, Cell surface HSPG anchor SDF-1? and enhance the local concentration of SDF-1? in the surrounding environment of CXCR4 receptor. FRET between EGFP-CXCR4 and SDF-1?-TR was dynamically studied by using confocal microscopy carried out at 19°C on living cells (HEK 293 EGFP-CXCR4). Binding of SDF-1?-TR to EGFP-CXCR4 was assessed by changes of EGFP fluorescence at 522 nm

, Simultaneously, assessment TR fluorescence permitted evaluation of both dependent and independent CXCR4 binding of the ligand

, Addition of a molar excess of SDF-1? (500 nM) reversed SDF-1?-TR interaction with EGFP-CXCR4 (image 11). b, time course of SDF-1?-TR interaction with EGFP-CXCR4 by FRET is shown as green traces for each experimental condition. Accumulation of SDF-1?-TR at cell surfaces is shown as red traces. Quantification of fluorescence for green and red traces was carried out by measuring fluorescence over the whole images. More detailed quantification was performed by measuring fluorescence from the indicated constant size areas (delimited by yellow rectangles in a), Cleavage of cell surface HS by heparitinase I did not affect CXCR4-dependent binding of SDF-1?-TR (images 5 and 6, before and after addition of SDF-1?-TR, respectively) but prevented accumulation of the ligand at cell surfaces as reflected by the absence of detectable TR fluorescence (images 7 and 8, before and after addition of SDF-1?-TR, respectively), vol.8

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