Information about cholesterol subcellular localization and transport pathways inside cells is essential for understanding and treatment of cholesterol-related diseases. However, there is a lack of reliable tools to monitor it. This work follows the fate of Sterolight, a BODIPY-labelled sterol, within the cell and demonstrates it as a suitable probe for visualization of sterol/lipid trafficking. Sterolight enters cells through an energy-independent process and knockdown experiments suggest caveolin-1 as its potential cellular carrier. Intracellular transport of Sterolight is a rapid process, and transfer from ER and mitochondria to lysosomes and later to lipid droplets requires the participation of active microtubules, as it can be inhibited by the microtubule disruptor nocodazole. Excess of the probe is actively exported from cells, in addition to being stored in lipid droplets, to re-establish the sterol balance. Efflux occurs through a mechanism requiring energy and may be selectively poisoned with verapamil or blocked in cells with mutated cholesterol transporter NPC1. Sterolight is efficiently transferred within and between different cell populations, making it suitable for monitoring numerous aspects of sterol biology, including the live tracking and visualization of intracellular and intercellular transport.

CZ OPENSCREEN Institute of Molecular Genetics of the Czech Academy of Sciences v v i Vídeňská 1083 142 20 Prague 4 Czech Republic

Light Microscopy Core Facility Institute of Molecular Genetics of the Czech Academy of Sciences v v i Vídeňská 1083 142 20 Prague 4 Czech Republic

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Brown MS, Goldstein JL. A receptor-mediated pathway for cholesterol homeostasis. Science. 1986;232:34–47. doi: 10.1126/science.3513311. PubMed DOI

Vance JE. Dysregulation of cholesterol balance in the brain: Contribution to neurodegenerative diseases. Dis. Model Mech. 2012;5:746–755. doi: 10.1242/dmm.010124. PubMed DOI PMC

Arenas F, Garcia-Ruiz C, Fernandez-Checa JC. Intracellular cholesterol trafficking and impact in neurodegeneration. Front. Mol. Neurosci. 2017;10:382. doi: 10.3389/fnmol.2017.00382. PubMed DOI PMC

Gidding SS, Allen NB. Cholesterol and atherosclerotic cardiovascular disease: A lifelong problem. J. Am. Heart Assoc. 2019;8:e012924. doi: 10.1161/JAHA.119.012924. PubMed DOI PMC

Gimpl G, Gehrig-Burger K. Probes for studying cholesterol binding and cell biology. Steroids. 2011;76:216–231. doi: 10.1016/j.steroids.2010.11.001. PubMed DOI

Maxfield FR, Wustner D. Analysis of cholesterol trafficking with fluorescent probes. Methods Cell. Biol. 2012;108:367–393. doi: 10.1016/B978-0-12-386487-1.00017-1. PubMed DOI PMC

Solanko KA, Modzel M, Solanko LM, Wustner D. Fluorescent sterols and cholesteryl esters as probes for intracellular cholesterol transport. Lipid Insights. 2015;8:95–114. doi: 10.4137/LPI.S31617. PubMed DOI PMC

Simons K, Sampaio JL. Membrane organization and lipid rafts. Cold Spring Harb. Perspect. Biol. 2011;3:a004697. doi: 10.1101/cshperspect.a004697. PubMed DOI PMC

Parton RG, Simons K. The multiple faces of caveolae. Nat. Rev. Mol. Cell Biol. 2007;8:185–194. doi: 10.1038/nrm2122. PubMed DOI

Pike LJ. Rafts defined: A report on the Keystone symposium on lipid rafts and cell function. J. Lipid Res. 2006;47:1597–1598. doi: 10.1194/jlr.E600002-JLR200. PubMed DOI

Ouweneel AB, Thomas MJ, Sorci-Thomas MG. The ins and outs of lipid rafts: Functions in intracellular cholesterol homeostasis, microparticles, and cell membranes. J. Lipid Res. 2020;61:676–686. doi: 10.1194/jlr.TR119000383. PubMed DOI PMC

