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Detecting and Controlling Dye Effects in Single-Virus Fusion Experiments
Univ Virginia, Dept Mol Physiol & Biol Phys, Charlottesville, VA 22908 USA;Univ Virginia, Dept Biomed Engn, Charlottesville, VA 22908 USA;Williams Coll, Dept Chem, Williamstown, MA 01267 USA.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics. Uppsala University, Science for Life Laboratory, SciLifeLab.
Stanford Univ, Dept Chem, Stanford, CA 94305 USA.
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics. Univ Virginia, Dept Mol Physiol & Biol Phys, Charlottesville, VA 22908 USA;Univ Virginia, Dept Biomed Engn, Charlottesville, VA 22908 USA.
2019 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 117, no 3, p. 445-452Article in journal (Refereed) Published
Abstract [en]

Fluorescent dye-dequenching assays provide a powerful and versatile means to monitor membrane fusion events. They have been used in bulk assays, for measuring single events in live cells, and for detailed analysis of fusion kinetics for liposomal, viral, and cellular fusion processes; however, the dyes used also have the potential to perturb membrane fusion. Here, using single-virus measurements of influenza membrane fusion, we show that fluorescent membrane probes can alter both the efficiency and the kinetics of lipid mixing in a dye- and illumination-dependent manner. R18, a dye that is commonly used to monitor lipid mixing between membranes, is particularly prone to these effects, whereas Texas Red is somewhat less sensitive. R18 further undergoes photoconjugation to viral proteins in an illumination-dependent manner that correlates with its inactivation of viral fusion. These results demonstrate how fluorescent probes can perturb measurements of biological activity and provide both data and a method for determining minimally perturbative measurement conditions.

Place, publisher, year, edition, pages
CELL PRESS , 2019. Vol. 117, no 3, p. 445-452
National Category
Biophysics
Identifiers
URN: urn:nbn:se:uu:diva-393335DOI: 10.1016/j.bpj.2019.06.022ISI: 000478966500005PubMedID: 31326109OAI: oai:DiVA.org:uu-393335DiVA, id: diva2:1355324
Funder
Wallenberg FoundationsNIH (National Institute of Health), R35 GM118044NIH (National Institute of Health), R01 GM098304Available from: 2019-09-27 Created: 2019-09-27 Last updated: 2019-09-27Bibliographically approved

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Giraldo, Ana M. VillamilKasson, Peter M.

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