Exploring Protein Superstructures and Dynamics in Live Bacterial Cells Using Single-Molecule and Superresolution Imaging

  • Julie S. Biteen
  • Lucy Shapiro
  • W. E. Moerner
Part of the Methods in Molecular Biology book series (MIMB, volume 783)


Single-molecule imaging enables biophysical measurements devoid of ensemble averaging, gives enhanced spatial resolution beyond the optical diffraction limit, and enables superresolution reconstruction of structures beyond the diffraction limit. This work summarizes how single-molecule and superresolution imaging can be applied to the study of protein dynamics and superstructures in live Caulobacter crescentus cells to illustrate the power of these methods in bacterial imaging. Based on these techniques, the diffusion coefficient and dynamics of the histidine protein kinase PleC, the localization behavior of the polar protein PopZ, and the treadmilling behavior and protein superstructure of the structural protein MreB are investigated with sub-40-nm spatial resolution, all in live cells.

Key words

Single-molecule imaging Live-cell imaging Live-cell PALM Superresolution imaging Superlocalization Caulobacter crescentus 



The authors warmly thank members of the Shapiro laboratory, specifically Ellen Judd, Harley McAdams, Grant Bowman, and Zemer Gitai, as well as members of the Moerner laboratory, specifically, Jason Deich, Marcelle Koenig, So Yeon Kim, Anika Kinkhabwala, Michael Thompson, and Nicole Tselentis, for their contributions to this work. This research was supported in part by DARPA Grant MDA-972-00-1-0032, NSF Grant MCB-0212503, Department of Energy Grants DE-FG02-04ER63777 and DE_FG02-05ER64136 9 (to LS), and NIH Grants P20-HG003638, R01-GM086196, R01-GM085437, R01-GM51426 (to LS), and R01-GM32506 (to LS).


