Heterocyclic sterol probes for live monitoring of sterol trafficking and lysosomal storage disorders

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.

Figure 1

Heterocyclic precursors (P1 and P2) and synthetic probes (FP-1 ‒ FP-14) for sterol sensing. R = Ac, H.

Figure 2

The intracellular conversion of FP-5. Cells loaded with FP-5 probe were harvested at various time points (0.5–24 h) and cellular extracts were subjected to MS analysis. The original acetylated form of FP-5 was gradually converted to hydroxyl-derivative as demonstrated by percentage of conversion during time.

Figure 3

The kinetics and localization of intracellular fluorescence of FP-5 and TF-Chol in U-2 OS cells. (A) Probes in DMSO solution were directly added to cultivation medium with 10% FCS at final concentration 0.5 μM and live fluorescence was recorded at indicated time points. Scale bar represents 10 μM. (B,C) Co-localization of FP-5 and organelle specific probes. Cells exposed to pulse with complex FP-5/MβCD (1 μg/ml) were chased for 10 min or 2 h, co-labelled with ER Tracker Red or LysoTracker Red and examined. Expansions of the regions indicated by the white boxes are shown on the upper right side or low left side. Localization of TF-Chol is included in Supplementary Fig. S15).

Figure 4

FP-5 fluorescence in cells with abnormal content of cholesterol. (A) Cholesterol transport in U-2 OS was inhibited by inhibitor U18666A (1 μg/ml) for 48 h and then cells were labelled with FP-5 (200 nM) for additional 24 h, fixed and stained with filipin (50 μg/ml). Expansion of the region indicated by the white box is shown on the low left side. (B) Human fibroblasts carrying mutations in NPC1 cholesterol transporter (clones GB03123E, GB18436) and control normal human fibroblasts (HDFa) were labelled with FP-5 (200 nM) for 6 h and examined. (C) Co-localization of FP-5 and filipin staining in mutant cell clones. (D) Differential kinetics of FP-5 and TF-Chol lysosomal labelling in NPC-GM18436 fibroblasts. Cells were incubated with FP-5 (200 nM) and TF-Chol (1 μM) for indicated times in medium containing 5% LPDS and imaged live. Scale bar represents 10 μM.

Figure 5

Schematic distribution of FP-5 signal in (A) U2-OS cells and (B) in Niemann-Pick fibroblasts following direct addition of FP-5 solution to cultivation medium. The FP-5 signal in U-2 OS cells progresses from plasmatic membrane to ER (time 0.5 h) and accumulates in lysosomes (2–24 h). In NPC1 fibroblasts with accumulated cholesterol, FP-5 signal appears in lysosomes quickly and intensifies within 0.5–2 h.