Poster R17 - Visualizing nuclear landscapes with multiplexed super-resolution fluorescence microscopy

Introduction: Nearly half of all human proteins localize to the cell nucleus where they perform diverse functions including DNA replication, ribosome biogenesis, and gene translation. How the nucleus organizes these phenomena, which require the coordinated assembly of multimeric protein complexes, remains poorly understood. This feat is especially challenging given that the nucleus lacks internal membranes to partition compartments and because this organization predominantly occurs below the resolution limit of conventional microscopes. To inform these organizational principles, we have developed a multiplexed super-resolution fluorescence microscopy system that enables simultaneous nanoscale visualization of DNA, epigenetic, and proteomic targets in single cells.

Materials and

Methods: We utilized oligonucleotide-guided points accumulation in nanoscale topography (Exchange-PAINT [1,2]) to label specific proteins and epigenetic marks and combined this with swept highly inclined and laminated optical sheet (HIST) microscopy [3] to perform optically sectioned single molecule imaging of the cell nucleus. We developed and validated a panel of 12 oligonucleotide imager strands for low-background labeling of nuclear targets and used them to image DNA [4], histone post-translational modifications, RNA Polymerase II CTD-tail phosphorylation variants, hPTM “readers” and “writers”, and nuclear landmarks such as nuclear speckles and the nuclear envelope (Fig. 1A, B).

Results, Conclusions, and Discussions: We image 13 nuclear labels with 10 nm lateral spatial resolution and 41 nm z-resolution in single nuclei across ~200 x 56 μm fields of view (containing ~8-10 interphase nuclei). Nanoscale features are observable with our reconstructed images (Fig. 1C, D) and further quantified for pairwise associations of various labels. We find that active and repressive regions within the nucleus begin to partition at approximately 1-micron. Below ~200 nm, proteins show distinctive variations in clustering which support unique biophysical interaction mechanisms. We also characterize target reorganization and chromatin structure upon histone acetyltransferase (HAT), histone deacetylase (HDAC), and transcriptional pharmacological inhibition in human cells. In conclusion, we have developed a multiplexed imaging approach to characterize nanoscale organization of proteins and DNA within the nucleus. We envision that this method will be useful to explore how the nuclear organization regulates diverse cellular processes from gene regulation to cell fate specification.

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