Single cell visualization of transcription kinetics variance of highly mobile identical genes using 3D nanoimaging
| Autor: | Annibale, Paolo, Gratton, Enrico |
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| Rok vydání: | 2014 |
| Předmět: | |
| Zdroj: | Scientific Reports, 5, 9258, 2015 |
| Druh dokumentu: | Working Paper |
| DOI: | 10.1038/srep09258 |
| Popis: | Both multi-cell biochemical assays and single cell fluorescence measurements have revealed that the elongation rate of Polymerase II (PolII) in eukaryotes varies largely across different cell types and genes. However, there is not yet a consensus whether intrinsic factors such as the position, local mobility or the engagement by an active molecular mechanism of a genetic locus could be the determinants of the observed heterogeneity. Employing high-speed 3D fluorescence nanoimaging we resolve here at the single cell level multiple, distinct regions of mRNA synthesis within a labeled transgene array. By employing phasor analysis, a fluorescence fluctuation spectroscopy technique, we demonstrate that these regions are active transcription sites that release mRNA molecules in the nucleoplasm, and we extract the local PolII elongation rate. While we detect a range of 10-100 bp/s for PolII elongation from cell to cell, we are now also able to measure up to a four-fold variation in the average elongation between identical copies of the same gene measured simultaneously within the same cell. Furthermore, we are able to visualize changes of PolII elongation as a function of time. We observe a correlation between the average elongation rate measured in a locus and its local mobility. Finally, by cross-correlating the transcriptional activity with the nm-sized movements of the active loci, we provide evidence of an active molecular mechanism determining displacements of the transcription sites concomitant to increases in transcriptional activity. Together these observations demonstrate that local factors, such as chromatin local mobility and the microenvironment of the transcription site, are an important source of transcription kinetics variability. Comment: 56 pages, 5 main figures and 10 supplementary figures |
| Databáze: | arXiv |
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