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An In Situ Fluorescence Assay for Cholesterol Transporter Activity of the Patched

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Hedgehog Signaling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2374))

Abstract

We recently developed a simultaneous in situ quantitative imaging technique for cholesterol in both leaflets of the plasma membrane of mammalian cells. This ratiometric fluorescence technique allows real-time monitoring of the cholesterol transporter activity of plasma membrane-resident proteins in living cells. When applied to the hedgehog signaling system, it enables direct quantitative measurement of the cholesterol transporter activity of Patched1 and the effect of the hedgehog ligand on this activity.

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Abbreviations

DMSO:

Dimethylsulfoxide

GUVs:

Giant unilamellar vesicles

Hh:

Hedgehog

IPM:

Inner leaflet of plasma membrane

OPM:

Outer leaflet of plasma membrane

PI:

Phosphatidylinositol

PM:

Plasma membrane

POPC:

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

POPE:

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine

POPS:

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine

PTCH1:

Patched1; PtdIns(4,5)P2, phosphatidylinositol-4,5-bisphosphate

References

  1. Yoon Y, Lee PJ, Kurilova S, Cho W (2011) In situ quantitative imaging of cellular lipids using molecular sensors. Nat Chem 3(11):868–874. nchem.1163 [pii]. https://doi.org/10.1038/nchem.1163

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Sharma A, Sun J, Singaram I, Ralko A, Lee D, Cho W (2020) Photostable and orthogonal solvatochromic fluorophores for simultaneous in situ quantification of multiple cellular signaling molecules. ACS Chem Biol 15(7):1913–1920. https://doi.org/10.1021/acschembio.0c00241

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Liu SL, Sheng R, O’Connor MJ, Cui Y, Yoon Y, Kurilova S, Lee D, Cho W (2014) Simultaneous in situ quantification of two cellular lipid pools using orthogonal fluorescent sensors. Angew Chem Int Ed Engl 53(52):14387–14391. https://doi.org/10.1002/anie.201408153

    Article  CAS  PubMed  Google Scholar 

  4. Liu SL, Sheng R, Jung JH, Wang L, Stec E, O’Connor MJ, Song S, Bikkavilli RK, Winn RA, Lee D, Baek K, Ueda K, Levitan I, Kim KP, Cho W (2017) Orthogonal lipid sensors identify transbilayer asymmetry of plasma membrane cholesterol. Nat Chem Biol 13(3):268–274. https://doi.org/10.1038/nchembio.2268

    Article  CAS  PubMed  Google Scholar 

  5. Liu SL, Wang ZG, Hu Y, Xin Y, Singaram I, Gorai S, Zhou X, Shim Y, Min JH, Gong LW, Hay N, Zhang J, Cho W (2018) Quantitative lipid imaging reveals a new signaling function of phosphatidylinositol-3,4-bisphophate: isoform- and site-specific activation of Akt. Mol Cell 71(6):1092–1104.e1095. https://doi.org/10.1016/j.molcel.2018.07.035

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Yeagle PL (1985) Cholesterol and the cell membrane. Biochim Biophys Acta 822(3–4):267–287

    Article  CAS  Google Scholar 

  7. Yeagle PL (1991) Modulation of membrane function by cholesterol. Biochimie 73(10):1303–1310. https://doi.org/10.1016/0300-9084(91)90093-g

    Article  CAS  PubMed  Google Scholar 

  8. Maxfield FR, Tabas I (2005) Role of cholesterol and lipid organization in disease. Nature 438(7068):612–621. nature04399 [pii]. https://doi.org/10.1038/nature04399

    Article  CAS  PubMed  Google Scholar 

  9. Ikonen E (2008) Cellular cholesterol trafficking and compartmentalization. Nat Rev Mol Cell Biol 9(2):125–138. https://doi.org/10.1038/nrm2336

    Article  CAS  PubMed  Google Scholar 

  10. Demel RA, Bruckdorfer KR, van Deenen LL (1972) The effect of sterol structure on the permeability of lipomes to glucose, glycerol and Rb +. Biochim Biophys Acta 255(1):321–330. https://doi.org/10.1016/0005-2736(72)90031-4

    Article  CAS  PubMed  Google Scholar 

  11. Lingwood D, Simons K (2010) Lipid rafts as a membrane-organizing principle. Science 327(5961):46–50. 327/5961/46 [pii]. https://doi.org/10.1126/science.1174621

    Article  CAS  PubMed  Google Scholar 

  12. Fantini J, Barrantes FJ (2013) How cholesterol interacts with membrane proteins: an exploration of cholesterol-binding sites including CRAC, CARC, and tilted domains. Front Physiol 4:31. https://doi.org/10.3389/fphys.2013.00031

