RESUMO
Development of single-molecule localization microscopy techniques has allowed nanometre scale localization accuracy inside cells, permitting the resolution of ultra-fine cell structure and the elucidation of crucial molecular mechanisms. Application of these methodologies to understanding processes underlying DNA replication and repair has been limited to defined in vitro biochemical analysis and prokaryotic cells. In order to expand these techniques to eukaryotic systems, we have further developed a photo-activated localization microscopy-based method to directly visualize DNA-associated proteins in unfixed eukaryotic cells. We demonstrate that motion blurring of fluorescence due to protein diffusivity can be used to selectively image the DNA-bound population of proteins. We designed and tested a simple methodology and show that it can be used to detect changes in DNA binding of a replicative helicase subunit, Mcm4, and the replication sliding clamp, PCNA, between different stages of the cell cycle and between distinct genetic backgrounds.
Assuntos
Proteínas de Ligação a DNA/análise , Microscopia de Fluorescência/métodos , Ciclo Celular , Replicação do DNA , Difusão , Componente 4 do Complexo de Manutenção de Minicromossomo/análise , Antígeno Nuclear de Célula em Proliferação/análise , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/análiseRESUMO
A technique for measuring distances between two or more fluorophores spaced in the 10-100 nm range is described. We identify a linear correlation between the intensity-amplitude in the difference-image of single molecules undergoing fluorescence fluctuations and their separation. The transform is used to map distances between coupled fluorophores.