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Reply to “Uncoupling diffusion and binding in FRAP experiments”

Nature Methods volume 6, pages 183–184 (2009)Cite this article

Brameshuber et al . reply: We thank Nevin Lambert for bringing this topic to discussion and pointing out the potential pitfalls in fluorescence recovery after photobleaching (FRAP) analysis1, which must also be considered in the case of our combined micropattern-FRAP method for the determination of interaction lifetimes2. As he pointed out, problems may arise when analyzing the binding of a mobile molecule to an immobile receptor3,4,5. Two scenarios for FRAP in the presence of binding may be distinguished: (i) diffusion-uncoupled FRAP, in which diffusion hardly affects the recovery dynamics (the recovery time, τ, equals the inverse of the off rate); and (ii) diffusion-coupled FRAP, in which probe molecules diffuse into the bleached spot and bind to the first free binding sites they encounter. Recovery proceeds as a succession of binding-unbinding and diffusion steps, and the resulting FRAP curve contains contributions from both diffusion and binding. Here we argue that, for our experimental conditions, the simplified assumption of diffusion-uncoupled FRAP is appropriate.

We provided one argument supporting this assumption in our original manuscript2. At the onset of the recovery process, we analyzed the mobility of the first Lck-CFP-YFP molecules entering the bait-captured spot in the photobleached area (Fig. 6 in ref. 2). We reasoned that, in the case of diffusion-coupled FRAP, the probe would move via a succession of short diffusive periods interrupted by transient binding to the bait; in the extreme case of very high on and off rates compared to the single-molecule observation time, a reduced effective diffusion constant would be obtained. However, we found no difference between Lck-CFP-YFP molecules moving in the bait-captured versus bait-free area. In particular, we observed no additional immobilization events in the bait-captured area, indicating that binding to unoccupied CD4 molecules is a very rare event, at least on the millisecond time scale within which the membrane-anchored Lck-CFP-YFP molecules moved over the photobleached spot.

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Figure 1: Spatial profile of the first Lck-CFP-YFP molecules entering the bleached spot.

References

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Acknowledgements

This work was supported by the Austrian Science Fund (project Y250-B10), the Competence Center for Biomolecular Therapeutics Research–Vienna and by the GEN-AU project of the Austrian Federal Ministry for Science and Research.

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Authors and Affiliations

  1. Biophysics Institute, Johannes Kepler University Linz, Altenbergerstr. 69, Linz, A-4040, Austria

    Mario Brameshuber, Michaela Schwarzenbacher, Martin Kaltenbrunner, Clemens Hesch, Julian Weghuber & Gerhard J Schütz

  2. Department of Molecular Immunology, Center for Physiology, Pathophysiology and Immunology, Medical University of Vienna, Lazarettgasse 19, Vienna, A-1090, Austria

    Wolfgang Paster & Hannes Stockinger

  3. Department of Knowledge-based Mathematical Systems, Johannes Kepler University Linz, Altenbergerstr. 69, Linz, A-4040, Austria

    Bettina Heise

  4. Center for Biomedical Nanotechnology, Upper Austrian Research GmbH, Scharitzerstr. 6-8, Linz, A-4020, Austria

    Alois Sonnleitner

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Correspondence to Hannes Stockinger or Gerhard J Schütz.

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Brameshuber, M., Schwarzenbacher, M., Kaltenbrunner, M. et al. Reply to “Uncoupling diffusion and binding in FRAP experiments”. Nat Methods 6, 183–184 (2009). https://doi.org/10.1038/nmeth0309-183b

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