown with higher magnification (C and D).</p

Effects of tetraspanins on CD9P1-induced cell motility on collagen I.

<p>(A) HEK/CD9P-1 cells were transfected with tetraspanins CD9, CD81, CD82, CD151 or CD9×82. Then, cells were plated on collagen I. Time-lapse videomicroscopy was performed at a rate of 1 frame per 15 min for 24h. The results correspond to six independent experiments; standard errors are shown. (B) Time-lapse videomicroscopy experiments were performed with HEK/CD9P-1 cells transfected with CD9 or CD82. The mAbs directed against integrins α1 (FB12) or α2 (Gi9) were added to the media (10 µg/ml). Three independent experiments were performed; standard errors are shown. (*) p<0.05, (**) p<0.01.</p

Functional and physical link between CD9P-1 and α2β1 integrin.

<p>(A) Time-lapse videomicroscopy experiments were performed with HEK-293 or HEK/CD9P-1 cells. Monoclonal antibodies directed against integrins α1 (FB12) or α2 (Gi9) were added in the media (10 µg/ml). Independent experiments were performed (n = 6); standard errors are shown; (*) p<0.01. (B) Cells were lysed using the mild detergent Brij58 and immunoprecipitation experiments were performed with mAbs directed against CD9P-1 (1F11), α2-integrin (Gi9), β1-integrin (9D6) and CD55. After electrophoresis and transfer to a membrane, Western blotting was performed using α2-integrin antibody H-293.</p

Expression of tetraspanins and integrins on HEK-293 and HEK/CD9P-1 cells.

<p>Fluorescence intensity: −<10; +: 10–50; ++: 50–100; +++: 100–300; ++++>300. Tr: transfection.</p

CD9P-1 overexpression affects HEK-293 cell migration.

<p>HEK-293 or HEK/CD9P-1 cells were plated on various matrices. The same field of cells was monitored over a period of 24h while being maintained in a microscope stage incubator. Time-lapse videomicroscopy was performed at a rate of 1 frame per 15 min. Independent experiments were performed: laminin-5 (n = 3), matrigel (n = 3), collagen I (n = 20), fibronectin (n = 11); standard errors are shown.</p

Structure-function relationship of CD9P-1 on cell motility.

<p>Wild type CD9P-1 exhibiting six Ig domains, transmembrane domain (TM) and a cytoplasmic domain (Cyt) is represented. CD9P-1 chimeric forms were generated by replacing the transmembrane and/or the cytoplasmic domain by the corresponding domains of HLA class I protein (shaded boxes). Time-lapse videomicroscopy experiments were performed on collagen I or fibronectin with cell lines expressing wild type or chimeric forms of CD9P-1. Three independent experiments were performed; standard errors are shown.</p

Effects of tetraspanins on CD9P1-induced cell motility on fibronectin.

<p>HEK/CD9P-1 cells were transfected with tetraspanins CD9, CD81, CD82 or CD9×82. Then, cells were plated on fibronectin. Time-lapse videomicroscopy was performed at a rate of 1 frame per 15 min for 24h. The results correspond to three independent experiments; standard errors are shown. (*) p<0.05, (**) p<0.01.</p