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A general approach to break the concentration barrier in single-molecule imaging

Nature Methods volume 9, pages 987–992 (2012)Cite this article

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Abstract

Single-molecule fluorescence imaging is often incompatible with physiological protein concentrations, as fluorescence background overwhelms an individual molecule's signal. We solve this problem with a new imaging approach called PhADE (PhotoActivation, Diffusion and Excitation). A protein of interest is fused to a photoactivatable protein (mKikGR) and introduced to its surface-immobilized substrate. After photoactivation of mKikGR near the surface, rapid diffusion of the unbound mKikGR fusion out of the detection volume eliminates background fluorescence, whereupon the bound molecules are imaged. We labeled the eukaryotic DNA replication protein flap endonuclease 1 with mKikGR and added it to replication-competent Xenopus laevis egg extracts. PhADE imaging of high concentrations of the fusion construct revealed its dynamics and micrometer-scale movements on individual, replicating DNA molecules. Because PhADE imaging is in principle compatible with any photoactivatable fluorophore, it should have broad applicability in revealing single-molecule dynamics and stoichiometry of macromolecular protein complexes at previously inaccessible fluorophore concentrations.

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Figure 1: Experimental design and validation of PhADE.
Figure 2: PhADE imaging of Fen1KikR visualizes the growth of individual replication bubbles.
Figure 3: During unperturbed replication, PhADE imaging of Fen1KikR reveals a high density of initiations.
Figure 4: PhADE reveals single molecules of Fen1KikGR at replication forks and measures Fen1KikGR off rate from DNA.

References

  1. Joo, C., Balci, H., Ishitsuka, Y., Buranachai, C. & Ha, T. Advances in single-molecule fluorescence methods for molecular biology. Annu. Rev. Biochem. 77, 51–76 (2008).

    Article  CAS  Google Scholar 

  2. van Oijen, A.M. Single-molecule approaches to characterizing kinetics of biomolecular interactions. Curr. Opin. Biotechnol. 22, 75–80 (2011).

    Article  CAS  Google Scholar 

  3. Klar, T.A. & Hell, S.W. Subdiffraction resolution in far-field fluorescence microscopy. Opt. Lett. 24, 954–956 (1999).

    Article  CAS  Google Scholar 

  4. Hell, S. & Stelzer, E.H.K. Properties of a 4Pi confocal fluorescence microscope. J. Opt. Soc. Am. A 9, 2159–2166 (1992).

    Article  Google Scholar 

  5. Levene, M.J. et al. Zero-mode waveguides for single-molecule analysis at high concentrations. Science 299, 682–686 (2003).

    Article  CAS  Google Scholar 

  6. Boukobza, E., Sonnenfeld, A. & Haran, G. Immobilization in surface-tethered lipid vesicles as a new tool for single biomolecule spectroscopy. J. Phys. Chem. B 105, 12165–12170 (2001).

    Article  CAS  Google Scholar 

  7. Habuchi, S., Tsutsui, H., Kochaniak, A.B., Miyawaki, A. & van Oijen, A.M. mKikGR, a monomeric photoswitchable fluorescent protein. PLoS ONE 3, e3944 (2008).

    Article  Google Scholar 

  8. Masai, H., Matsumoto, S., You, Z., Yoshizawa-Sugata, N. & Oda, M. Eukaryotic chromosome DNA replication: Where, when, and how? Annu. Rev. Biochem. 79, 89–130 (2010).

    Article  CAS  Google Scholar 

  9. Burgers, P.M.J. Polymerase dynamics at the eukaryotic DNA replication fork. J. Biol. Chem. 284, 4041–4045 (2009).

    Article  CAS  Google Scholar 

  10. Herrick, J. & Bensimon, A. Introduction to molecular combing: genomics, DNA replication, and cancer. in DNA Replication: Methods and Protocols (eds. Vengrova, S. & Dalgaard, J.Z.) Ch. 5, 71–101 (Humana, Totowa, NJ, 2009).

