Advertisement

Synthesis and Live-Cell Imaging of Fluorescent Sterols for Analysis of Intracellular Cholesterol Transport

  • Maciej Modzel
  • Frederik W. Lund
  • Daniel WüstnerEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1583)

Abstract

Cellular cholesterol homeostasis relies on precise control of the sterol content of organelle membranes. Obtaining insight into cholesterol trafficking pathways and kinetics by live-cell imaging relies on two conditions. First, one needs to develop suitable analogs that resemble cholesterol as closely as possible with respect to their biophysical and biochemical properties. Second, the cholesterol analogs should have good fluorescence properties. This interferes, however, often with the first requirement, such that the imaging instrumentation must be optimized to collect photons from suboptimal fluorophores, but good cholesterol mimics, such as the intrinsically fluorescent sterols, cholestatrienol (CTL) or dehydroergosterol (DHE). CTL differs from cholesterol only in having two additional double bonds in the ring system, which is why it is slightly fluorescent in the ultraviolet (UV). In the first part of this protocol, we describe how to synthesize and image CTL in living cells relative to caveolin, a structural component of caveolae. In the second part, we explain in detail how to perform time-lapse experiments of commercially available BODIPY-tagged cholesterol (TopFluor-cholesterol®; TF-Chol) in comparison to DHE. Finally, using two-photon time-lapse imaging data of TF-Chol, we demonstrate how to use our imaging toolbox SpatTrack for tracking sterol rich vesicles in living cells over time.

Key words

Cholesterol trafficking Fluorescent sterols Life-cell imaging SpatTrack 

Supplementary material

Supplemental Video 1

Dynamics of dehydroergosterol and TopFluor-cholesterol in CHO cells. CHO cells were co-labeled with DHE and TF-Chol, as described in the main text. Cells were washed, incubated in buffer medium for an additional 30 min at 37 °C and imaged on a UV-sensitive wide field microscope, as described in the main text. Cells were repeatedly imaged, first in the UV channel to follow the dynamics of DHE (left panel) followed by imaging in the green channel to follow TF-Chol (right panel), both at a 2 Hz acquisition rate. The grey-scale images were first normalized to keep the overall intensity constant . This is a crude correction for bleaching, which also amplifies noise for weak and decaying signals. Images were thereafter inverted for better visibility of small moving structures. Clearly, the DHE image is noisy and gets more noisy during the time lapse sequence, as the sterol bleaches for repeated acquisitions. The TF-Chol is much more photostable and allows for following the movement of small sterol-rich vesicles (AVI 1359 kb).

