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Towards mapping the 3D genome through high speed single-molecule tracking of functional transcription factors in single living cells.

ABSTRACT: 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.

SUBMITTER: Wollman AJM

PROVIDER: S-EPMC6971689 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Towards mapping the 3D genome through high speed single-molecule tracking of functional transcription factors in single living cells.

Wollman Adam J M AJM Hedlund Erik G EG Shashkova Sviatlana S Leake Mark C MC

Methods (San Diego, Calif.) 20190626

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 pr ...[more]

PMID: 31252059

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Project description:Soluble factors are an essential means of communication between cells and their environment. However, many molecules readily interact with extracellular matrix components, giving rise to multiple modes of diffusion. The molecular quantification of diffusion in situ is thus a challenging imaging frontier, requiring very high spatial and temporal resolution. Overcoming this methodological barrier is key to understanding the precise spatial patterning of the extracellular factors that regulate immune function. To address this, we have developed a high-speed light microscopy system capable of millisecond sampling in ex vivo tissue samples and submillisecond sampling in controlled in vitro samples to characterize molecular diffusion in a range of complex microenvironments. We demonstrate that this method outperforms competing tools for determining molecular mobility of fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) for evaluation of diffusion. We then apply this approach to study the chemokine CXCL13, a key determinant of lymphoid tissue architecture, and B-cell-mediated immunity. Super-resolution single-molecule tracking of fluorescently labeled CCL19 and CXCL13 in collagen matrix was used to assess the heterogeneity of chemokine mobility behaviors, with results indicating an immobile fraction and a mobile fraction for both molecules, with distinct diffusion rates of 8.4?±?0.2 and 6.2?±?0.3?µm2s-1, respectively. To better understand mobility behaviors in situ, we analyzed CXCL13-AF647 diffusion in murine lymph node tissue sections and observed both an immobile fraction and a mobile fraction with an example diffusion coefficient of 6.6?±?0.4?µm2s-1, suggesting that mobility within the follicle is also multimodal. In quantitatively studying mobility behaviors at the molecular level, we have obtained an increased understanding of CXCL13 bioavailability within the follicle. Our high-speed single-molecule tracking approach affords a novel perspective from which to understand the mobility of soluble factors relevant to the immune system.

Dataset Information

Towards mapping the 3D genome through high speed single-molecule tracking of functional transcription factors in single living cells.

Publications

Towards mapping the 3D genome through high speed single-molecule tracking of functional transcription factors in single living cells.

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