Figure 6.

A) Binding of Fl-TAT to RBCs and ghosts. Fl-TAT (5 µM) was added to 1% RBCs and the cells were observed by phase contrast and fluorescence microscopy. Fl-TAT appears to be diffusely distributed in solution and no accumulation of the peptide on the membrane of RBCs can be detected under these conditions. Addition of 0.1% Triton X results in rapid lysis of the RBCs. Fl-TAT is then observed to bind to the membrane of the formed ghosts. The set of images after addition of Triton X represent the same field of cells represented before addition and were acquired less than 10 sec after addition of the detergent. Fl-TAT also binds to ghost obtained by E5-TAT mediated hemolysis. RBCs were treated with 1 µM E5-TAT at pH 4.5 and Fl-TAT (5 µM) was added to the sample. While binding of Fl-TAT to the membrane of ghosts is clearly visible, the fraction of peptide that remains in solution appears much larger than in the Tritono X experiment. The data represented was obtained at pH 4.5 but similar results were obtained at pH 7 (data not shown). B) Annexin V binds to ghosts but no to intact RBCs. Cells were exposed to 1 µM E5-TAT at pH 6 to generate a mixture of intact RBCs and ghosts. The sample was treated with FITC-Annexin V and binding of Annexin V to membranes was detected by fluorescence microscopy (FITC image, pseudocolored green). C) Binding of TMR-TAT to GUVs. The fluorescent peptide binds to GUVs containing DPPS (+PS, GUV membrane staining) but not to GUVs lacking it (−PS, diffuse fluorescence in media). The scale bars represent 10 µm.