References

  1. 1.
    Solanko KA, Modzel M, Solanko LM, Wüstner D (2016) Fluorescent sterols and cholesteryl esters as probes for intracellular cholesterol transport. Lipid Insights 8(Suppl 1):95–114PubMedPubMedCentralGoogle Scholar
  2. 2.
    Wüstner D, Lund FW, Röhrl C, Stangl H (2015) Potential of BODIPY-cholesterol for analysis of cholesterol transport and diffusion in living cells. Chem Phys Lipids 194:12–28CrossRefPubMedGoogle Scholar
  3. 3.
    Li CH, Bai L, Li DD, Xia S, Xu T (2004) Dynamic tracking and mobility analysis of single GLUT4 storage vesicle in live 3T3-L1 cells. Cell Res 14(6):480–486CrossRefPubMedGoogle Scholar
  4. 4.
    Lizunov VA, Stenkula K, Troy A, Cushman SW, Zimmerberg J (2013) Insulin regulates Glut 4 confinement in plasma membrane clusters in adipose cells. PLoS One 8(3):e57559CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Hao X, Shang X, Wu J, Shan Y, Cai M, Jiang J et al (2011) Single-particle tracking of hepatitis B virus-like vesicle entry into cells. Small 7(9):1212–1218CrossRefPubMedGoogle Scholar
  6. 6.
    Lund FW, Lomholt MA, Solanko LM, Bittman R, Wüstner D (2012) Two-photon time-lapse microscopy of BODIPY-cholesterol reveals anomalous sterol diffusion in chinese hamster ovary cells. BMC Biophys 18:5–20Google Scholar
  7. 7.
    Chen H, Yang J, Low PS, Cheng JX (2008) Cholesterol level regulates endosome motility via Rab proteins. Biophys J 94(4):1508–1520CrossRefPubMedGoogle Scholar
  8. 8.
    Pentchev PG, Comly ME, Kruth HS, Tokoro T, Butler J, Sokol J et al (1987) Group C Niemann-Pick disease: faulty regulation of low-density lipoprotein uptake and cholesterol storage in cultured fibroblasts. FASEB J 1(1):40–45PubMedGoogle Scholar
  9. 9.
    Liscum L, Ruggiero RM, Faust JR (1989) The intracellular transport of low density lipoprotein-derived cholesterol is defective in Niemann-Pick type C fibroblasts. J Cell Biol 108(5):1625–1636CrossRefPubMedGoogle Scholar
  10. 10.
    Lloyd-Evans E, Morgan AJ, He X, Smith DA, Elliot-Smith E, Sillence DJ et al (2008) Niemann-Pick disease type C1 is a sphingosine storage disease that causes deregulation of lysosomal calcium. Nat Med 14(11):1247–1255CrossRefPubMedGoogle Scholar
  11. 11.
    Zervas M, Dobrenis K, Walkley SU (2001) Neurons in Niemann-Pick disease type C accumulate gangliosides as well as unesterified cholesterol and undergo dendritic and axonal alterations. J Neuropathol Exp Neurol 60(1):49–64CrossRefPubMedGoogle Scholar
  12. 12.
    Sleat DE, Wiseman JA, El-Banna M, Price SM, Verot L, Shen MM et al (2004) Genetic evidence for nonredundant functional cooperativity between NPC1 and NPC2 in lipid transport. Proc Natl Acad Sci U S A 101(16):5886–5891CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Vanier MT, Millat G (2003) Niemann-Pick disease type C. Clin Genet 64(4):269–281CrossRefPubMedGoogle Scholar
  14. 14.
    Lund FW, Jensen ML, Christensen T, Nielsen GK, Heegaard CW, Wustner D (2014) SpatTrack: an imaging toolbox for analysis of vesicle motility and distribution in living cells. Traffic 15(12):1406–1429CrossRefPubMedGoogle Scholar
  15. 15.
    Thompson RE, Larson DR, Webb WW (2002) Precise nanometer localization analysis for individual fluorescent probes. Biophys J 82(5):2775–2783CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Yamashiro DJ, Tycko B, Fluss SR, Maxfield FR (1984) Segregation of transferrin to a mildly acidic (pH 6.5) para-Golgi compartment in the recycling pathway. Cell 37:789–800CrossRefPubMedGoogle Scholar
  17. 17.
    Wüstner D, Solanko LM, Sokol E, Lund FW, Garvik O, Li Z et al (2011) Quantitative assessment of sterol traffic in living cells by dual labeling with dehydroergosterol and BODIPY-cholesterol. Chem Phys Lipids 164(3):221–235CrossRefPubMedGoogle Scholar
  18. 18.
    Mukherjee S, Soe TT, Maxfield FR (1999) Endocytic sorting of lipid analogues differing solely in the chemistry of their hydrophobic tails. J Cell Biol 144:1271–1284CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Wüstner D (2006) Quantification of polarized trafficking of transferrin and comparison with bulk membrane transport in hepatic cells. Biochem J 400:267–280CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Salzmann NH, Maxfield FR (1989) Fusion accessibility of endocytic compartments along the recycling and lysosomal endocytic pathways in intact cells. J Cell Biol 109:2097–2104CrossRefGoogle Scholar
  21. 21.
    Wüstner D, Herrmann A, Hao M, Maxfield FR (2002) Rapid nonvesicular transport of sterol between the plasma membrane domains of polarized hepatic cells. J Biol Chem 277:30325–30336CrossRefPubMedGoogle Scholar
  22. 22.
    Wüstner D, Færgeman NJ (2008) Chromatic aberration correction and deconvolution for UV sensitive imaging of fluorescent sterols in cytoplasmic lipid droplets. Cytometry A 73(8):727–744CrossRefPubMedGoogle Scholar
