Single Molecule Imaging of Transcription Factor Binding to DNA in Live Mammalian Cells

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Single Molecule Imaging of Transcription Factor Binding to DNA in Live Mammalian Cells

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Title: Single Molecule Imaging of Transcription Factor Binding to DNA in Live Mammalian Cells
Author: Gebhardt, Johann Christof manuel; Suter, David Michael; Roy, Rahul; Zhao, Zingqing W.; Chapman, Alec Randolph; Basu, Srinjan; Maniatis, Thomas P.; Xie, Xiaoliang Sunney

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Citation: Gebhardt, J. Christof M., David M. Suter, Rahul Roy, Ziqing W. Zhao, Alec R. Chapman, Srinjan Basu, Tom Maniatis, X. Sunnie Xie. Forthcoming. Single molecule imaging of transcription factor binding to DNA in live mammalian cells. Nature Methods.
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Abstract: Imaging single fluorescent proteins in living mammalian cells is challenged by out-of-focus fluorescence excitation. To reduce out-of-focus fluorescence we developed reflected light-sheet microscopy (RLSM), a fluorescence microscopy method allowing selective plane illumination throughout the nuclei of living mammalian cells. A thin light sheet parallel to the imaging plane and close to the sample surface is generated by reflecting an elliptical laser beam incident from the top by 90° with a small mirror. The thin light sheet allows for an increased signal-to-background ratio superior to that in previous illumination schemes and enables imaging of single fluorescent proteins with up to 100-Hz time resolution. We demonstrated the single-molecule sensitivity of RLSM by measuring the DNA-bound fraction of glucocorticoid receptor (GR) and determining the residence times on DNA of various oligomerization states and mutants of GR and estrogen receptor-α (ER), which permitted us to resolve different modes of DNA binding of GR. We demonstrated two-color single-molecule imaging by observing the spatiotemporal colocalization of two different protein pairs. Our single-molecule measurements and statistical analysis revealed dynamic properties of transcription factors.
Published Version: doi:10.1038/nmeth.2411
Terms of Use: This article is made available under the terms and conditions applicable to Open Access Policy Articles, as set forth at http://nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#OAP
Citable link to this page: http://nrs.harvard.edu/urn-3:HUL.InstRepos:10489393
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