Fluorescence lifetime imaging for studying DNA compaction and gene activities

Svitlana M. Levchenko; Artem Pliss; Xiao Peng; Paras N. Prasad; Junle Qu

doi:10.1038/s41377-021-00664-w

Go to Nature Website

Your Location：

Home >

Browse articles >

Fluorescence lifetime imaging for studying DNA compaction and gene activities

Articles

- Fluorescence lifetime imaging for studying DNA compaction and gene activities
- Light: Science & Applications Vol. 10, Issue 12, Pages: 2349-2359(2021)
- Affiliations：
  
  1.Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Physics and Optoelectronic Engineering, Shenzhen University, Shenzhen, Guangdong 518060, China
  2.Department of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387 Krakow, Poland
  3.Institute for Lasers, Photonics and Biophotonics, University at Buffalo, State University of New York, Buffalo, NY 14260-3000, USA
- Author bio：
  
  Paras N. Prasad (pnprasad@buffalo.edu)
  Junle Qu (jlqu@szu.edu.cn)
- Funds：
- DOI：10.1038/s41377-021-00664-w
  CLC：
- Published：31 December 2021，
  
  Published Online：02 November 2021，
  
  Received：25 March 2021，
  
  Revised：04 October 2021，
  
  Accepted：18 October 2021
- Accepted：
Scan QR Code
Levchenko, S. M. et al. Fluorescence lifetime imaging for studying DNA compaction and gene activities. Light: Science & Applications, 10, 2349-2359 (2021).
DOI：

Levchenko, S. M. et al. Fluorescence lifetime imaging for studying DNA compaction and gene activities. Light: Science & Applications, 10, 2349-2359 (2021). DOI： 10.1038/s41377-021-00664-w.

摘要

Abstract

Optical imaging is a most useful and widespread technique for the investigation of the structure and function of the cellular genomes. However

an analysis of immensely convoluted and irregularly compacted DNA polymer is highly challenging even by modern super-resolution microscopy approaches. Here we propose fluorescence lifetime imaging (FLIM) for the advancement of studies of genomic structure including DNA compaction

replication as well as monitoring of gene expression. The proposed FLIM assay employs two independent mechanisms for DNA compaction sensing. One mechanism relies on the inverse quadratic relation between the fluorescence lifetimes of fluorescence probes incorporated into DNA and their local refractive index

variable due to DNA compaction density. Another mechanism is based on the Förster resonance energy transfer (FRET) process between the donor and the acceptor fluorophores

both incorporated into DNA. Both these proposed mechanisms were validated in cultured cells. The obtained data unravel a significant difference in compaction of the gene-rich and gene-poor pools of genomic DNA. We show that the gene-rich DNA is loosely compacted compared to the dense DNA domains devoid of active genes.

关键词

Keywords

references

Stephens, D. J.&Allan, V. J. Light microscopy techniques for live cell imaging.Science300, 82–86 (2003)..

Xu, J. Q.&Liu, Y. A guide to visualizing the spatial epigenome with super-resolution microscopy.FEBS J.286, 3095–3109 (2019)..

Hoover, E. E.&Squier, J. A. Advances in multiphoton microscopy technology.Nat. Photonics7, 93–101 (2013)..

Huang, B., Babcock, H.&Zhuang, X. W. Breaking the diffraction barrier: super-resolution imaging of cells.Cell143, 1047–1058 (2010)..

Sigal, Y. M., Zhou, R. B.&Zhuang, X. W. Visualizing and discovering cellular structures with super-resolution microscopy.Science361, 880–887 (2018)..

Müller, T., Schumann, C.&Kraegeloh, A. STED microscopy and its applications: new insights into cellular processes on the nanoscale.ChemPhysChem13, 1986–2000 (2012)..

Liu, W. J. et al. Breaking the axial diffraction limit: a guide to axial super-resolution fluorescence microscopy.Laser Photonics Rev.12, 1700333 (2018)..

Huang, B. et al. Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy.Science319, 810–813 (2008)..

Audugé, N. et al. Chromatin condensation fluctuations rather than steady-state predict chromatin accessibility.Nucleic Acids Res.47, 6184–6194 (2019)..

Sparks, H. et al. Heterogeneity in tumor chromatin-doxorubicin binding revealed by in vivo fluorescence lifetime imaging confocal endomicroscopy.Nat. Commun.9, 2662 (2018)..

