STED nanoscopy combined with optical tweezers reveals protein dynamics on densely covered DNA

Abstract

Dense coverage of DNA by proteins is a ubiquitous feature of cellular processes such as DNA organization, replication and repair. We present a single-molecule approach capable of visualizing individual DNA-binding proteins on densely covered DNA and in the presence of high protein concentrations. Our approach combines optical tweezers with multicolor confocal and stimulated emission depletion (STED) fluorescence microscopy. Proteins on DNA are visualized at a resolution of 50 nm, a sixfold resolution improvement over that of confocal microscopy. High temporal resolution (<50 ms) is ensured by fast one-dimensional beam scanning. Individual trajectories of proteins translocating on DNA can thus be distinguished and tracked with high precision. We demonstrate our multimodal approach by visualizing the assembly of dense nucleoprotein filaments with unprecedented spatial resolution in real time. Experimental access to the force-dependent kinetics and motility of DNA-associating proteins at biologically relevant protein densities is essential for linking idealized in vitro experiments with the in vivo situation.

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Figure 1: Experimental layout.
Figure 2: Force spectroscopy and confocal fluorescence microscopy.
Figure 3: Characterization of STED nanoscopy of proteins on optically stretched DNA.
Figure 4: Characterization of localization precision.
Figure 5: STED nanoscopy of Sytox Red on optically stretched DNA.
Figure 6: TFAM binding and diffusion dynamics on optically stretched DNA.

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Acknowledgements

We thank A.S. Biebricher, A. Candelli and S. Berning for helpful discussions and advice; J. Dikic, E. Kroezinga, T. Hoekstra and S.E.D. Haene for biochemical support; and P. Noordeloos for technical support. This work is part of the research program of the Foundation for Fundamental Research on Matter (FOM) (E.J.G.P. and G.J.L.W.), which is part of the Netherlands Organisation for Scientific Research (NWO). We acknowledge support by NWO VENI (I.H.), VICI (E.J.G.P. and G.J.L.W.) and ECHO grants (G.J.L.W.) as well as a European Research Council (ERC) starting grant (G.J.L.W.).

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G.J.L.W. conceived the research and the multimodal approach to study mitochondrial transcription. I.H. conceived and designed the instrument and research. I.H. and G.S. built the instrument, wrote software, conceived and performed the experiments and analyzed all data. O.D.B. developed software. G.F. provided labeled TFAM and advised on experiments. W.W. and C.M. provided labeled restriction enzymes. S.W.H. advised on STED implementation and supplied phase plates. E.J.G.P. and G.J.L.W. advised on instrument design, experiments and analysis. I.H., G.S., S.W.H., E.J.G.P. and G.J.L.W. wrote the paper.

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Correspondence to Gijs J L Wuite.

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The authors declare no competing financial interests.

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Supplementary Figures 1–6 and Supplementary Notes 1 and 2 (PDF 971 kb)

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Heller, I., Sitters, G., Broekmans, O. et al. STED nanoscopy combined with optical tweezers reveals protein dynamics on densely covered DNA. Nat Methods 10, 910–916 (2013). https://doi.org/10.1038/nmeth.2599

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