Abstract
The monitoring of intracellular cholesterol homeostasis and trafficking is of great importance because their imbalance leads to many pathologies. Reliable tools for cholesterol detection are in demand. This study presents the design and synthesis of fluorescent probes for cholesterol recognition and demonstrates their selectivity by a variety of methods. The construction of dedicated library of 14 probes was based on heterocyclic (pyridine)-sterol derivatives with various attached fluorophores. The most promising probe, a P1-BODIPY conjugate FP-5, was analysed in detail and showed an intensive labelling of cellular membranes followed by intracellular redistribution into various cholesterol rich organelles and vesicles. FP-5 displayed a stronger signal, with faster kinetics, than the commercial TF-Chol probe. In addition, cells with pharmacologically disrupted cholesterol transport, or with a genetic mutation of cholesterol transporting protein NPC1, exhibited strong and fast FP-5 signal in the endo/lysosomal compartment, co-localizing with filipin staining of cholesterol. Hence, FP-5 has high potential as a new probe for monitoring cholesterol trafficking and its disorders.
Highlights
Cholesterol is a fundamental component of the plasma membrane in animal cells
Imbalance in cholesterol homeostasis leads to pathological processes of atherosclerosis, and deregulated cholesterol trafficking is involved in the pathogenesis of neurodegenerative diseases including Niemann-Pick’s disease type C (NPC), Alzheimer’s disease (AD), Parkinson’s diseases (PD), and possibly Huntington’s disease (HD)[9,10,11]
The fluorophores attached on a precursor P1 via 3-hydroxy group produced fluorescent probes FP-1 ‒ FP-4 or on pyridyl group via quaternization generated probes FP-5 ‒ FP-8 (Fig. 1, Supplementary Fig. S3A)
Summary
Cholesterol is a fundamental component of the plasma membrane in animal cells. It controls membrane structural integrity and fluidity, and modulates the activity of various membrane proteins. Cholesterol plays an essential role in the regulation of multiple signalling pathways It induces membrane packing in specific microdomains (lipid rafts) of the plasma membrane and provides a platform for a variety of membrane-associated signalling proteins[1]. Biosynthesis, as well as uptake of cholesterol from plasma via circulating lipoproteins, is strictly regulated This regulation involves several feedback loops that ensure the exact amount of cholesterol the cells need for their physiological function. Cholesterol homeostasis in cells is maintained by several mechanisms, including cellular uptake, synthesis, storage and efflux[3,4]. It is possible that some intracellular membranes are not as accessible to filipin as the plasma membrane[15] and the specificity of filipin for cholesterol is questionable[16]
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