1. Towards mapping the 3D genome through high speed single-molecule tracking of functional transcription factors in single living cells
- Author
-
Wollman, Adam J.M., Hedlund, Erik G., Shashkova, Sviatlana, and Leake, Mark C.
- Subjects
Saccharomyces cerevisiae Proteins ,EMCCD, electron multiplying charge-coupled device ,Intravital Microscopy ,FISH, fluorescence in situ hybridization ,Green Fluorescent Proteins ,PBS, phosphate-buffered saline ,Saccharomyces cerevisiae ,Article ,Imaging, Three-Dimensional ,Genes, Reporter ,Transcription factors ,GFP, green fluorescent protein ,Fluorescent Dyes ,Cell Nucleus ,Spatial Analysis ,Single-molecule ,ChIP-Seq, chromatin immunoprecipitation-sequencing ,Single Molecule Imaging ,Gene regulation ,Repressor Proteins ,Microscopy, Fluorescence ,Super-resolution ,RT, room temperature ,Nucleic Acid Conformation ,MSD, mean square displacement ,Chromosomes, Fungal ,Genome, Fungal ,Single-Cell Analysis ,PSF, point spread function ,Yeast genome ,Transcription - Abstract
Highlights • Single-molecule imaging to determine genome organization in live budding yeast. • Utilize astigmatism imaging to enable extraction of 3D position data. • Use a transcription factor Mig1 to mark the genome at target genes. • Map out 3D coordinates of target genes with millisecond sampling. • Observe differences to 3C structure indicating dynamic heterogeneity of the genome., How genomic DNA is organized in the nucleus is a long-standing question. We describe a single-molecule bioimaging method utilizing super-localization precision coupled to fully quantitative image analysis tools, towards determining snapshots of parts of the 3D genome architecture of model eukaryote budding yeast Saccharomyces cerevisiae with exceptional millisecond time resolution. We employ astigmatism imaging to enable robust extraction of 3D position data on genomically encoded fluorescent protein reporters that bind to DNA. Our relatively straightforward method enables snippets of 3D architectures of likely single genome conformations to be resolved captured via DNA-sequence specific binding proteins in single functional living cells.
- Published
- 2020