1 Department of Biochemistry and Molecular Biology, Faculty of Science, SDU2 SDU eScience Centre, Department of Mathematics and Computer Science (IMADA), Faculty of Science, SDU3 The Maersk Mc-Kinney Moller Institute, Faculty of Engineering, SDU4 Department of Chemistry and Biochemistry, Queens College, The City University of New York, Flushing, New York5 Max Planck Institute for Biophysical Chemistry, Department of NanoBiophotonics, Am Fassberg 11, Göttingen, Germany6 Department of Biochemistry and Molecular Biology, Faculty of Science, SDU7 The Maersk Mc-Kinney Moller Institute, Faculty of Engineering, SDU
Cholesterol tagged with the BODIPY fluorophore via the central difluoroboron moiety of the dye (B-Chol) is a promising probe for studying intracellular cholesterol dynamics. We synthesized a new BODIPY-cholesterol probe (B-P-Chol) with the fluorophore attached via one of its pyrrole rings to carbon-24 of cholesterol (B-P-Chol). Using two-photon fluorescence polarimetry in giant unilamellar vesicles and in the plasma membrane (PM) of living intact and actin-disrupted cells, we show that the BODIPY-groups in B-Chol and B-P-Chol are oriented perpendicular and almost parallel to the bilayer normal, respectively. B-Chol is in all three membrane systems much stronger oriented than B-P-Chol. Interestingly, we found that the lateral diffusion in the PM was two times slower for B-Chol than for B-P-Chol, although we found no difference in lateral diffusion in model membranes. Stimulated emission depletion microscopy, performed for the first time, to our knowledge, with fluorescent sterols, revealed that the difference in lateral diffusion of the BODIPY-cholesterol probes was not caused by anomalous subdiffusion, because diffusion of both analogs in the PM was free but not hindered. Our combined measurements show that the position and orientation of the BODIPY moiety in cholesterol analogs have a severe influence on lateral diffusion specifically in the PM of living cells.
Biophysical Journal, 2013, Vol 105, Issue 9, p. 2082-2092