Advertisement

Bacterial Chemoreceptor Imaging at High Spatiotemporal Resolution Using Photoconvertible Fluorescent Proteins

  • Jacopo Solari
  • Francois Anquez
  • Katharina M. Scherer
  • Thomas S. Shimizu
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1729)

Abstract

We describe two methods for high-resolution fluorescence imaging of the positioning and mobility of E. coli chemoreceptors fused to photoconvertible fluorescent proteins. Chemoreceptors such as Tar and Tsr are transmembrane proteins expressed at high levels (thousands of copies per cell). Together with their cognate cytosolic signaling proteins, they form clusters on the plasma membrane. Theoretical models imply that the size of these clusters is an important parameter for signaling, and recent PALM imaging has revealed a broad distribution of cluster sizes. We describe experimental setups and protocols for PALM imaging in fixed cells with ~10 nm spatial precision, which allows analysis of cluster-size distributions, and localized-photoactivation single-particle tracking (LPA-SPT) in live cells at ~10 ms temporal resolution, which allows for analysis of cluster mobility.

Keywords

Bacterial chemotaxis Superresolution microscopy Photoactivation localization microscopy (PALM) Single-particle tracking Receptor clustering Membrane protein mobility 

Notes

Acknowledgments

We thank J.S. Parkinson and G. Pinas for strains, plasmids, and helpful discussions; A.S.N. Seshasayee for strain HS1; and H.C. Berg and K.A. Fahrner for the gift of anti-FliC antibody. This work was supported by NWO/FOM and the Paul G. Allen Family Foundation.

References

  1. 1.
    Betzig E et al (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313:1642–1645CrossRefGoogle Scholar
  2. 2.
    Thompson RE, Larson DR, Webb WW (2002) Precise nanometer localization analysis for individual fluorescent probes. Biophys J 82:2775–2783CrossRefGoogle Scholar
  3. 3.
    Betzig E (2015) Single molecules, cells, and super-resolution optics (nobel lecture). Angew Chem Int Ed Engl 54:8034–8053CrossRefGoogle Scholar
  4. 4.
    Hell SW (2007) Far-field optical nanoscopy. Science 316:246–249CrossRefGoogle Scholar
  5. 5.
    Huang B, Bates M, Zhuang X (2009) Super-resolution fluorescence microscopy. Annu Rev Biochem 78:993–1016CrossRefGoogle Scholar
  6. 6.
    Betzig E (1995) Proposed method for molecular optical imaging. Opt Lett 20:237–239CrossRefGoogle Scholar
  7. 7.
    Greenfield D et al (2009) Self-organization of the Escherichia coli chemotaxis network imaged with super-resolution light microscopy. PLoS Biol 7:1–12CrossRefGoogle Scholar
  8. 8.
    Manley S et al (2008) High-density mapping of single-molecule trajectories with photoactivated localization microscopy. Nat Methods 5:155–157CrossRefGoogle Scholar
  9. 9.
    English BP, English BP, Hauryliuk V, Sanamrad A, Tankov S et al (2011) Single-molecule investigations of the stringent response machinery in living bacterial cells. Proc Natl Acad Sci U S A 108:E365–E373CrossRefGoogle Scholar
  10. 10.
    Niu L, Yu J (2008) Investigating intracellular dynamics of FtsZ cytoskeleton with photoactivation single-molecule tracking. Biophys J 95:2009–2016CrossRefGoogle Scholar
  11. 11.
    Stracy M, Lesterlin C, Garza de Leon F, Uphoff S, Zawadzki P et al (2015) Live-cell superresolution microscopy reveals the organization of RNA polymerase in the bacterial nucleoid. Proc Natl Acad Sci U S A 112:E4390–E4399CrossRefGoogle Scholar
  12. 12.
    Adler J (1966) Chemotaxis in bacteria. Science 153:708–716CrossRefGoogle Scholar
  13. 13.
    Sourjik V, Vaknin A, Shimizu TS, Berg HC (2007) In vivo measurement by FRET of pathway activity in bacterial chemotaxis. Methods Enzymol 423:365–391CrossRefGoogle Scholar
  14. 14.
    Berg HC, Block SM (1984) A miniature flow cell designed for rapid exchange of media under high-power microscope objectives. J Gen Microbiol 130:2915–2920Google Scholar
  15. 15.
    Ovesny M, Krizek P, Borkovec J, Svindrych Z, Hagen GM (2014) ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging. Bioinformatics 30:2389–2390CrossRefGoogle Scholar
  16. 16.
    Daszykowski M, Walczak B (2009) Density-based clustering methods. In: Reedijk J (ed) Reference module in chemistry, molecular sciences and chemical engineering, vol 2.29. Elsevier, Waltham, MA, pp 635–654Google Scholar
  17. 17.
    Colville K, Tompkins N, Rutenberg AD, Jericho MH (2010) Effects of poly(L-lysine) substrates on attached Escherichia coli bacteria. Langmuir 26:2639–2644CrossRefGoogle Scholar
  18. 18.
    Jaqaman K, Loerke D, Mettlen M, Kuwata H, Grinstein S (2008) l Robust single-particle tracking in live-cell time-lapse sequences. Nat Methods 5:695–702CrossRefGoogle Scholar
  19. 19.
    Thiem S, Kentner D, Sourjik V (2007) Positioning of chemosensory clusters in E. coli and its relation to cell division. EMBO J 26:1615–1623CrossRefGoogle Scholar
  20. 20.
    Amann E, Ochs B, Abel KJ (1988) Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene 69:301–315CrossRefGoogle Scholar
  21. 21.
    Guzman LM, Belin D, Carson MJ, Beckwith JJ (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177:4121–4130CrossRefGoogle Scholar
  22. 22.
    Guarente L (1983) Yeast promoters and lacZ fusions designed to study expression of cloned genes in yeast. Methods Enzymol 101:181–191CrossRefGoogle Scholar
  23. 23.
    Ames P, Studdert CA, Reiser RH, Parkinson JS (2002) Collaborative signaling by mixed chemoreceptor teams in Escherichia coli. Proc Natl Acad Sci U S A 99:7060–7065CrossRefGoogle Scholar
  24. 24.
    Endres RG, Oleksiuk O, Hansen CH, Meir Y, Sourjik V et al (2008) Variable sizes of Escherichia coli chemoreceptor signaling teams. Mol Syst Biol 4:211CrossRefGoogle Scholar
  25. 25.
    Frank V, Piñas GE, Cohen H, Parkinson JS, Vaknin A (2016) Networked chemoreceptors benefit bacterial chemotaxis performance. MBio 7:1–9CrossRefGoogle Scholar
  26. 26.
    Srinivasan R, Scolari VF, Lagomarsino MC, Seshasayee AS (2015) The genome-scale interplay amongst xenogene silencing, stress response and chromosome architecture in Escherichia coli. Nucleic Acids Res 43:295–308CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Jacopo Solari
    • 1
  • Francois Anquez
    • 2
  • Katharina M. Scherer
    • 3
  • Thomas S. Shimizu
    • 1
  1. 1.AMOLF InstituteAmsterdamThe Netherlands
  2. 2.Laboratoire de Physique des Lasers, Atomes et MoléculesUMR CNRS 8523, Université Lille 1Villeneuve d’AscqFrance
  3. 3.Laser Analytics Group, Department of Chemical Engineering and BiotechnologyUniversity of CambridgeCambridgeUK

Personalised recommendations