Advertisement

Optical Microscopy and Spectroscopy for Epigenetic Modifications in Single Living Cells

  • Yi Cui
  • Joseph IrudayarajEmail author
Protocol
  • 849 Downloads
Part of the Methods in Pharmacology and Toxicology book series (MIPT)

Abstract

Optical imaging with high spatiotemporal resolution and analytical accuracy is becoming the mainstay of tools capable of deciphering molecular dynamics and activities in single living cells. Over the past decades, information obtained by optical imaging has greatly enriched and reshaped our knowledge of biology and medicine. Investigating epigenetic modifications by optical microscopy and spectroscopy is expected to be the wave of the future or might even become the norm to complement biomedical practice. Independent of classical genetic mechanisms, epigenetics has recently drawn substantial attention due to its extensive involvement in physiological and pathological processes, as well as its reversibility. In order to understand the real-time behaviors of epigenetic regulation, nanoscale inspection at the sub-second timescale is imperative. In this chapter we discuss the basics of state-of-the-art optical methods for life science research and their potential applications in imaging live-cell epigenetics. Moreover, with established experience in single-molecule detection, we provide practical guidance on how to choose and adapt optical instrumentations for different applications. Last, recent advancements and representative examples in sensing live-cell epigenetics are reviewed.

Key words

Live-cell imaging Single-molecule detection Phototoxicity Spatiotemporal resolution Super-resolution microscopy Light-sheet microscopy FCS FRAP FRET DNA methylation Histone modification Chromatin dynamics 

Notes

Acknowledgement

The authors gratefully acknowledge funding from the W.M. Keck Foundation, National Science Foundation (#1249315), and Purdue Center for Cancer Research Core grant NIH-NCI P30CA023168.

