Quantitative assessment of sterol traffic in living cells by dual labeling with dehydroergosterol and BODIPY-cholesterol

Potential of BODIPY-cholesterol for analysis of cholesterol transport and diffusion in living cells

“…This result was recently confirmed in Chinese hamster ovarian (CHO) cells in which BChol strongly accumulated in lipid droplets independent of inhibition of acyl-CoA acyl transferase (ACAT) (Lee et al, 2015). In the same cells but also in adipocytes, droplet staining with filipin was low confirming our findings with DHE, namely that the free cholesterol content of lipid droplets is limited to the droplet membrane under normal and fatty-acid loaded conditions (Du et al, 2011;Prattes, 2000;Wüstner and Faergeman, 2008a;Wüstner et al, 2011b). Thus, accumulation of BChol in lipid droplets is artificially high due to the BODIPY moiety which is itself a droplet marker (Listenberger and Brown, 2007;Spandl et al, 2009).…”

“…Close resemblance of cholesterol was found for CTL and DHE also in recent molecular simulation studies (Pourmousa et al, 2014;Robalo et al, 2013), while larger deviation was found for BChol (Hölttä-Vuori et al, 2008). In experiments with model membrane systems, CTL and DHE partition strongly into the lo phase in expense of the ld phase, at least fourfold more efficient than BChol (Baumgart et al, 2007;Garvik et al, 2009;Sezgin et al, 2012;Wüstner et al, 2011b). All -to our knowledge -known sterol transfer proteins bind and transfer DHE or CTL, while some do not recognize or transfer BChol and other tagged cholesterol analogs, as nitrobenzoxadiazole (NBD)-cholesterol (Wüstner and Solanko, 2015).…”

“…All -to our knowledge -known sterol transfer proteins bind and transfer DHE or CTL, while some do not recognize or transfer BChol and other tagged cholesterol analogs, as nitrobenzoxadiazole (NBD)-cholesterol (Wüstner and Solanko, 2015). In addition the attached fluorophore can directly bias the intracellular targeting of the cholesterol probes, as we have shown for the artificially high transport of BChol to lipid droplets in fatty-acid loaded Baby hamster kidney and HeLa cells (Wüstner et al, 2011b). This result was recently confirmed in Chinese hamster ovarian (CHO) cells in which BChol strongly accumulated in lipid droplets independent of inhibition of acyl-CoA acyl transferase (ACAT) (Lee et al, 2015).…”

A comparative study on fluorescent cholesterol analogs as versatile cellular reporters

“…Consequently, it is not surprising that the %Lo values of all other dye-tagged analogs are smaller. Yet, TF-Chol and the PEG2000-linked Star512-and KK114-PEG-Chol revealed almost similar Lo preference with values of %Lo у 50% in both GUVs and GPMVs, as indicated before ( 21,26,28,42,55 ). All other analogs, i.e., those labeled with NBD or dansyl, mostly avoided the more ordered environments order in the Lo environment and, thus, the order difference between Lo and Ld environments is less in GPMVs than in GUVs, leading, in general, to an easier access of more ordered environments and, consequently, to larger %Lo values in GPMVs compared with GUVs ( 28,51,53 ).…”

“…For such investigations, fl uorescent analogs of Chol are required. As has been shown before in various reports ( 10,11,(14)(15)(16)(17)(18)(19)(20)(21), natural Chol analogs such as DHE and CTL are, in principle, the most suitable molecules to mimic Chol. They can be inserted specifi cally into the plasma membrane or delivered to cells as part of lipoproteins for subsequent analysis of their transport itineraries and metabolism.…”