Synthesis and characterization of a novel rhodamine labeled cholesterol reporter

• Published in: Biochimica et biophysica acta. Biomembranes Vol. 1859; no. 6; pp. 1099 - 1113
• Main Authors: Maiwald, Alexander, Bauer, Olivia, Gimpl, Gerald
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.06.2017
• Subjects: 3T3-L1 Cells; Animals; beta-Cyclodextrins - chemistry; Cell Fractionation; CHO Cells; Cholestanols - chemistry; Cholestanols - metabolism; Cholesterol; Cholesterol - chemistry; Cricetulus; Endosomes - chemistry; Endosomes - metabolism; Fluorescent Dyes - chemical synthesis; Fluorescent Dyes - metabolism; Fluorescent probe; HEK293 Cells; Humans; Lysosomes - chemistry; Lysosomes - metabolism; Membrane domains; Membrane Microdomains; Mice; Microscopy; Molecular Probes - chemical synthesis; Molecular Probes - metabolism; Octoxynol - chemistry; Optical Imaging; Rhodamines - chemistry; Unilamellar Liposomes - chemistry; Unilamellar Liposomes - metabolism; NPC; BODIPY; Membrane domains; BChol; GUV; ACAT; Cholesterol; LPDS; BSA; HDL; NBD; Microscopy; LDL; MβCD; RChol; Fluorescent probe; DChol; PVA; DOPC
• ISSN: 0005-2736; 1879-2642
• DOI: 10.1016/j.bbamem.2017.02.018

We introduce the novel fluorescent cholesterol probe RChol in which a sulforhodamine group is linked to the sixth carbon atom of the steroid backbone of cholesterol. The same position has recently been selected to generate the fluorescent reporter 6-dansyl-cholestanol (DChol) and the photoreactive 6-azi-cholestanol. In comparison with DChol, RChol is brighter, much more photostable, and requires less energy for excitation, i.e. favorable conditions for microscopical imaging. RChol easily incorporates into methyl-β-cyclodextrin forming a water-soluble inclusion complex that acts as an efficient sterol donor for cells and membranes. Like cholesterol, RChol possesses a free 3′OH group, a prerequisite to undergo intracellular esterification. RChol was also able to support the growth of cholesterol auxotrophic cells and can therefore substitute for cholesterol as a major component of the plasma membrane. According to subcellular fractionation, slight amounts of RChol (~12%) were determined in low-density Triton-insoluble fractions whereas the majority of RChol was localized in non-rafts fractions. In phase-separated giant unilamellar vesicles, RChol preferentially partitions in liquid-disordered membrane domains. Intracellular RChol was transferred to extracellular sterol acceptors such as high density lipoproteins in a dose-dependent manner. Unlike DChol, RChol was not delivered to the cholesterol storage pathway. Instead, it translocated to endosomes/lysosomes with some transient contacts to peroxisomes. Thus, RChol is considered as a useful probe to study the endosomal/lysosomal pathway of cholesterol. [Display omitted] •A novel fluorescent cholesterol termed RChol was synthesized and characterized.•RChol is bright, photostable and can be complexed with cyclodextrin.•RChol supports the growth of cholesterol auxotrophic cells.•The probe is useful to visualize the endosomal/lysosomal pathway.