Wallrabe, H.&Periasamy, A. Imaging protein molecules using FRET and FLIM microscopy.Curr. Opin. Biotechnol.16, 19–27 (2005)..

Llères, D. et al. Quantitative analysis of chromatin compaction in living cells using FLIM-FRET.J. Cell Biol.187, 481–496 (2009)..

Becker, W.Advanced Time-Correlated Single Photon Counting Applications(New York: Springer, 2015).

Sherrard, A. et al. Streamlined histone-based fluorescence lifetime imaging microscopy (FLIM) for studying chromatin organisation.Biol. Open7, bio031476 (2018)..

Strickler, S. J.&Berg, R. A. Relationship between absorption intensity and fluorescence lifetime of molecules.J. Chem. Phys.37, 814–822 (1962)..

Pliss, A. et al. Fluorescence lifetime of fluorescent proteins as an intracellular environment probe sensing the cell cycle progression.ACS Chem. Biol.7, 1385–1392 (2012)..

Pliss, A. et al. Single cell assay for molecular diagnostics and medicine: monitoring intracellular concentrations of macromolecules by two-photon fluorescence lifetime imaging.Theranostics5, 919–930 (2015)..

Pliss, A. et al. Cycles of protein condensation and discharge in nuclear organelles studied by fluorescence lifetime imaging.Nat. Commun.10, 455 (2019)..

Suhling, K. et al. Imaging the environment of green fluorescent protein.Biophys. J.83, 3589–3595 (2002)..

Pliss, A.&Prasad, P. N. High resolution mapping of subcellular refractive index by Fluorescence Lifetime Imaging: a next frontier in quantitative cell science?Methods Appl. Fluorescence8, 032001 (2020)..

Fritz, A. J. et al. Chromosome territories and the global regulation of the genome.Genes, Chromosomes Cancer58, 407–426 (2019)..

Hübner, M. R., Eckersley-Maslin, M. A.&Spector, D. L. Chromatin organization and transcriptional regulation.Curr. Opin. Genet. Dev.23, 89–95 (2013)..

Oudet, P., Gross-Bellard, M.&Chambon, P. Electron microscopic and biochemical evidence that chromatin structure is a repeating unit.Cell4, 281–300 (1975)..

Osborne, C. S. et al. Active genes dynamically colocalize to shared sites of ongoing transcription.Nat. Genet.36, 1065–1071 (2004)..

Volpi, E. V. et al. Large-scale chromatin organization of the major histocompatibility complex and other regions of human chromosome 6 and its response to interferon in interphase nuclei.J. Cell Sci.113, 1565–1576 (2000)..

Fritz, A. J. et al. Chromosome territories and the global regulation of the genome.Genes, Chromosomes Cancer58, 407–426 (2019)..

Eltsov, M. et al. Analysis of cryo-electron microscopy images does not support the existence of 30-nm chromatin fibers in mitotic chromosomes in situ.Proc. Natl Acad. Sci. USA105, 19732–19737 (2008)..

Ma, H. et al. Spatial and temporal dynamics of DNA replication sites in mammalian cells.J. Cell Biol.143, 1415–1425 (1998)..

Koberna, K. et al. Electron microscopy of DNA replication in 3-D: evidence for similar-sized replication foci throughout S-phase.J. Cell. Biochem.94, 126–138 (2005)..

Pliss, A. et al. Chromatin dynamics is correlated with replication timing.Chromosoma118, 459–470 (2009)..

Pliss, A. et al. Chromatin dynamics in living cells: Identification of oscillatory motion.J. Cell Physiol.228, 609–616 (2013)..

Luger, K. et al. Crystal structure of the nucleosome core particle at 2.8 Å resolution.Nature389, 251–260 (1997)..

Flors, C.&Earnshaw, W. C. Super-resolution fluorescence microscopy as a tool to study the nanoscale organization of chromosomes.Curr. Opin. Chem. Biol.15, 838–844 (2011)..

Schwarzer, W. et al. Two independent modes of chromatin organization revealed by cohesin removal.Nature551, 51–56 (2017)..

Mir, M. et al. Chromatin topology, condensates and gene regulation: shifting paradigms or just a phase?Development146, dev182766 (2019)..