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Sheng R, Chen Y, Yung Gee H, Stec E, Melowic HR, Blatner NR, Tun MP, Kim Y, Kallberg M, Fujiwara TK, Hye Hong J, Pyo Kim K, Lu H, Kusumi A, Goo Lee M, Cho W (2012) Cholesterol modulates cell signaling and protein networking by specifically interacting with PDZ domain-containing scaffold proteins. Nat Commun 3:1249. ncomms2221 [pii]. https://doi.org/10.1038/ncomms2221

    Article  CAS  PubMed  Google Scholar 

  14. Sheng R, Kim H, Lee H, Xin Y, Chen Y, Tian W, Cui Y, Choi JC, Doh J, Han JK, Cho W (2014) Cholesterol selectively activates canonical Wnt signalling over non-canonical Wnt signalling. Nat Commun 5:4393. https://doi.org/10.1038/ncomms5393

    Article  CAS  PubMed  Google Scholar 

  15. Francis KR, Ton AN, Xin Y, O’Halloran PE, Wassif CA, Malik N, Williams IM, Cluzeau CV, Trivedi NS, Pavan WJ, Cho W, Westphal H, Porter FD (2016) Modeling Smith-Lemli-Opitz syndrome with induced pluripotent stem cells reveals a causal role for Wnt/beta-catenin defects in neuronal cholesterol synthesis phenotypes. Nat Med 22(4):388–396. https://doi.org/10.1038/nm.4067

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Huang P, Nedelcu D, Watanabe M, Jao C, Kim Y, Liu J, Salic A (2016) Cellular cholesterol directly activates smoothened in Hedgehog signaling. Cell 166(5):1176–1187.e1114. https://doi.org/10.1016/j.cell.2016.08.003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Huang P, Zheng S, Wierbowski BM, Kim Y, Nedelcu D, Aravena L, Liu J, Kruse AC, Salic A (2018) Structural basis of smoothened activation in Hedgehog signaling. Cell 174(2):312–324.e316. https://doi.org/10.1016/j.cell.2018.04.029

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Beachy PA, Hymowitz SG, Lazarus RA, Leahy DJ, Siebold C (2010) Interactions between Hedgehog proteins and their binding partners come into view. Genes Dev 24(18):2001–2012. https://doi.org/10.1101/gad.1951710

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Zhang Y, Bulkley DP, Xin Y, Roberts KJ, Asarnow DE, Sharma A, Myers BR, Cho W, Cheng Y, Beachy PA (2018) Structural basis for cholesterol transport-like activity of the Hedgehog receptor patched. Cell 175(5):1352–1364.e1314. https://doi.org/10.1016/j.cell.2018.10.026

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Bagatolli LA, Gratton E (1999) Two-photon fluorescence microscopy observation of shape changes at the phase transition in phospholipid giant unilamellar vesicles. Biophys J 77(4):2090–2101

    Article  CAS  Google Scholar 

  21. Yoon Y, Tong J, Lee PJ, Albanese A, Bhardwaj N, Kallberg M, Digman MA, Lu H, Gratton E, Shin YK, Cho W (2010) Molecular basis of the potent membrane-remodeling activity of the epsin 1 N-terminal homology domain. J Biol Chem 285(1):531–540. . M109.068015 [pii]. https://doi.org/10.1074/jbc.M109.068015

    Article  CAS  PubMed  Google Scholar 

  22. Yoon Y, Zhang X, Cho W (2012) Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) specifically induces membrane penetration and deformation by Bin/amphiphysin/Rvs (BAR) domains. J Biol Chem 287(41):34078–34090. https://doi.org/10.1074/jbc.M112.372789

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

The work is supported by a National Institutes of Health grant R35GM122530.

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Authors and Affiliations

  1. Department of Chemistry, University of Illinois at Chicago, Chicago, IL, USA

    Wonhwa Cho, Arthur Ralko & Ashutosh Sharma

Authors
  1. Wonhwa Cho
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  2. Arthur Ralko
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  3. Ashutosh Sharma
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Corresponding author

Correspondence to Wonhwa Cho .

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Editors and Affiliations

  1. Department of Molecular Genetics and Biophysics, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Xiaochun Li

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© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Cho, W., Ralko, A., Sharma, A. (2022). An In Situ Fluorescence Assay for Cholesterol Transporter Activity of the Patched. In: Li, X. (eds) Hedgehog Signaling. Methods in Molecular Biology, vol 2374. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1701-4_4

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  • DOI: https://doi.org/10.1007/978-1-0716-1701-4_4

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1700-7

  • Online ISBN: 978-1-0716-1701-4

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