Fielding PE, Fielding CJ. Intracellular transport of low density lipoprotein derived free cholesterol begins at clathrin-coated pits and terminates at cell surface caveolae. Biochemistry. 1996;35:14932–14938. doi: 10.1021/bi9613382. PubMed DOI

Fielding CJ, Fielding PE. Relationship between cholesterol trafficking and signaling in rafts and caveolae. Biochim. Biophys. Acta. 2003;1610:219–228. doi: 10.1016/S0005-2736(03)00020-8. PubMed DOI

Mobius W, et al. Recycling compartments and the internal vesicles of multivesicular bodies harbor most of the cholesterol found in the endocytic pathway. Traffic. 2003;4:222–231. doi: 10.1034/j.1600-0854.2003.00072.x. PubMed DOI

Ikonen E. Cellular cholesterol trafficking and compartmentalization. Nat. Rev. Mol. Cell Biol. 2008;9:125–138. doi: 10.1038/nrm2336. PubMed DOI

Hao M, et al. Vesicular and non-vesicular sterol transport in living cells. The endocytic recycling compartment is a major sterol storage organelle. J. Biol. Chem. 2002;277:609–617. doi: 10.1074/jbc.M108861200. PubMed DOI

Litvinov DY, Savushkin EV, Dergunov AD. Intracellular and plasma membrane events in cholesterol transport and homeostasis. J. Lipids. 2018;2018:3965054. doi: 10.1155/2018/3965054. PubMed DOI PMC

Prinz WA. Non-vesicular sterol transport in cells. Prog. Lipid Res. 2007;46:297–314. doi: 10.1016/j.plipres.2007.06.002. PubMed DOI PMC

Mesmin B, Antonny B, Drin G. Insights into the mechanisms of sterol transport between organelles. Cell. Mol. Life Sci. 2013;70:3405–3421. doi: 10.1007/s00018-012-1247-3. PubMed DOI PMC

Plochberger B, et al. Lipoprotein particles interact with membranes and transfer their cargo without receptors. Biochemistry. 2020;59:4421–4428. doi: 10.1021/acs.biochem.0c00748. PubMed DOI PMC

Wustner D, Solanko K. How cholesterol interacts with proteins and lipids during its intracellular transport. BBA-Biomembranes. 1848;1908–1926:2015. doi: 10.1016/j.bbamem.2015.05.010. PubMed DOI

Tong J, Manik MK, Im YJ. Structural basis of sterol recognition and nonvesicular transport by lipid transfer proteins anchored at membrane contact sites. Proc. Natl. Acad. Sci. U.S.A. 2018;115:E856–E865. doi: 10.1073/pnas.1719709115. PubMed DOI PMC

Luo J, Jiang LY, Yang HY, Song BL, et al. Intracellular cholesterol transport by sterol transfer proteins at membrane contact sites. Trends Biochem. Sci. . 2019;44:273–292. doi: 10.1016/j.tibs.2018.10.001. PubMed DOI

Maxfield FR, van Meer G. Cholesterol, the central lipid of mammalian cells. Curr. Opin. Cell Biol. 2010;22:422–429. doi: 10.1016/j.ceb.2010.05.004. PubMed DOI PMC

Infante RE, Radhakrishnan A. Continuous transport of a small fraction of plasma membrane cholesterol to endoplasmic reticulum regulates total cellular cholesterol. Elife. 2017;6:466. doi: 10.7554/eLife.25466. PubMed DOI PMC

Lange Y, Ye J, Rigney M, Steck TL. Regulation of endoplasmic reticulum cholesterol by plasma membrane cholesterol. J. Lipid Res. 1999;40:2264–2270. doi: 10.1016/S0022-2275(20)32101-5. PubMed DOI

Lange Y, Steck TL. Active membrane cholesterol as a physiological effector. Chem. Phys. Lipids. 2016;199:74–93. doi: 10.1016/j.chemphyslip.2016.02.003. PubMed DOI

Phillips MC. Molecular mechanisms of cellular cholesterol efflux. J. Biol. Chem. 2014;289:24020–24029. doi: 10.1074/jbc.R114.583658. PubMed DOI PMC