  23. 23.
    Thevenaz P, Ruttimann UE, Unser E (1998) A pyramid approach to subpixel registration based on intensity. IEEE Trans Image Process 7:27–41CrossRefPubMedGoogle Scholar
  24. 24.
    Hao M, Lin SX, Karylowski OJ, Wüstner D, McGraw TE, Maxfield FR (2002) Vesicular and non-vesicular sterol transport in living cells. The endocytic recycling compartment is a major sterol storage organelle. J Biol Chem 277:609–617CrossRefPubMedGoogle Scholar
  25. 25.
    Mondal M, Mesmin B, Mukherjee S, Maxfield FR (2009) Sterols are mainly in the cytoplasmic leaflet of the plasma membrane and the endocytic recycling compartment in CHO cells. Mol Biol Cell 20(2):581–588CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Mesmin B, Pipalia NH, Lund FW, Ramlall TF, Sokolov A, Eliezer D et al (2011) STARD4 abundance regulates sterol transport and sensing. Mol Biol Cell 22(21):4004–4015CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Lund FW, Lomholt MA, Solanko LM, Wüstner D (2012) Two-photon time-lapse microscopy of BODIPY-cholesterol reveals anomalous sterol diffusion in Chinese hamster ovary cells. BMC Biophys 5:20CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Lin SX, Grant B, Hirsh D, Maxfield FR (2001) Rme-1 regulates the distribution and function of the endocytic recycling compartment in mammalian cells. Nat Cell Biol 3:567–572CrossRefPubMedGoogle Scholar
  29. 29.
    Ikonen E, Parton RG (2000) Caveolins and cellular cholesterol balance. Traffic 1:212–217CrossRefPubMedGoogle Scholar
  30. 30.
    Sharma DK, Brown JC, Choudhury A, Peterson TE, Holicky E, Marks DL et al (2004) Selective stimulation of caveolar endocytosis by glycosphingolipids and cholesterol. Mol Biol Cell 15:3114–3122CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Pol A, Luetterforst R, Lindsay M, Heino S, Ikonen E, Parton RG (2001) A caveolin dominant negative mutant associates with lipid bodies and induces intracellular cholesterol imbalance. J Cell Biol 152:1057–1070CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Baumgart T, Hunt G, Farkas ER, Webb WW, Feigenson GW (2007) Fluorescence probe partitioning between Lo/Ld phases in lipid membranes. Biochim Biophys Acta 1768(9):2182–2194CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Spink CH, Yeager MD, Feigenson GW (1990) Partitioning behavior of indocarbocyanine probes between coexisting gel and fluid phases in model membranes. Biochim Biophys Acta 1023:25–33CrossRefPubMedGoogle Scholar
  34. 34.
    Pitas RE, Innerarity TL, Weinstein JN, Mahley RW (1981) Acetoacetylated lipoproteins used to distinguish fibroblasts from macrophages in vitro by fluorescence microscopy. Arteriosclerosis 1:177–185CrossRefPubMedGoogle Scholar
  35. 35.
    Tabas I, Lim S, Xu XX, Maxfield FR (1990) Endocytosed beta-VLDL and LDL are delivered to different intracellular vesicles in mouse peritoneal macrophages. J Cell Biol 111:929–940CrossRefPubMedGoogle Scholar
  36. 36.
    Ghosh RN, Webb WW (1994) Automated detection and tracking of individual and clustered cell surface low density lipoprotein receptor molecules. Biophys J 66(5):1301–1318CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Garvik O, Benediktson P, Simonsen AC, Ipsen JH, Wüstner D (2009) The fluorescent cholesterol analog dehydroergosterol induces liquid-ordered domains in model membranes. Chem Phys Lipids 159(2):114–118CrossRefPubMedGoogle Scholar
  38. 38.
    Hao M, Mukherjee S, Sun Y, Maxfield FR (2004) Effects of cholesterol depletion and increased lipid unsaturation on the properties of endocytic membranes. J Biol Chem 279:14171–14178CrossRefPubMedGoogle Scholar
  39. 39.
    Wüstner D, Færgeman NJ (2008) Spatiotemporal analysis of endocytosis and membrane distribution of fluorescent sterols in living cells. Histochem Cell Biol 130(5):891–908CrossRefPubMedGoogle Scholar
  40. 40.
    Wüstner D (2007) Plasma membrane sterol distribution resembles the surface topography of living cells. Mol Biol Cell 18:211–228CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Shvets E, Bitsikas V, Howard G, Hansen CG, Nichols BJ (2015) Dynamic caveolae exclude bulk membrane proteins and are required for sorting of excess glycosphingolipids. Nat Commun 6:6867CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Du X, Kumar J, Ferguson C, Schulz TA, Ong YS, Hong W et al (2011) A role for oxysterol-binding protein-related protein 5 in endosomal cholesterol trafficking. J Cell Biol 192(1):121–135CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Lee HJ, Zhang W, Zhang D, Yang Y, Liu B, Barker EL et al (2015) Assessing cholesterol storage in live cells and C. elegans by stimulated Raman scattering imaging of phenyl-Diyne cholesterol. Sci Rep 5:7930CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Crocker JC, Grier DG (1996) Methods of digital video microscopy for colloidal studies. J Colloid Interface Sci 179:298–311CrossRefGoogle Scholar
