Partitioning and Single-Molecule Diffusion Dynamics of Bodipy-FTY720 in Biomembranes

Abstract

FTY720, a synthetic analog of sphingosine that has immunosuppressive properties, is the first oral drug to be approved by the U.S. FDA for treatment of multiple sclerosis (under the trade name Gilenya). We have examined the partitioning and diffusion properties of a fluorescent (Bodipy) FTY720 analog (Bdp-FTY720) in the plasma membrane (PM) of epithelial cells (HTB126) and model membranes. Based on two-channel confocal co-localization imaging with DiI-C12, a liquid-disordered phase marker, Bdp-FTY720 has an affinity for the liquid-disordered lipid phase in both binary and ternary phase GUVs. Bdp-FTY720 resides in both the cytoplasm and the plasma membrane of HTB126 cells. The translational diffusion of single Bdp-FTY720 molecules was investigated in the PM of intact cells and giant PM vesicles (GPMVs) using fluorescence correlation spectroscopy. In GPMVs, the two-dimensional anomalous (α=0.9) diffusion of Bdp-FTY720 is 3×10−8 cm2/s. The corresponding cytosolic Bdp-FTY720 diffuses mostly as a fast species (3.5×10−7 cm2/s); a small population (∼2%) diffuses at a slow rate (1.5×10−8 cm2/s). On the nanosecond time scale, the Bdp-FTY720 anisotropy in GPMVs decayed as a biexponential (f1=40 ± 20 ns, β1 = 0.10 ± 0.04, f2=2.4 ± 0.9 ns, β2=0.06 ± 0.02) with an estimated order parameter of 0.62 in the lipid bilayer. In the cytoplasm, the Bdp-FTY720 rotational motion is significantly slower (f1=120 ± 50 ns, β2 = 0.13 ± 0.01, f2=4.4 ± 0.7 ns, and β1 = 0.088 ± 0.009). These results indicate heterogeneous Bdp-FTY720 interactions in the cytoplasm compared with a hindered diffusion in the PM. These findings elucidate the biophysical characteristics of this new FTY720 analog and set the stage for its application in cell biology.