Monitoring Transcription Factor Oligomerization in Single Living Cells by Number and Brightness Analysis

  • Eugenia Cammarota
  • Davide MazzaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2038)


One key step in the activation of inducible transcription factors is their homooligomerization, which can be measured in individual living cells by a fluorescence microscopy technique called Number and Brightness analysis (N&B). In this chapter we describe how to acquire and analyze confocal microscopy time-series to provide information about transcription factor oligomerization in living cells using this technique.

Key words

Number and brightness Oligomerization Transcription factor Live cell imaging Image analysis 



We are grateful to Dr. Valeria Caiolfa and Dr. Moreno Zamai for their support on N&B analysis. Confocal imaging was carried out in ALEMBIC, an advanced microscopy laboratory established by IRCCS Ospedale San Raffaele and Università Vita-Salute San Raffaele. This work was supported by Fondazione Cariplo (E.C. and D.M.: 2014-1157).


  1. 1.
    Hager GL, McNally JG, Misteli T (2009) Transcription dynamics. Mol Cell 35:741–753. Scholar
  2. 2.
    Perkins ND (2007) Integrating cell-signalling pathways with NF-κB and IKK function. Nat Rev Mol Cell Biol 8:49–62. Scholar
  3. 3.
    Purvis JE, Karhohs KW, Mock C et al (2012) p53 dynamics control cell fate. Science 336:1440–1444. Scholar
  4. 4.
    Funnell APW, Crossley M (2012) Homo- and heterodimerization in transcriptional regulation. Adv Exp Med Biol 747:105–121. Scholar
  5. 5.
    Kitayner M, Rozenberg H, Kessler N et al (2006) Structural basis of DNA recognition by p53 tetramers. Mol Cell 22:741–753. Scholar
  6. 6.
    Gaglia G, Guan Y, Shah JV, Lahav G (2013) Activation and control of p53 tetramerization in individual living cells. Proc Natl Acad Sci 110:15497–15501. Scholar
  7. 7.
    Filtz TM, Vogel WK, Leid M (2014) Regulation of transcription factor activity by interconnected post-translational modifications. Trends Pharmacol Sci 35:76–85. Scholar
  8. 8.
    Loffreda A, Jacchetti E, Antunes S et al (2017) Live-cell p53 single-molecule binding is modulated by C-terminal acetylation and correlates with transcriptional activity. Nat Commun 8:313. Scholar
  9. 9.
    Liu Z, Tjian R (2018) Visualizing transcription factor dynamics in living cells. J Cell Biol 217:1181–1191. Scholar
  10. 10.
    Mueller F, Karpova TS, Mazza D, McNally JG (2012) Monitoring dynamic binding of chromatin proteins in vivo by fluorescence recovery after photobleaching. Methods Mol Biol 833:153–176. Scholar
  11. 11.
    Digman MA, Brown CM, Horwitz AR et al (2008) Paxillin dynamics measured during adhesion assembly and disassembly by correlation spectroscopy. Biophys J 94:2819–2831. Scholar
  12. 12.
    Mazza D, Stasevich TJ, Karpova TS, McNally JG (2012) Monitoring dynamic binding of chromatin proteins in vivo by fluorescence correlation spectroscopy. Methods Mol Biol 833:177–200. Scholar
  13. 13.
    Mazza D, Ganguly S, McNally JG (2013) Monitoring dynamic binding of chromatin proteins in vivo by single-molecule tracking. Methods Mol Biol. Scholar
  14. 14.
    Digman MA, Dalal R, Horwitz AF, Gratton E (2008) Mapping the number of molecules and brightness in the laser scanning microscope. Biophys J 94:2320–2332. Scholar
  15. 15.
    Presman DM, Ganguly S, Schiltz RL et al (2016) DNA binding triggers tetramerization of the glucocorticoid receptor in live cells. Proc Natl Acad Sci 113:8236–8241. Scholar
  16. 16.
    Day RN (2014) Measuring protein interactions using Förster resonance energy transfer and fluorescence lifetime imaging microscopy. Methods 66:200–207. Scholar
  17. 17.
    Bader AN, Hofman EG, Voortman J et al (2009) Homo-FRET imaging enables quantification of protein cluster sizes with subcellular resolution. Biophys J 97:2613–2622. Scholar
  18. 18.
    Liu Z, Xing D, Su QP et al (2014) Super-resolution imaging and tracking of protein–protein interactions in sub-diffraction cellular space. Nat Commun 5:4443. Scholar
  19. 19.
    Liesche C, Grußmayer KS, Ludwig M et al (2015) Automated analysis of single-molecule photobleaching data by statistical modeling of spot populations. Biophys J 109:2352–2362. Scholar
  20. 20.
    Chen B, Gilbert LA, Cimini BA et al (2013) Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155:1479–1491. Scholar
  21. 21.
    Zacharias DA, Violin JD, Newton AC, Tsien RY (2002) Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296:913–916. Scholar
  22. 22.
    Los GV, Encell LP, McDougall MG et al (2008) HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem Biol 3:373–382. Scholar
  23. 23.
    Grimm JB, English BP, Chen J et al (2015) A general method to improve fluorophores for live-cell and single-molecule microscopy. Nat Methods 12:244–250. Scholar
  24. 24.
    Dalal RB, Digman MA, Horwitz AF et al (2008) Determination of particle number and brightness using a laser scanning confocal microscope operating in the analog mode. Microsc Res Tech 71:69–81. Scholar
  25. 25.
    Unruh JR, Gratton E (2008) Analysis of molecular concentration and brightness from fluorescence fluctuation data with an electron multiplied CCD camera. Biophys J 95:5385–5398. Scholar
  26. 26.
    Nolan R, Iliopoulou M, Alvarez L, Padilla-Parra S (2018) Detecting protein aggregation and interaction in live cells: a guide to number and brightness. Methods 140–141:172–177. Scholar
  27. 27.
    Hellriegel C, Caiolfa VR, Corti V et al (2011) Number and brightness image analysis reveals ATF-induced dimerization kinetics of uPAR in the cell membrane. FASEB J 25:2883–2897. Scholar
  28. 28.
    Nolan R, Alvarez LAJ, Elegheert J et al (2017) nandb—number and brightness in R with a novel automatic detrending algorithm. Bioinformatics 33:3508–3510. Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Istituto Scientifico Ospedale San Raffaele, Centro di Imaging SperimentaleMilanItaly
  2. 2.Fondazione CEN, European Center for NanomedicineMilanItaly

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