Fluorescent Sterols and Cholesteryl Esters as Probes for Intracellular Cholesterol Transport

Abstract

Cholesterol transport between cellular organelles comprised vesicular trafficking and nonvesicular exchange; these processes are often studied by quantitative fluorescence microscopy. A major challenge for using this approach is producing analogs of cholesterol with suitable brightness and structural and chemical properties comparable with those of cholesterol. This review surveys currently used fluorescent sterols with respect to their behavior in model membranes, their photophysical properties, as well as their transport and metabolism in cells. In the first part, several intrinsically fluorescent sterols, such as dehydroergosterol or cholestatrienol, are discussed. These polyene sterols (P-sterols) contain three conjugated double bonds in the steroid ring system, giving them slight fluorescence in ultraviolet light. We discuss the properties of P-sterols relative to cholesterol, outline their chemical synthesis, and explain how to image them in living cells and organisms. In particular, we show that P-sterol esters inserted into low-density lipoprotein can be tracked in the fibroblasts of Niemann-Pick disease using high-resolution deconvolution microscopy. We also describe fluorophore-tagged cholesterol probes, such as BODIPY-, NBD-, Dansyl-, or Pyrene-tagged cholesterol, and eventual esters of these analogs. Finally, we survey the latest developments in the synthesis and use of alkyne cholesterol analogs to be labeled with fluorophores by click chemistry and discuss the potential of all approaches for future applications.

Keywords: endocytosis; fluorescent sterols; imaging; metabolism; optical microscopy; sterol trafficking.

Figure 1

Structure and biosynthesis of cholesterol biosynthesis. The chemical structure with atom numbering (A) and a space-filling representation (B) (Structure shown above kindly supplied by Avanti Polar Lipids, Inc.). An overview of cholesterol’s biosynthesis (C).

Figure 2

Schematic illustration of cholesterol transport. Cholesterol uptake via LDL particles, its liberation in late endosomes and lysosomes (LE/LYSs), and trafficking from the PM to the endocytic recycling compartment (ERC), the TGN, the ER, or to mitochondria, as happening in steroidogenic cells, is shown. Between these membranes, cholesterol moves by vesicular (lined arrows) and/or nonvesicular pathways (dashed arrows). See the text for more details. Adapted from Wüstner D, Solanko LM and Lund FW. 2012. Cholesterol trafficking and distribution between cellular membranes., in: I.a.B. Levitan F. (Ed.) Cholesterol regulation of ion channels and receptors., John Wiley & Sons Inc., pp. 3–25. © 2012 John Wiley & Sons, Inc.

Figure 3

Schematic pathway of DHE synthesis and possible side pathways. The chemical structure of ergosterol (A) and that of DHE having only one additional double located in the C-ring (B). (C) Synthesis of DHE from ergosterol and the possible side reactions leading to impurities: (1) ergosterol; (2) ergosteryl acetate; (3) dehydroergosteryl acetate; (4) ergosteryl D acetate; (5) ergosterol D; and (6) DHE. Adapted from Ref. .

Figure 4

Three-photon imaging and PURE-LET image denoising for visualization of DHE in polarized HepG2 cells and in nematodes. A, B, HepG2 cells were labeled for 2 minutes with DHE from a DHE/cyclodextrin complex, washed and chased for 30 minutes at 37°C. A field with polarized cells forming an intercellular BC was selected and imaged on a home-built multiphoton microscope using a femtosecond-pulsed Ti:Sapphire laser emitting at 930 nm as excitation source. (A) Selected intensity-averaged frames acquired along the optical axis and (B) corresponding maximum intensity projection of all six intensity-averaged images from 10 acquisitions each, either prior to (upper panels) or after PURE-LET denoising (lower panels). (C, upper row) Adult glomutant nematodes grown on agar plates containing DHE and imaged on the same system and (C, lower row) result after PURE-LET denoising. Images are shown color-coded with a FIRE-LUT. See text and Refs. , , and for further details.

Figure 5

CTL and its esters. (A) The chemical structure of CTL differing from cholesterol only in having two additional double bonds that are located in the B- and C-rings, respectively. (B) CTL-linoleate and (C) CTL-oleate. (D) Parinaric acid is an intrinsically fluorescent polyunsaturated fatty acid, which has been esterified to cholesterol as a sensor of lipoprotein structure. See text for details and references.

Figure 6

Direct observation of CTL ester processing after ingestion of LDL in Niemann–Pick C2 fibroblasts. (A) The esterification reaction of CTL (1) with linoleate (2) to CTL-linoleate (3) and water followed by reconstitution of CTL-linoleate into LDL (4) for imaging in cells (5). NPC2 disease fibroblasts were incubated in the medium containing CTL-linoleate reconstituted into LDL as the only lipoprotein source for 24 hours. Cells were washed and imaged on a UV-sensitive wide field microscope equipped with z-stacking capability for acquiring multiple images along the optical axis. (B, upper row, “Image”): CTL image as acquired (“Raw”) or after various iterations of RL deconvolution. Especially after deconvolution, CTL fluorescence in LE/LYSs, the perinuclear region, and the PM can be discerned. In addition, the inset being enlarged in the middle panel (B, “Zoom”) reveals many small peripheral vesicles containing CTL, again clearly visible after RL deconvolution for 20–40 iterations. The improved image quality can also be judged from the frequency representation of the images, ie, their Fourier transform (B, lower panel, “FFT”). Herein, the contribution of higher spatial frequencies (ie, higher values in the periphery of the FFT maps) is apparent after deconvolution. Higher frequencies in the FFT maps correspond to finer details in the images, indicating that maximum-likelihood deconvolution efficiently removes out-of-focus blur, resulting in higher contrast and resolution. This is essential for detecting sterol in small vesicles and other structures. Images are shown color-coded with a FIRE-LUT.

Figure 7

Structure of BODIPY-tagged cholesterol analogs, their esters, and BODIPY-fatty acids, which can be esterified to cholesterol. Commercially available BODIPY-cholesterol (A, TopFluor-cholesterol from Avanti), its ester with linoleate (B), or with oleate (C). Hydrolysis of such esters in cells allows one to follow the fate of the tagged cholesterol moiety. BODIPY-P-cholesterol (D) is a probe in which the BODIPY group is linked to cholesterol’s side chain via one of the pyrrole rings of the dye. Three different BODIPY-labeled fatty acids have been linked to cholesterol to produce a fluorescent ester (E). Hydrolysis of such esters in cells enables one to follow the fate of the tagged fatty acid moiety.

Figure 8

Structure of additional extrinsically fluorescent cholesterol analogs. 6-Dansyl-cholesterol (A, DChol), 22-NBD-cholesterol (B), 25-NBD-cholesterol (C), pyrene-tagged cholesterol analogs (D, when n = 2, it is called Pyr-met-Chol as described in the text). (E) Alkyne cholesterol can be chemically linked to various fluorescent dyes including BODIPY when provided as azido derivative. See text for further explanations.