Advertisement

Super-Resolution Microscopy and Tracking of DNA-Binding Proteins in Bacterial Cells

  • Stephan UphoffEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1431)

Abstract

The ability to detect individual fluorescent molecules inside living cells has enabled a range of powerful microscopy techniques that resolve biological processes on the molecular scale. These methods have also transformed the study of bacterial cell biology, which was previously obstructed by the limited spatial resolution of conventional microscopy. In the case of DNA-binding proteins, super-resolution microscopy can visualize the detailed spatial organization of DNA replication, transcription, and repair processes by reconstructing a map of single-molecule localizations. Furthermore, DNA-binding activities can be observed directly by tracking protein movement in real time. This allows identifying subpopulations of DNA-bound and diffusing proteins, and can be used to measure DNA-binding times in vivo. This chapter provides a detailed protocol for super-resolution microscopy and tracking of DNA-binding proteins in Escherichia coli cells. The protocol covers the construction of cell strains and describes data acquisition and analysis procedures, such as super-resolution image reconstruction, mapping single-molecule tracks, computing diffusion coefficients to identify molecular subpopulations with different mobility, and analysis of DNA-binding kinetics. While the focus is on the study of bacterial chromosome biology, these approaches are generally applicable to other molecular processes and cell types.

Key words

Super-resolution fluorescence microscopy Single-molecule imaging Single-particle tracking DNA-binding proteins DNA repair Lambda red recombination Escherichia coli 

Notes

Acknowledgments

Rodrigo Reyes-Lamothe, David J. Sherratt, and Achillefs N. Kapanidis helped with the original development of this protocol. Katarzyna Ginda and David J. Sherratt are thanked for their comments on the manuscript. Stephan Uphoff was funded by a Sir Henry Wellcome Postdoctoral Fellowship by the Wellcome Trust (101636/Z/13/Z) and a Junior Research Fellowship at St. John’s College, Oxford. Microscopy at Micron Oxford was supported by a Wellcome Trust Strategic Award (091911) and MRC grant (MR/K01577X/1).