  11. Walter, J., Sun, L. & Newport, J. Regulated chromosomal DNA replication in the absence of a nucleus. Mol. Cell 1, 519–529 (1998).

    Article  CAS  Google Scholar 

  12. Strausfeld, U.P. et al. Cip1 blocks the initiation of DNA replication in Xenopus extracts by inhibition of cyclin-dependent kinases. Curr. Biol. 4, 876–883 (1994).

    Article  CAS  Google Scholar 

  13. Blow, J.J. Control of chromosomal DNA replication in the early Xenopus embryo. EMBO J. 20, 3293–3297 (2001).

    Article  CAS  Google Scholar 

  14. Lebofsky, R., Takahashi, T. & Walter, J.C. DNA replication in nucleus-free Xenopus egg extracts. Methods Mol. Biol. 521, 229–252 (2009).

    Article  CAS  Google Scholar 

  15. Yardimci, H., Loveland, A.B., Habuchi, S., van Oijen, A.M. & Walter, J.C. Uncoupling of sister replisomes during eukaryotic DNA replication. Mol. Cell 40, 834–840 (2010).

    Article  CAS  Google Scholar 

  16. Gary, R., Kim, K., Cornelius, H.L., Park, M.S. & Matsumoto, Y. Proliferating cell nuclear antigen facilitates excision in long-patch base excision repair. J. Biol. Chem. 274, 4354–4363 (1999).

    Article  CAS  Google Scholar 

  17. Harrington, J.J. & Lieber, M.R. The characterization of a mammalian DNA structure-specific endonuclease. EMBO J. 13, 1235–1246 (1994).

    Article  CAS  Google Scholar 

  18. Shen, B., Nolan, J.P., Sklar, L.A. & Park, M.S. Functional analysis of point mutations in human flap endonuclease-1 active site. Nucleic Acids Res. 25, 3332–3338 (1997).

    Article  CAS  Google Scholar 

  19. Blow, J.J. & Laskey, R.A. Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs. Cell 47, 577–587 (1986).

    Article  CAS  Google Scholar 

  20. Walter, J. & Newport, J.W. Regulation of replicon size in Xenopus egg extracts. Science 275, 993–995 (1997).

    Article  CAS  Google Scholar 

  21. Herrick, J., Stanislawski, P., Hyrien, O. & Bensimon, A. Replication fork density increases during DNA synthesis in X. laevis egg extracts. J. Mol. Biol. 300, 1133–1142 (2000).

    Article  CAS  Google Scholar 

  22. Lucas, I., Chevrier-Miller, M., Sogo, J.M. & Hyrien, O. Mechanisms ensuring rapid and complete DNA replication despite random initiation in Xenopus early embryos. J. Mol. Biol. 296, 769–786 (2000).

    Article  CAS  Google Scholar 

  23. Blow, J.J., Gillespie, P.J., Francis, D. & Jackson, D.A. Replication origins in Xenopus egg extract are 5–15 kb apart and are activated in clusters that fire at different times. J. Cell Biol. 152, 15–26 (2001).

    Article  CAS  Google Scholar 

  24. Marheineke, K. & Hyrien, O. Control of replication origin density and firing time in Xenopus egg extracts. J. Biol. Chem. 279, 28071–28081 (2004).

    Article  CAS  Google Scholar 

  25. Marheineke, K. & Hyrien, O. Aphidicolin triggers a block to replication origin firing in Xenopus egg extracts. J. Biol. Chem. 276, 17092–17100 (2001).

    Article  CAS  Google Scholar 

  26. Ridelis, I. et al. Use of Kikume green-red fusions to study the influence of pharmacological chaperones on trafficking of G protein–coupled receptors. FEBS Lett. 586, 784–791 (2012).

