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Information Center for Mathematical Science

세미나

Information Center for Mathematical Science

세미나

2018 KAIST Physics Seminar
Title Capturing protein cluster dynamics and gene expression output in live cells
Date 2018-10-16
Speaker Won-Ki Cho (MIT)
Sponsors 2018-10-16
Host KAIST
Place #1323, E6-2
Abstract Detecting biomolecular interactions in the single-molecule level has been achieved using up-todate in vitro fluorescence-based single-molecule techniques. However, visualizing molecular dynamics of biomolecules in living cells has been tackled by some problems, like back-ground noise, diffraction-limit and toxicity of fluorescent probes. Here, to overcome the limitations, we developed a live-cell single-molecule imaging approach using photo-activated localization microscopy (PALM). To investigate gene expression in living cells, we labeled a photoactivatable fluorescent protein to RNA Polymerase II (Pol II) protein that directly synthesizes messenger RNA (mRNA) molecules from DNA during transcription, and also labeled mRNA molecules of a specific gene for dual-color live-cell imaging. We found that Pol II proteins form clusters at an active gene locus and moreover lifetime of Pol II cluster is proportional to the mRNA output. More recently, to address biological processes upstream of transcription, we developed a livecell assay to observe cooperative dynamics of enhancers, which are gene-expression regulatory DNA elements, and transcription sites. Gene activation is thought to involve a multistep process whereby transcription factors bind to distal enhancer sites and recruit a Mediator protein complex which contacts the Pol II protein at the gene loci. The interaction of Mediator and Pol II has yet to be observed in the nucleus of living cells and the dynamics of this interaction are not yet elucidated. We used quantitative PALM imaging and lattice-light-sheet imaging to study the organization and dynamics of Mediator and Pol II. We found that Mediator and Pol II form large stable clusters and also observed that these clusters have properties expected for phase-separated biomolecular condensates. Moreover, labeling a specific gene, we observed protein condensates dynamically contact with the active gene.