Adorni MP, et al. The roles of different pathways in the release of cholesterol from macrophages. J. Lipid Res. 2007;48:2453–2462. doi: 10.1194/jlr.M700274-JLR200. PubMed DOI

Dutta D, Donaldson JG. Search for inhibitors of endocytosis: Intended specificity and unintended consequences. Cell. Logist. 2012;2:203–208. doi: 10.4161/cl.23967. PubMed DOI PMC

Mukherjee S, Zha X, Tabas I, Maxfield FR. Cholesterol distribution in living cells: Fluorescence imaging using dehydroergosterol as a fluorescent cholesterol analog. Biophys. J. 1998;75:1915–1925. doi: 10.1016/S0006-3495(98)77632-5. PubMed DOI PMC

Wustner D. Fluorescent sterols as tools in membrane biophysics and cell biology. Chem. Phys. Lipids. 2007;146:1–25. doi: 10.1016/j.chemphyslip.2006.12.004. PubMed DOI

McIntosh AL, et al. Fluorescence techniques using dehydroergosterol to study cholesterol trafficking. Lipids. 2008;43:1185–1208. doi: 10.1007/s11745-008-3194-1. PubMed DOI PMC

Marks DL, Bittman R, Pagano RE. Use of Bodipy-labeled sphingolipid and cholesterol analogs to examine membrane microdomains in cells. Histochem. Cell Biol. 2008;130:819–832. doi: 10.1007/s00418-008-0509-5. PubMed DOI PMC

Holtta-Vuori M, et al. BODIPY-cholesterol: A new tool to visualize sterol trafficking in living cells and organisms. Traffic. 2008;9:1839–1849. doi: 10.1111/j.1600-0854.2008.00801.x. PubMed DOI

Hofmann K, et al. A novel alkyne cholesterol to trace cellular cholesterol metabolism and localization. J. Lipid Res. 2014;55:583–591. doi: 10.1194/jlr.D044727. PubMed DOI PMC

Jao CY, et al. Bioorthogonal probes for imaging sterols in cells. ChemBioChem. 2015;16:611–617. doi: 10.1002/cbic.201402715. PubMed DOI PMC

Feltes M, et al. Synthesis and characterization of diazirine alkyne probes for the study of intracellular cholesterol trafficking. J. Lipid Res. 2019;60:707–716. doi: 10.1194/jlr.D091470. PubMed DOI PMC

Fujimoto T, Hayashi M, Iwamoto M, Ohno-Iwashita Y. Crosslinked plasmalemmal cholesterol is sequestered to caveolae: Analysis with a new cytochemical probe. J. Histochem. Cytochem. 1997;45:1197–1205. doi: 10.1177/002215549704500903. PubMed DOI

Shimada Y, Maruya M, Iwashita S, Ohno-Iwashita Y. The C-terminal domain of perfringolysin O is an essential cholesterol-binding unit targeting to cholesterol-rich microdomains. Eur. J. Biochem. 2002;269:6195–6203. doi: 10.1046/j.1432-1033.2002.03338.x. PubMed DOI

Ohno-Iwashita Y, et al. Cholesterol-binding toxins and anti-cholesterol antibodies as structural probes for cholesterol localization. Subcell. Biochem. 2010;51:597–621. doi: 10.1007/978-90-481-8622-8_22. PubMed DOI

Sezgin E, et al. A comparative study on fluorescent cholesterol analogs as versatile cellular reporters. J. Lipid Res. 2016;57:299–309. doi: 10.1194/jlr.M065326. PubMed DOI PMC

Schoop V, Martello A, Eden ER, Hoglinger D. Cellular cholesterol and how to find it. Biochim. Biophys. Acta Mol. Cell Biol. Lipids. 2021;1866:158989. doi: 10.1016/j.bbalip.2021.158989. PubMed DOI

Matos ALL, et al. CHIMs are versatile cholesterol analogs mimicking and visualizing cholesterol behavior in lipid bilayers and cells. Commun. Biol. 2021;4:720. doi: 10.1038/s42003-021-02252-5. PubMed DOI PMC

