A novel alkyne cholesterol to trace cellular cholesterol metabolism and localization

Abstract

the cellular fate of cholesterol by fl uorescence microscopy, various fl uorescent analogs ( 13-16 ), sterol binding toxins ( 17 ), and anti-cholesterol antibodies ( 18 ) have been used. Isotope-labeled cholesterol ( 1 ) and isotope-labeled toxins ( 19 ) have been employed to follow metabolism or organization of cholesterol in membranes, respectively. A probe suitable for conveniently tracing both cholesterol metabolism and localization would be of great value.With the advent of bioorthogonal chemistry ( 20 ), including click chemistry ( 21 ), the sensitive and specifi c detection of compounds containing azido groups or terminal alkynes has become possible. Lipids containing an alkyne moiety have been used to trace their metabolism and distribution ( 22-26 ) and to monitor protein lipidation ( 27-30 ) or protein-lipid interaction ( 31 ). Thus far, no alkyne-bearing analog of cholesterol has been used to trace cholesterol in mammalian cells.Here we report on the synthesis and use of alkyne cholesterol to study cholesterol metabolism by in vitro assays, and in vivo by using various cells. These data are supplemented by cellular localization studies of the probe. MATERIALS AND METHODSFilipin and azide-PEG3-biotin conjugate were obtained from Sigma. Borondifl uorodipyrromethene (Bodipy)-cholesterol [23-(dipyrrometheneboron-difl uoride)-24-norcholesterol] was from 26,26,27,27, H 6 , and 4-cholesten-3-one were from C/D/N Isotopes and Steraloids, respectively. LD540 was described before ( 32 ). Antibodies against calnexin (Stressgen, SPA-860), EGFR (Epitomics, 2235-1), GM130 (Epitomics, 1837-1), and HSP60 (Stressgen, SPA-806) were used.Abstract Cholesterol is an important lipid of mammalian cells and plays a fundamental role in many biological processes. Its concentration in the various cellular membranes differs and is tightly regulated. Here, we present a novel alkyne cholesterol analog suitable for tracing both cholesterol metabolism and localization. This probe can be detected by click chemistry employing various reporter azides. Alkyne cholesterol is accepted by cellular enzymes from different biological species (Brevibacterium, yeast, rat, human) and these enzymes include cholesterol oxidases, hydroxylases, and acyl transferases that generate the expected metabolites in in vitro and in vivo assays. Using fl uorescence microscopy, we studied the distribution of cholesterol at subcellular resolution, detecting the lipid in the Golgi and at the plasma membrane, but also in the endoplasmic reticulum and mitochondria. In summary, alkyne cholesterol represents a versatile, sensitive, and easy-to-use tool for tracking cellular cholesterol metabolism and localization as it allows for manifold detection methods including mass spectrometry, thin-layer chromatography/fl uorography, and fl uorescence microscopy. Cholesterol is the major sterol of mammalian cell membranes ( 1 ). Beyond its role for discrete membrane structures such as caveolae ( 2 ) or microdomains ( 3 ), cholesterol also specifi cally interacts with numerous...

A novel intrinsically fluorescent probe for study of uptake and trafficking of 25-hydroxycholesterol

“…This distribution is similar to that of the fl uorescent cholesterol probes, DHE ( 32 ) and CTL ( 46 ), yet markedly different from the previously reported 20(S)-hydroxycholesterol alkyne probe ( 40 ). This fi nding suggests that 25-HCTL, like other sterol probes ( 40,44 ), initially transits through the lysosome and then redistributes among membranes (e.g., ERC and plasma membrane) to achieve a steady-state distribution similar to that of cholesterol. 25-HCTL is also esterifi ed and incorporated into lipid droplets, indicating that, like 25-HC, the analog traffi cs to the ER.…”

“…This is supported by experiments demonstrating that 25-HCTL partitions into serum lipoproteins or purifi ed LDL and enters the cell via LDLR, rather than through passive diffusion. This mechanism may be shared with other oxysterol probes that also initially transit through the lysosome and then redistribute to other organelles ( 40,44 ).…”

“…approaches have been successfully used. One approach is the introduction of an alkyne moiety to lipids that can be conjugated to a fl uorophore using bio-orthognal chemistry ( 40,44,45 ). Although the alkyne moiety does not greatly alter the biophysical properties of the sterols, it precludes live cell imaging due to the conjugation requirements ( 40,44 ).…”

“…One approach is the introduction of an alkyne moiety to lipids that can be conjugated to a fl uorophore using bio-orthognal chemistry ( 40,44,45 ). Although the alkyne moiety does not greatly alter the biophysical properties of the sterols, it precludes live cell imaging due to the conjugation requirements ( 40,44 ). An alternative approach takes advantage of intrinsically fl uorescent sterols, including the naturally occurring cholesterol analog, dehydroergosterol (DHE) ( 32 ), or a synthetic sterol, CTL ( 46 ).…”

A highly sensitive protocol for microscopy of alkyne lipids and fluorescently tagged or immunostained proteins

“…A robust, basic protocol for alkyne lipid imaging in fixed cells has been described before (18). Based on the published protocol we have now developed three optimized protocol variants to enhance the sensitivity of alkyne lipid microscopy (variant A), and/ or to combine imaging of alkyne lipids and fluorescent fusion proteins (variant B), or to allow for parallel protein detection by immunocytochemistry (variant C).…”