  45. 45.
    Lund FW, Wustner D (2013) A comparison of single particle tracking and temporal image correlation spectroscopy for quantitative analysis of endosome motility. J Microsc 252(2):169–188CrossRefPubMedGoogle Scholar
  46. 46.
    Dixit R, Cyr R (2003) Cell damage and reactive oxygen species production induced by fluorescence microscopy: effect on mitosis and guidelines for non-invasive fluorescence microscopy. Plant J 36(2):280–290CrossRefPubMedGoogle Scholar
  47. 47.
    Saxton MJ (1993) Lateral diffusion in an archipelago. Single-particle diffusion. Biophys J 64:1766–1780CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Rudnick J, Gaspari G (1987) The shapes of random walks. Science 237(4813):384–389CrossRefPubMedGoogle Scholar
  49. 49.
    Wüstner D (2005) Improved visualization and quantitative analysis of fluorescent membrane sterol in polarized hepatic cells. J Microsc 220:47–64CrossRefPubMedGoogle Scholar
  50. 50.
    Prattes S, Horl G, Hammer A, Blaschitz A, Graier WF, Sattler W, Zechner R, Steyrer E (2000) Intracellular distribution and mobilization of unesterified cholesterol in adipocytes: triglyceride droplets are surrounded by cholesterol-rich ER-like surface layer structures. J Cell Sci 113:2977–2989PubMedGoogle Scholar
  51. 51.
    McGookey DJ, Anderson RW (1983) Morphological characterization of the cholesteryl ester cycle in cultured mouse macrophage foam cells. J Cell Biol 97:1156–1168CrossRefPubMedGoogle Scholar
  52. 52.
    Brown MS, Goldstein JL, Krieger M, Ho YK, Anderson RG (1979) Reversible accumulation of cholesteryl esters in macrophages incubated with acetylated lipoproteins. J Cell Biol 82:597–613CrossRefPubMedGoogle Scholar
  53. 53.
    Wüstner D, Mondal M, Tabas I, Maxfield FR (2005) Direct observation of rapid internalization and intracellular transport of sterol by macrophage foam cells. Traffic 6:396–412CrossRefPubMedGoogle Scholar
  54. 54.
    Li Q-T, Sawyer WH (1993) Effect of cholesteryl ester on the distribution of fluorescent cholesterol analogues in triacylglycerol-rich emulsions. Biochim Biophys Acta 1166:145–153CrossRefPubMedGoogle Scholar
  55. 55.
    Saito H, Minamida T, Arimoto I, Handa T, Miyajima K (1996) Physical states of surface and core lipids in lipid emulsions and apolipoprotein binding to the emulsion surface. J Biol Chem 271:15515–15520CrossRefPubMedGoogle Scholar
  56. 56.
    Listenberger LL, & Brown DA (2007) Fluorescent detection of lipid droplets and associated proteins. Curr Protoc Cell Biol 24(24.2)Google Scholar
  57. 57.
    Spandl J, White DJ, Peychl J, Thiele C (2009) Live cell multicolor imaging of lipid droplets with a new dye, LD540. Traffic 10(11):1579–1584CrossRefPubMedGoogle Scholar
  58. 58.
    Sezgin E, Betul Can F, Schneider F, Clausen MP, Galiani S, Stanly TA et al (2016) A comparative study on fluorescent cholesterol analogs as versatile cellular reporters. J Lipid Res 57(2):299–309PubMedPubMedCentralGoogle Scholar
  59. 59.
    Wustner D, Christensen T, Solanko LM, Sage D (2014) Photobleaching kinetics and time-integrated emission of fluorescent probes in cellular membranes. Molecules 19(8):11096–11130Google Scholar
  60. 60.
    Saxton MJ (1997) Single-particle tracking: the distribution of diffusion coefficients. Biophys J 72(4):1744–1753CrossRefPubMedPubMedCentralGoogle Scholar
  61. 61.
    Ernst D, Kohler J (2013) Measuring a diffusion coefficient by single-particle tracking: statistical analysis of experimental mean squared displacement curves. Phys Chem Chem Phys 15(3):845–849CrossRefPubMedGoogle Scholar
  62. 62.
    Davies M (2014) Long-lived reactive species formed on proteins induce changes in protein and lipid turnover. Free Radic Biol Med 75(Suppl 1):S6–S7Google Scholar
  63. 63.
    Qian H, Sheetz MP, Elson EL (1991) Single particle tracking. Analysis of diffusion and flow in two-dimensional systems. Biophys J 60(4):910–921CrossRefPubMedPubMedCentralGoogle Scholar
  64. 64.
    Umansky M, Weihs D (2012) Novel algorithm and MATLAB-based program for automated power law analysis of single particle, time-dependent mean-square displacement. Comput Phys Commun 183:1783–1792CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Maciej Modzel
    • 1
  • Frederik W. Lund
    • 1
    • 2
  • Daniel Wüstner
    • 1
    Email author
  1. 1.Department of Biochemistry and Molecular BiologyUniversity of Southern DenmarkOdense MDenmark
  2. 2.Department of BiochemistryWeill Medical College of Cornell UniversityNew YorkUSA

Personalised recommendations