References

  1. 1.
    Cui Y, Irudayaraj J (2015) Inside single cells: quantitative analysis with advanced optics and nanomaterials. Wiley Interdiscip Rev Nanomed Nanobiotechnol 7:387–407CrossRefPubMedGoogle Scholar
  2. 2.
    Magidson V, Khodjakov A (2013) Circumventing photodamage in live-cell microscopy. Method Cell Biol 114:545–560CrossRefGoogle Scholar
  3. 3.
    Huisken J, Stainier DYR (2009) Selective plane illumination microscopy techniques in developmental biology. Development 136:1963–1975CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Gomez D, Shankman LS, Nguyen AT, Owens GK (2013) Detection of histone modifications at specific gene loci in single cells in histological sections. Nat Methods 10:171–177CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Miyanari Y, Ziegler-Birling C, Torres-Padilla ME (2013) Live visualization of chromatin dynamics with fluorescent TALEs. Nat Struct Mol Biol 20:1321–1324CrossRefPubMedGoogle Scholar
  6. 6.
    Anton T, Bultmann S, Leonhardt H, Markaki Y (2014) Visualization of specific DNA sequences in living mouse embryonic stem cells with a programmable fluorescent CRISPR/Cas system. Nucleus 5:163–172CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Magde D, Webb WW, Elson E (1972) Thermodynamic fluctuations in a reacting system—measurement by fluorescence correlation spectroscopy. Phys Rev Lett 29:705CrossRefGoogle Scholar
  8. 8.
    Chen Y, Muller JD, So PT, Gratton E (1999) The photon counting histogram in fluorescence fluctuation spectroscopy. Biophys J 77:553–567CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Chen J, Nag S, Vidi PA, Irudayaraj J (2011) Single molecule in vivo analysis of toll-like receptor 9 and CpG DNA interaction. Plos One 6, e17991CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Cui Y, Irudayaraj J (2015) Dissecting the behavior and function of MBD3 in DNA methylation homeostasis by single-molecule spectroscopy and microscopy. Nucleic Acids Res 43:3046–3055CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Yamagata K, Yamazaki T, Yamashita M, Hara Y, Ogonuki N, Ogura A (2005) Noninvasive visualization of molecular events in the mammalian zygote. Genesis 43:71–79CrossRefPubMedGoogle Scholar
  12. 12.
    Yamazaki T, Yamagata K, Baba T (2007) Time-lapse and retrospective analysis of DNA methylation in mouse preimplantation embryos by live cell imaging. Dev Biol 304:409–419CrossRefPubMedGoogle Scholar
  13. 13.
    Yamagata K (2008) Capturing epigenetic dynamics during pre-implantation development using live cell imaging. J Biochem 143:279–286CrossRefPubMedGoogle Scholar
  14. 14.
    Cui Y, Cho IH, Chowdhury B, Irudayaraj J (2013) Real-time dynamics of methyl-CpG-binding domain protein 3 and its role in DNA demethylation by fluorescence correlation spectroscopy. Epigenetics 8:1089–1100CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Kapusta P, Machan R, Benda A, Hof M (2012) Fluorescence lifetime correlation spectroscopy (FLCS): concepts, applications and outlook. Int J Mol Sci 13:12890–12910CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Frauer C, Rottach A, Meilinger D, Bultmann S, Fellinger K, Hasenoder S, Wang MX, Qin WH, Soding J, Spada F, Leonhardt H (2011) Different binding properties and function of CXXC zinc finger domains in Dnmt1 and Tet1. Plos One 6Google Scholar
  17. 17.
    Schneider K, Fuchs C, Dobay A, Rottach A, Qin W, Wolf P, Alvarez-Castro JM, Nalaskowski MM, Kremmer E, Schmid V, Leonhardt H, Schermelleh L (2013) Dissection of cell cycle-dependent dynamics of Dnmt1 by FRAP and diffusion-coupled modeling. Nucleic Acids Res 41:4860–4876CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Chowdhury B, McGovern A, Cui Y, Choudhury SR, Cho IH, Cooper B, Chevassut T, Lossie AC, Irudayaraj J (2015) The hypomethylating agent Decitabine causes a paradoxical increase in 5-hydroxymethylcytosine in human leukemia cells. Sci Rep 5:9281CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Lin CW, Jao CY, Ting AY (2004) Genetically encoded fluorescent reporters of histone methylation in living cells. J Am Chem Soc 126:5982–5983CrossRefPubMedGoogle Scholar
  20. 20.
    Lin CW, Ting AY (2004) A genetically encoded fluorescent reporter of histone phosphorylation in living cells. Angew Chem Int Ed Engl 43:2940–2943CrossRefPubMedGoogle Scholar
  21. 21.
    Sasaki K, Ito T, Nishino N, Khochbin S, Yoshida M (2009) Real-time imaging of histone H4 hyperacetylation in living cells. Proc Natl Acad Sci U S A 106:16257–16262CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Ito T, Umehara T, Sasaki K, Nakamura Y, Nishino N, Terada T, Shirouzu M, Padmanabhan B, Yokoyama S, Ito A, Yoshida M (2011) Real-time imaging of histone H4K12-specific acetylation determines the modes of action of histone deacetylase and bromodomain inhibitors. Chem Biol 18:495–507CrossRefPubMedGoogle Scholar
  23. 23.
    Sasaki K, Ito A, Yoshida M (2012) Development of live-cell imaging probes for monitoring histone modifications. Bioorg Med Chem 20:1887–1892CrossRefPubMedGoogle Scholar
  24. 24.
    Erickson HP (2009) Size and shape of protein molecules at the nanometer level determined by sedimentation, gel filtration, and electron microscopy. Biol Proced Online 11:32–51CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Hayashi-Takanaka Y, Yamagata K, Nozaki N, Kimura H (2009) Visualizing histone modifications in living cells: spatiotemporal dynamics of H3 phosphorylation during interphase. J Cell Biol 187:781–790CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Johansen KM, Johansen J (2006) Regulation of chromatin structure by histone H3S10 phosphorylation. Chromosome Res 14:393–404CrossRefPubMedGoogle Scholar
  27. 27.
    Hayashi-Takanaka Y, Yamagata K, Wakayama T, Stasevich TJ, Kainuma T, Tsurimoto T, Tachibana M, Shinkai Y, Kurumizaka H, Nozaki N, Kimura H (2011) Tracking epigenetic histone modifications in single cells using Fab-based live endogenous modification labeling. Nucleic Acids Res 39:6475–6488CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Sato Y, Mukai M, Ueda J, Muraki M, Stasevich TJ, Horikoshi N, Kujirai T, Kita H, Kimura T, Hira S, Okada Y, Hayashi-Takanaka Y, Obuse C, Kurumizaka H, Kawahara A, Yamagata K, Nozaki N, Kimura H (2013) Genetically encoded system to track histone modification in vivo. Sci Rep 3:2436PubMedPubMedCentralGoogle Scholar
  29. 29.
    Lleres D, James J, Swift S, Norman DG, Lamond AI (2009) Quantitative analysis of chromatin compaction in living cells using FLIM-FRET. J Cell Biol 187:481–496CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Recamier V, Izeddin I, Bosanac L, Dahan M, Proux F, Darzacq X (2014) Single cell correlation fractal dimension of chromatin A framework to interpret 3D single molecule super-resolution. Nucleus 5:75–84CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Hu YS, Zhu Q, Elkins K, Tse K, Li Y, Fitzpatrick JA, Verma IM, Cang H (2013) Light-sheet Bayesian microscopy enables deep-cell super-resolution imaging of heterochromatin in live human embryonic stem cells. Opt Nanoscopy 2:7CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Liu J, Vidi PA, Lelievre SA, Irudayaraj JM (2015) Nanoscale histone localization in live cells reveals reduced chromatin mobility in response to DNA damage. J Cell Sci 128:599–604CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Hihara S, Pack CG, Kaizu K, Tani T, Hanafusa T, Nozaki T, Takemoto S, Yoshimi T, Yokota H, Imamoto N, Sako Y, Kinjo M, Takahashi K, Nagai T, Maeshima K (2012) Local nucleosome dynamics facilitate chromatin accessibility in living mammalian cells. Cell Rep 2:1645–1656CrossRefPubMedGoogle Scholar
  34. 34.
    Nozaki T, Kaizu K, Pack CG, Tamura S, Tani T, Hihara S, Nagai T, Takahashi K, Maeshima K (2013) Flexible and dynamic nucleosome fiber in living mammalian cells. Nucleus 4:349–356CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of Agricultural and Biological Engineering, Bindley Bioscience Centre, Purdue Center for Cancer ResearchPurdue UniversityWest LafayetteUSA

Personalised recommendations