Single Protein Dynamics in Living Cells
Real-time visualization of cellular dynamics
Transcription is one of the most fundamental processes in cells. Most studies on transcription have been done outside cells using purified RNA polymerase and DNA. Due to this limitation, the mechanisms of transcription in live cells are still unknown. We are using high resolution fluorescence microscopy to study the dynamics of transcription in live cells (Yang et al, Nat comm 10, 5131, 2019). Our goal is to understand the mechanism of transcriptional regulation in space and time in cells. To achieve this, we develop various methods based on single-molecule fluorescence microscopy to simultaneously visualize DNA, mRNA, and RNA polymerase in a living cell.
Super-resolution microscopy for membrane protein dynamics
PALM (photoactivated localization microscopy) is one of the most promising super-resolution techniques (Nobel prize in Chemistry 2014) that use stochastic photo-activation (or photo–switching) of fluorescent proteins. This method is adequate for studying the localization of the specific target proteins in living cells. However, PALM is generally used for understanding only the static view of biomolecules. Compared with PALM, sptPALM (single-particle tracking PALM) has been developed for studying dynamic behaviors of biomolecules in living cells. In our lab, we are actively pursuing to improve PALM & sptPALM technique for studying the dynamic behavior of membrane proteins in living cells and in vitro system (Cho et al, J. Am. Chem. Soc. 143, 14125-14135, 2021).
In Vitro Single-molecule Dynamics
Single-molecule FRET for sub-millisecond and DNA bending dynamics
We are using alternating-laser excitation(ALEX) confocal microscopy for observing single-molecule FRET. The advantage of ALEX is that it can analyze the single-molecule FRET with high temporal resolution with accurate FRET values. It showed that ATP-independent SSB displacement by RecO in Deinococcus radiodurans at single-molecule level (Hwang et al, eLife 9, e50945, 2020). We also found highly bent DNA dynamics which interconverts two types of local melting, namely, a kink in the middle and forks at the ends using ALEX (Kim et al, Angew. Chem. Int. Ed. 31, 8943, 2015). Furthermore, we figured out that the effect of bending on the formation of Z-DNA using single-molecule FRET (Yi et al, J. Am. Chem. Soc. 144, 29, 2022).
Parkinson’s disease(PD) is the most popular neurodegenerative disease after Alzheimer’s disease. α-Synuclein, the main component of Lewy body, is believed to be the toxic species in PD. However, the detailed molecular pathogenesis of PD is still unclear. Our group investigates the pathogenic behaviors of α-Synuclein oligomers on SNARE-mediated vesicle fusion, i.e., the fundamental process of neurotransmission (Yoo et al, J. Mol. Biol., in press, 2022). For this analysis, we utilize ALEX (alternating laser excitation) –FRET technique, which enables efficient observation of the vesicle reaction in the presence of α-Synuclein oligomers. In our previous study, we suggested that α-Synuclein oligomers inhibit vesicle fusion by blocking SNARE complex formation, which is believed to destruct normal dopamine release in neurons (Yoo et al, PNAS 110, 4087, 2013).