    Article  CAS  Google Scholar 

  27. Herrick, J., Jun, S., Bechhoefer, J. & Bensimon, A. Kinetic model of DNA replication in eukaryotic organisms. J. Mol. Biol. 320, 741–750 (2002).

    Article  CAS  Google Scholar 

  28. Ayyagari, R., Gomes, X.V., Gordenin, D.A. & Burgers, P.M.J. Okazaki fragment maturation in yeast. I. Distribution of functions between FEN1 and DNA2. J. Biol. Chem. 278, 1618–1625 (2003).

    Article  CAS  Google Scholar 

  29. Hoskins, A.A., Gelles, J. & Moore, M.J. New insights into the spliceosome by single molecule fluorescence microscopy. Curr. Opin. Chem. Biol. 15, 864–870 (2011).

    Article  CAS  Google Scholar 

  30. Jain, A. et al. Probing cellular protein complexes using single-molecule pull-down. Nature 473, 484–488 (2011).

    Article  CAS  Google Scholar 

  31. Thompson, M.A., Biteen, J.S., Lord, S.J., Conley, N.R. & Moerner, W.E. Molecules and methods for super-resolution imaging. in Single Molecule Tools, Part B: Super-Resolution, Particle Tracking, Multiparameter, and Force Based Methods (ed. Walter, N.G.) Ch. 2, 27–59 (Academic Press, 2010).

  32. Lord, S.J. et al. Azido push-pull fluorogens photoactivate to produce bright fluorescent labels. J. Phys. Chem. B 114, 14157–14167 (2010).

    Article  CAS  Google Scholar 

  33. Bates, M., Blosser, T.R. & Zhuang, X. Short-range spectroscopic ruler based on a single-molecule optical switch. Phys. Rev. Lett. 94, 108101 (2005).

    Article  Google Scholar 

  34. Conley, N.R., Biteen, J.S. & Moerner, W.E. Cy3-Cy5 covalent heterodimers for single-molecule photoswitching. J. Phys. Chem. B 112, 11878–11880 (2008).

    Article  CAS  Google Scholar 

  35. Patterson, G., Davidson, M., Manley, S. & Lippincott-Schwartz, J. Superresolution imaging using single-molecule localization. Annu. Rev. Phys. Chem. 61, 345–367 (2010).

    Article  CAS  Google Scholar 

  36. Loparo, J.J., Kulczyk, A.W., Richardson, C.C. & van Oijen, A.M. Simultaneous single-molecule measurements of phage T7 replisome composition and function reveal the mechanism of polymerase exchange. Proc. Natl. Acad. Sci. USA 108, 3584–3589 (2011).

    Article  CAS  Google Scholar 

  37. Havens, C.G. & Walter, J.C. Docking of a specialized PIP box onto chromatin-bound PCNA creates a degron for the ubiquitin ligase CRL4Cdt2. Mol. Cell 35, 93–104 (2009).

    Article  CAS  Google Scholar 

  38. Bibikova, M. et al. Characterization of FEN-1 from Xenopus laevis. J. Biol. Chem. 273, 34222–34229 (1998).

    Article  CAS  Google Scholar 

  39. Walter, J.C. Evidence for sequential action of cdc7 and cdk2 protein kinases during initiation of DNA replication in Xenopus egg extracts. J. Biol. Chem. 275, 39773–39778 (2000).

    Article  CAS  Google Scholar 

  40. Wohlschlegel, J.A. et al. Inhibition of eukaryotic DNA replication by geminin binding to Cdt1. Science 290, 2309–2312 (2000).

    Article  CAS  Google Scholar 

  41. Yardimci, H., Loveland, A.B., van Oijen, A.M. & Walter, J.C. Single-molecule analysis of DNA replication in Xenopus egg extracts. Methods. published online, doi:10.1016/j.ymeth.2012.03.033 (6 April 2012).

  42. Lebofsky, R., van Oijen, A.M. & Walter, J.C. DNA is a co-factor for its own replication in Xenopus egg extracts. Nucleic Acids Res. 39, 545–555 (2011).