Kralova J, et al. Heterocyclic sterol probes for live monitoring of sterol trafficking and lysosomal storage disorders. Sci. Rep. 2018;8:14428. doi: 10.1038/s41598-018-32776-6. PubMed DOI PMC

Mayle KM, Le AM, Kamei DT. The intracellular trafficking pathway of transferrin. Biochim. Biophys. Acta. 1820;264–281:2012. doi: 10.1016/j.bbagen.2011.09.009. PubMed DOI PMC

Vercauteren D, et al. The use of inhibitors to study endocytic pathways of gene carriers: Optimization and pitfalls. Mol. Ther. 2010;18:561–569. doi: 10.1038/mt.2009.281. PubMed DOI PMC

Macia E, et al. Dynasore, a cell-permeable inhibitor of dynamin. Dev. Cell. 2006;10:839–850. doi: 10.1016/j.devcel.2006.04.002. PubMed DOI

Dutta D, Williamson CD, Cole NB, Donaldson JG. Pitstop 2 is a potent inhibitor of clathrin-independent endocytosis. PLoS ONE. 2012;7:e45799. doi: 10.1371/journal.pone.0045799. PubMed DOI PMC

Willox AK, Sahraoui YM, Royle SJ. Non-specificity of Pitstop 2 in clathrin-mediated endocytosis. Biol. Open. 2014;3:326–331. doi: 10.1242/bio.20147955. PubMed DOI PMC

Zhu XD, et al. Caveolae-dependent endocytosis is required for class A macrophage scavenger receptor-mediated apoptosis in macrophages. J. Biol. Chem. 2011;286:8231–8239. doi: 10.1074/jbc.M110.145888. PubMed DOI PMC

Simons K, Ikonen E. Cell biology—How cells handle cholesterol. Science. 2000;290:1721–1726. doi: 10.1126/science.290.5497.1721. PubMed DOI

Pelkmans L, Helenius A. Endocytosis via caveolae. Traffic. 2002;3:311–320. doi: 10.1034/j.1600-0854.2002.30501.x. PubMed DOI

Yao Q, et al. Caveolin-1 interacts directly with dynamin-2. J. Mol. Biol. 2005;348:491–501. doi: 10.1016/j.jmb.2005.02.003. PubMed DOI

Smart EJ, Ying Y, Donzell WC, Anderson RG. A role for caveolin in transport of cholesterol from endoplasmic reticulum to plasma membrane. J. Biol. Chem. 1996;271:29427–29435. doi: 10.1074/jbc.271.46.29427. PubMed DOI

Fielding CJ, Fielding PE. Cholesterol and caveolae: Structural and functional relationships. Biochim. Biophys. Acta. 2000;1529:210–222. doi: 10.1016/s1388-1981(00)00150-5. PubMed DOI

Sohn J, Lin H, Fritch MR, Tuan RS. Influence of cholesterol/caveolin-1/caveolae homeostasis on membrane properties and substrate adhesion characteristics of adult human mesenchymal stem cells. Stem Cell Res. Ther. 2018;9:86. doi: 10.1186/s13287-018-0830-4. PubMed DOI PMC

Nagai N, Ogata F, Otake H, Nakazawa Y, Kawasaki N. Energy-dependent endocytosis is responsible for drug transcorneal penetration following the instillation of ophthalmic formulations containing indomethacin nanoparticles. Int. J. Nanomed. 2019;14:1213–1227. doi: 10.2147/Ijn.S196681. PubMed DOI PMC

Matthaeus C, Taraska JW. Energy and dynamics of caveolae trafficking. Front. Cell Dev. Biol. 2020;8:614472. doi: 10.3389/fcell.2020.614472. PubMed DOI PMC

Kaplan MR, Simoni RD. Transport of cholesterol from the endoplasmic reticulum to the plasma membrane. J. Cell Biol. 1985;101:446–453. doi: 10.1083/jcb.101.2.446. PubMed DOI PMC