  1. 1.
    Ambrose W. P., Moerner W. E. (1991) Fluorescence spectroscopy and spectral diffusion of single impurity molecules in a crystal Nature 349, 225–7.CrossRefGoogle Scholar
  2. 2.
    Thompson R. E., Larson D. R., Webb W. W. (2002) Precise nanometer localization analysis for individual fluorescent probes Biophys J 82, 2775–83.PubMedCrossRefGoogle Scholar
  3. 3.
    Schütz G. J., Kada G., Pastushenko V. P., Schindler H. (2000) Properties of lipid microdomains in a muscle cell membrane visualized by single molecule microscopy EMBO J 19, 892–901.PubMedCrossRefGoogle Scholar
  4. 4.
    Sako Y., Minoghchi S., Yanagida T. (2000) Single-molecule imaging of EGFR signalling on the surface of living cells Nat Cell Biol 2, 168–72.PubMedCrossRefGoogle Scholar
  5. 5.
    Moerner W. E. (2003) Optical measurements of single molecules in cells Trends Anal Chem 22, 544–8.CrossRefGoogle Scholar
  6. 6.
    Vrljic M., Nishimura S. Y., Moerner W. E., McConnell H. M. (2005) Cholesterol Depletion Suppresses the Translational Diffusion of Class II Major Histocompatibility Complex Proteins in the Plasma Membrane Biophys J 88, 334–47.PubMedCrossRefGoogle Scholar
  7. 7.
    Vrljic M., Nishimura S. Y., Brasselet S., Moerner W. E., McConnell H. M. (2002) Translational Diffusion of Individual Class II MHC Membrane Proteins in Cells Biophys J 83, 2681–92.PubMedCrossRefGoogle Scholar
  8. 8.
    Lee H. D., Dubikovskaya E. A., Hwang H., Semyonov A. N., Wang H., Jones L. R., Twieg R. J., Moerner W. E., Wender P. A. (2008) Single-Molecule Motions of Oligoarginine Transporter Conjugates on the Plasma Membrane of Chinese Hamster Ovary Cells J Am Chem Soc 130, 9364–70.PubMedCrossRefGoogle Scholar
  9. 9.
    Harms G. S., Cognet L., Lommerse P. H. M., Blab G. A., Schmidt T. (2001) Autofluorescent Proteins in Single-Molecule Research: Applications to Live Cell Imaging Microscopy Biophys J 80, 2396–408.PubMedCrossRefGoogle Scholar
  10. 10.
    Deich J., Judd E. M., McAdams H. H., Moerner W. E. (2004) Visualization of the movement of single histidine kinase molecules in live Caulobacter cells Proc Nat Acad Sci USA 101, 15921–6.PubMedCrossRefGoogle Scholar
  11. 11.
    Kim S. Y., Gitai Z., Kinkhabwala A., Shapiro L., Moerner W. E. (2006) Single molecules of the bacterial actin MreB undergo directed treadmilling motion in Caulobacter crescentus Proc Nat Acad Sci USA 103, 10929–34.PubMedCrossRefGoogle Scholar
  12. 12.
    Xie X. S., Choi P. J., Li G. W., Lee N. K., Lia G. (2008) Single-Molecule Approach to Molecular Biology in Living Bacterial Cells Annu Rev Biophys 37, 417–44.PubMedCrossRefGoogle Scholar
  13. 13.
    Conley N. R., Biteen J. S., Moerner W. E. (2008) Cy3-Cy5 Covalent Heterodimers for Single-Molecule Photoswitching J Phys Chem B 112, 11878–80.PubMedCrossRefGoogle Scholar
  14. 14.
    Bowman G. R., Comolli L. R., Zhu J., Eckart M., Koenig M., Downing K. H., Moerner W. E., Earnest T., Shapiro L. (2008) A Polymeric Protein Anchors the Chromosomal Origin/ParB Complex at a Bacterial Cell Pole Cell 134, 945–55.PubMedCrossRefGoogle Scholar
  15. 15.
    Biteen J. S., Thompson M. A., Tselentis N. K., Bowman G. R., Shapiro L., Moerner W. E. (2008) Super-resolution imaging in live Caulobacter crescentus cells using photoswitchable EYFP Nat Methods 5, 947–9.PubMedCrossRefGoogle Scholar
  16. 16.
    Moerner W. E., Fromm D. P. (2003) Methods of Single-Molecule Fluorescence Spectroscopy and Microscopy Rev Sci Instrum 74, 3597–619.CrossRefGoogle Scholar
  17. 17.
    Betzig E., Patterson G. H., Sougrat R., Lindwasser O. W., Olenych S., Bonifacino J. S., Davidson M. W., Lippincott-Schwartz J., Hess H. F. (2006) Imaging intracellular fluorescent proteins at nanometer resolution Science 313, 1642–5.PubMedCrossRefGoogle Scholar
  18. 18.
    Rust M. J., Bates M., Zhuang X. (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM) Nat Methods 3, 793–5.PubMedCrossRefGoogle Scholar
  19. 19.
    Hess S. T., Girirajan T. P. K., Mason M. D. (2006) Ultra-high resolution imaging by fluorescence photoactivation localization microscopy Biophys J 91, 4258–72.PubMedCrossRefGoogle Scholar
  20. 20.
