Spatial organization of RNA polymerase II inside a mammalian cell nucleus revealed by reflected light-sheet superresolution microscopy
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Date
August 7, 2015
Author(s)
Gebhardt, J. Christof M.
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Citation
Zhaoa, Ziqing W., Rahul Roya, J. Christof M. Gebhardta, David M. Sutera, Alec R. Chapmana, and X. Sunney Xiea. 2013. Spatial organization of RNA polymerase II inside
a mammalian cell nucleus revealed by reflected
light-sheet superresolution microscopy. PNAS 111 (2), 681–686. doi:10.1073/pnas.1318496111/-/DCSupplemental.
a mammalian cell nucleus revealed by reflected
light-sheet superresolution microscopy. PNAS 111 (2), 681–686. doi:10.1073/pnas.1318496111/-/DCSupplemental.
Abstract
Superresolution microscopy based on single-molecule centroid determination has been widely applied to cellular imaging in recent years. However, quantitative imaging of the mammalian nucleus has been challenging due to the lack of 3D optical sectioning methods for normal-sized cells, as well as the inability to accurately count the absolute copy numbers of biomolecules in highly dense structures. Here we report a reflected light-sheet superresolution microscopy method capable of imaging inside the mammalian nucleus with superior signal-to-background ratio as well as molecular counting with single-copy accuracy. Using reflected light-sheet superresolution microscopy, we probed the spatial organization of transcription by RNA polymerase II (RNAP II) molecules and quantified their global extent of clustering inside the mammalian nucleus. Spatiotemporal clustering analysis that leverages on the blinking photophysics of specific organic dyes showed that the majority (>70%) of the transcription foci originate from single RNAP II molecules, and no significant clustering between RNAP II molecules was detected within the length scale of the reported diameter of “transcription factories.” Colocalization measurements of RNAP II molecules equally labeled by two spectrally distinct dyes confirmed the primarily unclustered distribution, arguing against a prevalent existence of transcription factories in the mammalian nucleus as previously proposed. The methods developed in our study pave the way for quantitative mapping and stoichiometric characterization of key biomolecular species deep inside mammalian cells.
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