Razin S. Cholesterol uptake is dependent on membrane fluidity in mycoplasmas. Biochim. Biophys. Acta. 1978;513:401–404. doi: 10.1016/0005-2736(78)90208-0. PubMed DOI

Rothblat GH, Hartzell RW, Jr, Mialhe H, Kritchevsky D. The uptake of cholesterol by L5178Y tissue-culture cells: Studies with free cholesterol. Biochim. Biophys. Acta. 1966;116:133–145. doi: 10.1016/0005-2760(66)90099-3. PubMed DOI

Gimpl G, Gehrig-Burger K. Cholesterol reporter molecules. Biosci. Rep. 2007;27:335–358. doi: 10.1007/s10540-007-9060-1. PubMed DOI

Casley-Smith JR. Endocytosis: The different energy requirements for the uptake of particles by small and large vesicles into peritoneal macrophages. J. Microsc. 1969;90:15–30. doi: 10.1111/j.1365-2818.1969.tb00691.x. DOI

Fiorentino I, et al. Energy independent uptake and release of polystyrene nanoparticles in primary mammalian cell cultures. Exp. Cell Res. 2015;330:240–247. doi: 10.1016/j.yexcr.2014.09.017. PubMed DOI

Skiba PJ, Zha X, Maxfield FR, Schissel SL, Tabas I. The distal pathway of lipoprotein-induced cholesterol esterification, but not sphingomyelinase-induced cholesterol esterification, is energy-dependent. J. Biol. Chem. 1996;271:13392–13400. doi: 10.1074/jbc.271.23.13392. PubMed DOI

Romer W, et al. Shiga toxin induces tubular membrane invaginations for its uptake into cells. Nature. 2007;450:670–675. doi: 10.1038/nature05996. PubMed DOI

Johannes L. Shiga Toxin-A model for glycolipid-dependent and lectin-driven endocytosis. Toxins (Basel) 2017;9:340. doi: 10.3390/toxins9110340. PubMed DOI PMC

Maniti O, Blanchard E, Trugnan G, Lamaziere A, Ayala-Sanmartin J. Metabolic energy-independent mechanism of internalization for the cell penetrating peptide penetratin. Int. J. Biochem. Cell Biol. 2012;44:869–875. doi: 10.1016/j.biocel.2012.02.010. PubMed DOI

Miaczynska M, Stenmark H. Mechanisms and functions of endocytosis. J. Cell Biol. 2008;180:7–11. doi: 10.1083/jcb.200711073. PubMed DOI PMC

Bosch M, Mari M, Gross SP, Fernandez-Checa JC, Pol A. Mitochondrial cholesterol: A connection between caveolin, metabolism, and disease. Traffic. 2011;12:1483–1489. doi: 10.1111/j.1600-0854.2011.01259.x. PubMed DOI PMC

Fridolfsson HN, Roth DM, Insel PA, Patel HH. Regulation of intracellular signaling and function by caveolin. FASEB J. 2014;28:3823–3831. doi: 10.1096/fj.14-252320. PubMed DOI PMC

Pol A, Morales-Paytuvi F, Bosch M, Parton RG. Non-caveolar caveolins—Duties outside the caves. J. Cell Sci. 2020;133:241562. doi: 10.1242/jcs.241562. PubMed DOI

Murata M, et al. VIP21/caveolin is a cholesterol-binding protein. Proc. Natl. Acad. Sci. U.S.A. 1995;92:10339–10343. doi: 10.1073/pnas.92.22.10339. PubMed DOI PMC

Lange Y. Cholesterol movement from plasma membrane to rough endoplasmic reticulum. Inhibition by progesterone. J. Biol. Chem. 1994;269:3411–3414. doi: 10.1016/S0021-9258(17)41877-1. PubMed DOI

Maxfield FR, Wustner D. Intracellular cholesterol transport. J. Clin. Invest. 2002;110:891–898. doi: 10.1172/JCI16500. PubMed DOI PMC

Steck TL, Lange Y. Cell cholesterol homeostasis: Mediation by active cholesterol. Trends Cell Biol. 2010;20:680–687. doi: 10.1016/j.tcb.2010.08.007. PubMed DOI PMC

