Real-Time Imaging of a Single Gene Reveals Transcription-Initiated Local Confinement.

Genome dynamics are intimately linked to the regulation of gene expression, the most fundamental mechanism in biology, yet we still do not know whether the very process of transcription drives spatial organization at specific gene loci. Here, we have optimized the ANCHOR/ParB DNA-labeling system for real-time imaging of a single-copy, estrogen-inducible transgene in human cells. Motion of an ANCHOR3-tagged DNA locus was recorded in the same cell before and during the appearance of nascent MS2-labeled mRNA. We found that transcription initiation by RNA polymerase 2 resulted in confinement of the mRNA-producing gene domain within minutes. Transcription-induced confinement occurred in each single cell independently of initial, highly heterogeneous mobility. Constrained mobility was maintained even when inhibiting polymerase elongation. Chromatin motion at constant step size within a largely confined area hence leads to increased collisions that are compatible with the formation of gene-specific chromatin domains, and reflect the assembly of functional protein hubs and DNA processing during the rate-limiting steps of transcription.

Quantitative nanoscale imaging of orientational order in biological filaments by polarized superresolution microscopy.

Essential cellular functions as diverse as genome maintenance and tissue morphogenesis rely on the dynamic organization of filamentous assemblies. For example, the precise structural organization of DNA filaments has profound consequences on all DNA-mediated processes including gene expression, whereas control over the precise spatial arrangement of cytoskeletal protein filaments is key for mechanical force generation driving animal tissue morphogenesis. Polarized fluorescence is currently used to extract structural organization of fluorescently labeled biological filaments by determining the orientation of fluorescent labels, however with a strong drawback: polarized fluorescence imaging is indeed spatially limited by optical diffraction, and is thus unable to discriminate between the intrinsic orientational mobility of the fluorophore labels and the real structural disorder of the labeled biomolecules. Here, we demonstrate that quantitative single-molecule polarized detection in biological filament assemblies allows not only to correct for the rotational flexibility of the label but also to image orientational order of filaments at the nanoscale using superresolution capabilities. The method is based on polarized direct stochastic optical reconstruction microscopy, using dedicated optical scheme and image analysis to determine both molecular localization and orientation with high precision. We apply this method to double-stranded DNA in vitro and microtubules and actin stress fibers in whole cells.