5T4 Regulates CXCR4 Chemotaxis Figure 4. Role of 5T4 expression in the CXC12/CXCR4 axis in MEF. (A), MEF derived from wild-type, (WT, black columns), 5T4 heterozygote, (HET, grey columns) and 5T4 null, (5T4KO, white columns) embryos show 5T4 gene dose related CXCLl2 chemotaxis. (B), Chemotaxis of 5T4KO MEF following mock infection, (black columns), or infection with RAd-eGFP, (grey columns), or RAd-m5T4, (white columns); CXCL12 chemotaxis is only restored by RAd-m5T4. (+ or – 30 ng CXCL12). (C), Pattern of expression of CXCR4, (green) and 5T4, (red) in WT and 5T4KO MEF. In WT cells, CXCR4 and 5T4 are seen at the cell surface and clearly co-localize (CXCR4 = green; 5T4 = red; composite: co-localization = yellow; co-localized areas shown in separate channel) while in 5T4KO cells CXCR4 is located intracellularly around the nucleus; compare to DAPI labeling (blue). (D), 5T4KO MEF infected with RAd-m5T4 exhibit cell surface expression of both 5T4 and CXCR4 also displayed by co-localization (5T4 = red; CXCR4 = green; composite co localization = yellow; co-localized areas shown in separate channel). RAd-GFP had no effect on CXCR4 expression (not shown). doi:10.1371/journal.pone.0009982.g004 human tumor cells which exhibit higher levels of 5T4 expression and where pulldown and reciprocal western analyses suggest association of some 5T4 and CXCR4 molecules in non-ionic detergent solubilized lysates (Southgate et al unpublished). These observations need further study using cross-linked membrane preparations as well as FRET studies. In order to distinguish where the CXCR4 and 5T4 interaction might occur, we studied the effects of cytoskeleton, microtubule and Golgi disruption on the co-localization pattern of the two molecules in MEF. CXCR4 intracellular trafficking to the cell surface post Golgi is dependent on the microtubules and not the actin cytoskeleton. Once at the plasma membrane, co-localized CXCR4 PLoS ONE | www.plosone.org 8 April 2010 | Volume 5 | Issue 4 | e9982