Conrad PA, Smart EJ, Ying YS, Anderson RG, Bloom GS. Caveolin cycles between plasma membrane caveolae and the Golgi complex by microtubule-dependent and microtubule-independent steps. J. Cell Biol. 1995;131:1421–1433. doi: 10.1083/jcb.131.6.1421. PubMed DOI PMC

Wiegand V, Chang TY, Strauss JF, 3rd, Fahrenholz F, Gimpl G. Transport of plasma membrane-derived cholesterol and the function of Niemann-Pick C1 Protein. FASEB J. 2003;17:782–784. doi: 10.1096/fj.02-0818fje. PubMed DOI

Maiwald A, Bauer O, Gimpl G. Synthesis and characterization of a novel rhodamine labeled cholesterol reporter. Biochim. Biophys. Acta Biomembr. 1859;1099–1113:2017. doi: 10.1016/j.bbamem.2017.02.018. PubMed DOI

Mendez AJ. Cholesterol efflux mediated by apolipoproteins is an active cellular process distinct from efflux mediated by passive diffusion. J. Lipid Res. 1997;38:1807–1821. doi: 10.1016/S0022-2275(20)37155-8. PubMed DOI

Sankaranarayanan S, et al. Serum albumin acts as a shuttle to enhance cholesterol efflux from cells. J. Lipid Res. 2013;54:671–676. doi: 10.1194/jlr.M031336. PubMed DOI PMC

Chen W, et al. Preferential ATP-binding cassette transporter A1-mediated cholesterol efflux from late endosomes/lysosomes. J. Biol. Chem. 2001;276:43564–43569. doi: 10.1074/jbc.M107938200. PubMed DOI

Lusa S, et al. Depletion of rafts in late endocytic membranes is controlled by NPC1-dependent recycling of cholesterol to the plasma membrane. J. Cell Sci. 2001;114:1893–1900. doi: 10.1242/jcs.114.10.1893. PubMed DOI

Davis DM, Sowinski S. Membrane nanotubes: Dynamic long-distance connections between animal cells. Nat. Rev. Mol. Cell Biol. 2008;9:431–436. doi: 10.1038/nrm2399. PubMed DOI

Crespo AC, et al. Decidual NK cells transfer granulysin to selectively kill bacteria in trophoblasts. Cell. 2020;182:1125–1139. doi: 10.1016/j.cell.2020.07.019. PubMed DOI PMC

Souriant S, et al. Tuberculosis exacerbates HIV-1 infection through IL-10/STAT3-dependent tunneling nanotube formation in macrophages. Cell Rep. 2019;26:3586–3599. doi: 10.1016/j.celrep.2019.02.091. PubMed DOI PMC

Strauss K, et al. Exosome secretion ameliorates lysosomal storage of cholesterol in Niemann-Pick type C disease. J. Biol. Chem. 2010;285:26279–26288. doi: 10.1074/jbc.M110.134775. PubMed DOI PMC

Wustner D, Mondal M, Tabas I, Maxfield FR. Direct observation of rapid internalization and intracellular transport of sterol by macrophage foam cells. Traffic. 2005;6:396–412. doi: 10.1111/j.1600-0854.2005.00285.x. PubMed DOI

Brasaemle DL, Wolins NE. Isolation of lipid droplets from cells by density gradient centrifugation. Curr. Protoc. Cell Biol. 2016;72:31511–131513. doi: 10.1002/cpcb.10. PubMed DOI PMC

McDonald JG, Thompson BM, McCrum EC, Russell DW. Extraction and analysis of sterols in biological matrices by high performance liquid chromatography electrospray ionization mass spectrometry. Lipidomics and Bioactive Lipids: Mass-Spectrometry-Based Lipid Analysis. 2007;432:145–170. doi: 10.1016/S0076-6879(07)32006-5. PubMed DOI

Li CH, Tam PKS. An iterative algorithm for minimum cross entropy thresholding. Pattern Recognit. Lett. 1998;19:771–776. doi: 10.1016/S0167-8655(98)00057-9. DOI