Dans, P. D. et al. Multiscale simulation of DNA.Curr. Opin. Struct. Biol.37, 29–45 (2016)..

Maeshima, K., Hihara, S.&Takata, H. New insight into the mitotic chromosome structure: irregular folding of nucleosome fibers without 30-nm chromatin structure.Cold Spring Harb. Symposia Quant. Biol.75, 439–444 (2010)..

Maeshima, K., Hihara, S.&Eltsov, M. Chromatin structure: does the 30-nm fibre exist in vivo?Curr. Opin. Cell Biol.22, 291–297 (2010)..

Spagnol, S. T.&Dahl, K. N. Spatially resolved quantification of chromatin condensation through differential local rheology in cell nuclei fluorescence lifetime imaging.PLoS ONE11, e0146244 (2016)..

Abdollahi, E., Taucher-Scholz, G.&Jakob, B. Application of fluorescence lifetime imaging microscopy of DNA binding dyes to assess radiation-induced chromatin compaction changes.Int. J. Mol. Sci.19, 2399 (2018)..

Auksorius, E. et al. Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging.Opt. Lett.33, 113–115 (2008)..

Lesoine, M.D. et al. Supercontinuum stimulated emission depletion fluorescence lifetime imaging.J. Phys. Chem. B116, 7821–7826 (2012)..

Allshire, R. C.&Madhani, H. D. Ten principles of heterochromatin formation and function.Nat. Rev. Mol. Cell Biol.19, 229–244 (2018)..

Salzman, N. P., Moore, D. E.&Mendelsohn, J. Isolation and characterization of human metaphase chromosomes.Proc. Natl Acad. Sci. USA56, 1449–1456 (1966)..

Di Primo, C.&Lebars, I. Determination of refractive index increment ratios for protein-nucleic acid complexes by surface plasmon resonance.Anal. Biochem.368, 148–155 (2007)..

Pedrali-Noy, G. et al. Synchronization of HeLa cell cultures by inhibition of DNA polymeraseawith aphidicolin.Nucleic Acids Res.8, 377–387 (1980)..

Raska, I. et al. Ultrastructural cryoimmunocytochemistry is a convenient tool for the study of DNA replication in cultured cells.J. Electron Microsc. Tech.18, 91–105 (1991)..

Nakayasu, H.&Berezney, R. Mapping replicational sites in the eucaryotic cell nucleus.J. Cell Biol.108, 1–11 (1989)..

Dimitrova, D. S.&Berezney, R. The spatio-temporal organization of DNA replication sites is identical in primary, immortalized and transformed mammalian cells.J. Cell Sci.115, 4037–4051 (2002)..

Zink, D. et al. Structure and dynamics of human interphase chromosome territories in vivo.Hum. Genet.102, 241–251 (1998)..

Kurz, A. et al. Active and inactive genes localize preferentially in the periphery of chromosome territories.J. Cell Biol.135, 1195–1205 (1996)..

Aten, J. A. et al. DNA double labelling with IdUrd and cldurd for spatial and temporal analysis of cell proliferation and DNA replication.Histochemical J.24, 251–259 (1992)..

Koberna, K. et al. Nuclear organization studied with the help of a hypotonic shift: its use permits hydrophilic molecules to enter into living cells.Chromosoma108, 325–335 (1999)..

Llères, D. et al. Quantitative FLIM-FRET microscopy to monitor nanoscale chromatin compaction in vivo reveals structural roles of condensin complexes.Cell Rep.18, 1791–1803 (2017)..

Berezney, R., Dubey, D. D.&Huberman, J. A. Heterogeneity of eukaryotic replicons, replicon clusters, and replication foci.Chromosoma108, 471–484 (2000)..

Palazzo, A. F.&Gregory, T. R. The case for junk DNA.PLoS Genet.10, e1004351 (2014)..

Levchenko, S. M., Pliss, A.&Qu, J. L. Fluorescence lifetime imaging of fluorescent proteins as an effective quantitative tool for noninvasive study of intracellular processes.J. Innovative Optical Health Sci.11, 1730009 (2018)..

Becker, W.The bh TCSPC Handbook. 8th edn. 954 (Becker&Hickl, 2019).

Views

Downloads

CSCD

Alert me when the article has been cited

Submit

Tools

Publicity Resources

No data

Related Author

No data

Related Institution

No data

⁰