    Article  CAS  Google Scholar 

  43. Edwards, M.C. et al. MCM2–7 complexes bind chromatin in a distributed pattern surrounding the origin recognition complex in Xenopus egg extracts. J. Biol. Chem. 277, 33049–33057 (2002).

    Article  CAS  Google Scholar 

  44. Abràmoff, M.D., Magalhães, P.J. & Ram, S.J. Image processing with ImageJ. Biophotonics Int. 11, 36–42 (2004).

    Google Scholar 

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Acknowledgements

We thank H. Yardimci and J.J. Loparo for assistance with experiments and critical reading of the manuscript and C.G. Havens (Harvard University) for sharing advice and reagents. This work was supported by grants from the US National Institutes of Health (NIH) (GM077248), American Cancer Society (RSG0823401GMC) and Netherlands Organization for Scientific Research (NWO; Vici 680-47-607) to A.M.v.O. as well as by grants from the NIH (GM62267) and American Cancer Society (RSG0823401GMC) to J.C.W. A.B.L. was supported by the NIH and National Institute of General Medical Sciences Molecular Biophysics Training Grant (T32 GM008313).

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Author notes

  1. Anna B Loveland, Satoshi Habuchi & Antoine M van Oijen

    Present address: Present addresses: Department of Biochemistry, Brandeis University, Waltham, Massachusetts, USA (A.B.L.), Division of Chemical and Life Sciences and Engineering, King Abdullah University of Science and Technology, Thuwal, Kingdom of Saudi Arabia (S.H.) and The Zernike Institute for Advanced Materials, University of Groningen, Groningen, The Netherlands (A.M.v.O.).,

  2. Johannes C Walter and Antoine M van Oijen: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA

    Anna B Loveland, Satoshi Habuchi, Johannes C Walter & Antoine M van Oijen

  2. Graduate Program in Biophysics, Harvard University, Cambridge, Massachusetts, USA

    Anna B Loveland

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  1. Anna B Loveland
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Contributions

A.B.L. performed all experiments. A.M.v.O. and S.H. designed the microscope. A.B.L., A.M.v.O. and J.C.W. designed experiments and performed data analysis. A.B.L., A.M.v.O. and J.C.W. prepared the manuscript.

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Correspondence to Johannes C Walter or Antoine M van Oijen.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Tables 1–3 and Supplementary Methods (PDF 1007 kb)

PhADE imaging of Fen1KikR under conditions when only one origin fires reveals the growth rate of individual replication bubbles.

D179A Fen1KikR was imaged (as in Fig. 2b) using PhADE between 5 and 25 min after NPE addition (time stamps). During this time, D179A Fen1KikR signals appeared, grew in length, and some eventually split as forks traveled far apart. Some D179A Fen1KikR signals were lost as DNA tethers broke and DNA compacted. The extracts were washed out 25 min after NPE addition, dig-dUTP incorporation was detected and DNA was stained with an intercalating dye. (AVI 5321 kb)

PhADE imaging of D179A Fen1KikR reveals the timing and distribution of initiation events.

D179A Fen1KikR was imaged using PhADE (as in Fig. 3a) between 2.5 and 10 min after NPE addition (time stamps). During this time, D179A Fen1KikR signals appeared, grew and merged, which we interpret as replication initiation, elongation and termination. Some molecules were lost as DNA tethers broke and DNA compacted. A kymograph of the molecule labeled #1 in the first frame of the movie is shown in Figure 3a. All molecules highlighted with cyan boxes in the first frame were analyzed for Fig. 3b–d. Arrows indicate QDot 605-Biotin that are used as fiduciary markers. (AVI 1942 kb)

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Loveland, A., Habuchi, S., Walter, J. et al. A general approach to break the concentration barrier in single-molecule imaging. Nat Methods 9, 987–992 (2012). https://doi.org/10.1038/nmeth.2174

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