Spatial organization of RNA polymerase II inside a mammalian cell nucleus revealed by reflected light-sheet superresolution microscopy

Ziqing W. Zhao; Rahul Roy; J. Christof M. Gebhardt; David M. Suter; Alec R. Chapman; X. Sunney Xie

doi:10.1073/pnas.1318496111

Edited by Robert H. Singer, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY, and approved December 6, 2013 (received for review October 1, 2013)

Significance

We developed an optical imaging technique that combines reflected light-sheet illumination with superresolution microscopy, allowing us to image inside mammalian nuclei at subdiffraction-limit resolution and to count biomolecules with single-copy accuracy. Applying this technique to probe the spatial organization of RNA polymerase II-mediated transcription, we found that the majority of the transcription foci consist of only one RNAP II molecule, contrary to previous proposals. By quantifying the global extent of clustering across RNAP II molecules in the nucleus, we provide clear and convincing answers to the controversy surrounding the prevalent existence of “transcription factories.” Moreover, our work presents imaging and analysis tools for the quantitative characterization of nuclear structures, which could be generally applied to probe many other mammalian systems.

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.

Footnotes

↵¹Z.W.Z., R.R., J.C.M.G., and D.M.S. contributed equally to this work.
↵²Present address: Department of Chemical Engineering, Indian Institute of Science, Bangalore 560012, Karnataka, India.
↵³Present address: Department of Physics, University of Ulm, 89081 Ulm, Germany.
↵⁴Present address: The Institute of Bioengineering, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
↵⁵To whom correspondence should be addressed. E-mail: xie{at}chemistry.harvard.edu.

Author contributions: Z.W.Z., R.R., J.C.M.G., D.M.S., and X.S.X. designed research; Z.W.Z., R.R., J.C.M.G., and D.M.S. performed research; A.R.C. contributed new reagents/analytic tools; Z.W.Z., R.R., and J.C.M.G. analyzed data; and Z.W.Z., R.R., J.C.M.G., D.M.S., and X.S.X. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1318496111/-/DCSupplemental.