References

  1. 1.
    Hell SW (2007) Far-field optical nanoscopy. Science 316:1153–1158CrossRefPubMedGoogle Scholar
  2. 2.
    Betzig E, Patterson GH, Sougrat R et al (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313:1642–5CrossRefPubMedGoogle Scholar
  3. 3.
    Rust MJ, Bates M, Zhuang X (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3:793–795CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Biteen JS, Thompson MA, Tselentis NK et al (2008) Super-resolution imaging in live Caulobacter crescentus cells using photoswitchable EYFP. Nat Methods 5:947–949CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    English BP, Hauryliuk V, Sanamrad A et al (2011) Single-molecule investigations of the stringent response machinery in living bacterial cells. Proc Natl Acad Sci U S A 108:E365–373CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Garner EC, Bernard R, Wang W et al (2011) Coupled, circumferential motions of the cell wall synthesis machinery and MreB filaments in B. subtilis. Science 333:222–225CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Greenfield D, McEvoy AL, Shroff H et al (2009) Self-organization of the Escherichia coli chemotaxis network imaged with super-resolution light microscopy. PLoS Biol 7, e1000137CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Badrinarayanan A, Reyes-Lamothe R, Uphoff S et al (2012) In vivo architecture and action of bacterial structural maintenance of chromosome proteins. Science 338:528–531CrossRefPubMedGoogle Scholar
  9. 9.
    Uphoff S, Reyes-Lamothe R, de Leon FG et al (2013) Single-molecule DNA repair in live bacteria. Proc Natl Acad Sci U S A 110:8063–8068CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Endesfelder U, Finan K, Holden SJ et al (2013) Multiscale spatial organization of RNA polymerase in Escherichia coli. Biophys J 105:172–181CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Bakshi S, Dalrymple RM, Li W et al (2013) Partitioning of RNA polymerase activity in live Escherichia coli from analysis of single-molecule diffusive trajectories. Biophys J 105:2676–2686CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Fiche J-B, Cattoni DI, Diekmann N et al (2013) Recruitment, assembly, and molecular architecture of the SpoIIIE DNA pump revealed by superresolution microscopy. PLoS Biol 11, e1001557CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Holden SJ, Pengo T, Meibom KL et al (2014) High throughput 3D super-resolution microscopy reveals Caulobacter crescentus in vivo Z-ring organization. Proc Natl Acad Sci U S A 111:4566–4571CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Diepold A, Kudryashev M, Delalez NJ et al (2015) Composition, formation, and regulation of the cytosolic c-ring, a dynamic component of the type III secretion injectisome. PLoS Biol 13, e1002039CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Stracy M, Lesterlin C, de Leon FG et al (2015) Live-cell superresolution microscopy reveals the organization of RNA polymerase in the bacterial nucleoid. Proc Natl Acad Sci U S A 201507592Google Scholar
  16. 16.
    Saxton MJ, Jacobson K (1997) Single-particle tracking: applications to membrane dynamics. Annu Rev Biophys Biomol Struct 26:373–399CrossRefPubMedGoogle Scholar
  17. 17.
    Manley S, Gillette JM, Patterson GH et al (2008) High-density mapping of single-molecule trajectories with photoactivated localization microscopy. Nat Methods 5:155–157CrossRefPubMedGoogle Scholar
  18. 18.
    Gebhardt JCM, Suter DM, Roy R et al (2013) Single-molecule imaging of transcription factor binding to DNA in live mammalian cells. Nat Methods 10:421–426CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Etheridge TJ, Boulineau RL, Herbert A et al (2014) Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy. Nucleic Acids Res 42:e146–e146CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97:6640–6645CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Subach FV, Patterson GH, Manley S et al (2009) Photoactivatable mCherry for high-resolution two-color fluorescence microscopy. Nat Meth 6:153–159CrossRefGoogle Scholar
  22. 22.
    Reyes-Lamothe R, Possoz C, Danilova O et al (2008) Independent positioning and action of Escherichia coli replisomes in live cells. Cell 133:90–102CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Roy R, Hohng S, Ha T (2008) A practical guide to single-molecule FRET. Nat Methods 5:507–516CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Uphoff S, Sherratt DJ, Kapanidis AN (2014) Visualizing protein-DNA interactions in live bacterial cells using photoactivated single-molecule tracking. J Vis Exp 85:24638084Google Scholar
  25. 25.
    Thomason LC, Costantino N, Court DL (2007) E. coli genome manipulation by P1 transduction. Curr Protoc Mol Biol Chapter 1:Unit 1.17PubMedGoogle Scholar
  26. 26.
    Holden SJ, Uphoff S, Hohlbein J et al (2010) Defining the limits of single-molecule FRET resolution in TIRF microscopy. Biophys J 99:3102–3111CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Mortensen KI, Churchman LS, Spudich JA et al (2010) Optimized localization analysis for single-molecule tracking and super-resolution microscopy. Nat Methods 7:377–381CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Smith CS, Joseph N, Rieger B et al (2010) Fast, single-molecule localization that achieves theoretically minimum uncertainty. Nat Methods 7:373–375CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Bakshi S, Siryaporn A, Goulian M et al (2012) Superresolution imaging of ribosomes and RNA polymerase in live Escherichia coli cells. Mol Microbiol 85:21–38CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Crocker JC, Grier DG (1996) Methods of digital video microscopy for colloidal studies. J Colloid Interface Sci 179:298–310CrossRefGoogle Scholar
  31. 31.
    Persson F, Lindén M, Unoson C et al (2013) Extracting intracellular diffusive states and transition rates from single-molecule tracking data. Nat Methods 10:265–269CrossRefPubMedGoogle Scholar
  32. 32.
    Veatch SL, Machta BB, Shelby SA et al (2012) Correlation functions quantify super-resolution images and estimate apparent clustering due to over-counting. PLoS One 7, e31457CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Sengupta P, Jovanovic-Talisman T, Lippincott-Schwartz J (2013) Quantifying spatial organization in point-localization superresolution images using pair correlation analysis. Nat Protoc 8:345–354CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Baba T, Ara T, Hasegawa M et al (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2:2006.0008CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Landgraf D, Okumus B, Chien P et al (2012) Segregation of molecules at cell division reveals native protein localization. Nat Methods 9:480–482CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Wang S, Moffitt JR, Dempsey GT et al (2014) Characterization and development of photoactivatable fluorescent proteins for single-molecule-based superresolution imaging. Proc Natl Acad Sci U S A 111:8452–8457CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Lee S-H, Shin JY, Lee A et al (2012) Counting single photoactivatable fluorescent molecules by photoactivated localization microscopy (PALM). Proc Natl Acad Sci U S A 109:17436–17441CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Durisic N, Laparra-Cuervo L, Sandoval-Álvarez Á et al (2014) Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate. Nat Methods 11:156–162CrossRefPubMedGoogle Scholar
  39. 39.
    Small A, Stahlheber S (2014) Fluorophore localization algorithms for super-resolution microscopy. Nat Methods 11:267–279CrossRefPubMedGoogle Scholar
  40. 40.
    Thompson RE, Larson DR, Webb WW (2002) Precise nanometer localization analysis for individual fluorescent probes. Biophys J 82:2775–2783CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Holden SJ, Uphoff S, Kapanidis AN (2011) DAOSTORM: an algorithm for high- density super-resolution microscopy. Nat Methods 8:279–280CrossRefPubMedGoogle Scholar
  42. 42.
    Zhu L, Zhang W, Elnatan D et al (2012) Faster STORM using compressed sensing. Nat Methods 9:721–723CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of BiochemistryUniversity of OxfordOxfordUK

Personalised recommendations