    Thompson M. A., Biteen J. S., Lord S. J., Conley N. R., Moerner W. E. (2010) Molecules and Methods for Super-resolution Imaging Methods Enzymol 475, 27–59.PubMedCrossRefGoogle Scholar
  21. 21.
    Wheeler R. T., Shapiro L. (1999) Differential localization of two histidine kinases controlling bacterial cell differentiation Mol Cell 4, 683–94.PubMedCrossRefGoogle Scholar
  22. 22.
    Shapiro L., McAdams H., Losick R. (2002) Generating and exploiting polarity in bacteria Science 298, 1942–6.PubMedCrossRefGoogle Scholar
  23. 23.
    Rudner D. Z., Pan Q., Losick R. M. (2002) Evidence that subcellular localization of a bacterial membrane protein is achieved by diffusion and capture Proc Nat Acad Sci USA 99, 8701–6.PubMedGoogle Scholar
  24. 24.
    Steinhauer J., Agha R., Pham T., Varga A. W., Goldberg M. B. (1999) The unipolar Shigella surface protein IcsA is targeted directly to the bacterial old pole: IcsP cleavage of IcsA occurs over the entire bacterial surface Mol Microbiol 32, 367–77.PubMedCrossRefGoogle Scholar
  25. 25.
    Robbins J. R., Monack D., McCallum S. J., Vegas A., Pham E., Goldberg M. B., Theriot J. A. (2001) The making of a gradient: IcsA (VirG) polarity in Shigella flexneri Mol Microbiol 41, 861–72.PubMedCrossRefGoogle Scholar
  26. 26.
    Gitai Z., Dye N., Shapiro L. (2004) An actin-like gene can determine cell polarity in bacteria Proc Nat Acad Sci USA 101, 8643–8.PubMedCrossRefGoogle Scholar
  27. 27.
    Gitai Z., Dye N. A., Reisenauer A., Wachi M., Shapiro L. (2005) MreB actin-mediated segregation of a specific region of a bacterial chromosome Cell 120, 329–41.PubMedCrossRefGoogle Scholar
  28. 28.
    Dye N. A., Pincus Z., Theriot J. A., Shapiro L., Gitai Z. (2005) Two independent spiral structures control cell shape in Caulobacter Proc Nat Acad Sci USA 102, 18608–13.PubMedCrossRefGoogle Scholar
  29. 29.
    Van Den Ent E., Amos L. A., Lowe J. (2001) Prokaryotic origin of the actin cytoskelecton Nature 413, 39–44.PubMedCrossRefGoogle Scholar
  30. 30.
    Figge R. M., Divakaruni A. V., Gober J. W. (2004) MreB, the cell shape-determining bacterial actin homologue, co-ordinates cell wall morphogenesis in Caulobacter crescentus Mol Microbiol 51, 1321–32.PubMedCrossRefGoogle Scholar
  31. 31.
    Dickson R. M., Cubitt A. B., Tsien R. Y., Moerner W. E. (1997) On/Off Blinking and Switching Behavior of Single Green Fluorescent Protein Molecules Nature 388, 355–8.PubMedCrossRefGoogle Scholar
  32. 32.
    Shroff H., Galbraith C. G., Galbraith J. A., Betzig E. (2008) Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics Nat Methods 5, 417–23.PubMedCrossRefGoogle Scholar
  33. 33.
    Juette M. F., Gould T. J., Lessard M. D., Mlodzianoski M. J., Nagpure B. S., Bennett B. T., Hess S. T., Bewersdorf J. (2008) Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples. Nat Meth 5, 527–9.Google Scholar
  34. 34.
    Huang B., Wang W., Bates M., Zhuang X.(2008) Three-Dimensional Super-Resolution Imaging by Stochastic Optical Reconstruction Microscopy Science 319, 810–3.PubMedCrossRefGoogle Scholar
  35. 35.
    Pavani S. R. P., Thompson M. A., Biteen J. S., Lord S. J., Liu N., Twieg R. J., Piestun R., Moerner W. E. (2009) Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function Proc Nat Acad Sci USA 106, 2995–9.Google Scholar
  36. 36.
    Shroff H., Galbraith C. G., Galbraith J. A., White H., Gillette J., Olenych S., Davidson M. W., Betzig E. (2007) Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes Proc Nat Acad Sci USA 104, 20308–13.PubMedCrossRefGoogle Scholar
  37. 37.
    Bates M., Huang B., Dempsey G. T., Zhuang X. (2007) Multicolor super-resolution imaging with photo-switchable fluorescent probes Science 317, 1749–53.PubMedCrossRefGoogle Scholar
  38. 38.
    Vaziri A., Tang J., Shroff H., Shank C. V. (2008) Multilayer three-dimensional super resolution imaging of thick biological samples Proc Nat Acad Sci USA 105, 20221–6.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Julie S. Biteen
    • 1
  • Lucy Shapiro
    • 2
  • W. E. Moerner
    • 3
  1. 1.Department of ChemistryUniversity of MichiganAnn ArborUSA
  2. 2.Department of Developmental BiologyStanford UniversityStanfordUSA
  3. 3.Department of ChemistryStanford UniversityStanfordUSA

Personalised recommendations