title: Synthesis and characterization of a novel rhodamine labeled cholesterol reporter.
abstract: 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.
authors.affiliation: Institute of Pharmacy and Biochemistry, Gutenberg-University Mainz, Johann-Joachim Becherweg 30, D-55128 Mainz, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Maiwald
authors.affiliation: Institute of Pharmacy and Biochemistry, Gutenberg-University Mainz, Johann-Joachim Becherweg 30, D-55128 Mainz, Germany.
authors.forename: Olivia
authors.initials: O
authors.lastname: Bauer
authors.affiliation: Institute of Pharmacy and Biochemistry, Gutenberg-University Mainz, Johann-Joachim Becherweg 30, D-55128 Mainz, Germany. Electronic address: gimpl@uni-mainz.de.
authors.forename: Gerald
authors.initials: G
authors.lastname: Gimpl
mesh_terms: D041721:3T3-L1 Cells; D000818:Animals; D016466:CHO Cells; D002458:Cell Fractionation; D002777:Cholestanols / Q000737:chemistry / Q000378:metabolism; D002784:Cholesterol / Q000737:chemistry*; D003412:Cricetulus; D011992:Endosomes / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008247:Lysosomes / Q000737:chemistry / Q000378:metabolism*; D021962:Membrane Microdomains; D051379:Mice; D015335:Molecular Probes / Q000138:chemical synthesis / Q000378:metabolism*; D017830:Octoxynol / Q000737:chemistry; D061848:Optical Imaging; D012235:Rhodamines / Q000737:chemistry*; D053835:Unilamellar Liposomes / Q000737:chemistry / Q000378:metabolism; D047392:beta-Cyclodextrins / Q000737:chemistry
chemical_list: C473152:6-dansylcholestanol; D002777:Cholestanols; D005456:Fluorescent Dyes; D015335:Molecular Probes; D012235:Rhodamines; D053835:Unilamellar Liposomes; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D017830:Octoxynol; D002784:Cholesterol
keywords: Cholesterol; Fluorescent probe; Membrane domains; Microscopy