Seeing is believing. Physics and Light are two wonderful tools to investigate living systems. Our lab develops state-of-the-art single-molecule biophysics and bioimaging techniques (e.g. super-resolution imaging, translation imaging, single-molecule FRET, single-molecule pull-down, single-molecule optogenetics, single-molecule force spectroscopy) and apply them to study complex biological systems.
1. Super-resolution imaging (STED, PALM, STORM)
Seeing is believing. Imaging with light has been an enormously successful method in biology to visualize tissues, cells and some intracellular structures. However, there is a fundamental limitation in optical imaging resolution, i.e., it is impossible to separate objects beyond the optical diffraction limit. Typically, it is difficult to image objects smaller than 250-300 nm with visible light.
Nature Reviews Molecular Cell Biology 2008
One obvious solution is increasing the energy of probe particle: from photons to X-ray or electrons. Although electron microscopy can achieve nanometer resolution, it requires demanding sample preparation, no molecular identification is possible, and most of all, EM cannot image in real-time or in live cells.
Super-resolution optical microscopy, such as STED or PALM/STORM, breaks this diffraction limit by using novel, unconventional modifications. STED uses an additional light beam (a donut beam) to deplete the fluorescence to break the diffraction limit. PALM/STORM uses the fact that the fluorophore’s center can be much more precisely located if the fluorophores are well separated. PALM/STORM uses photoactivatable fluorophores to separate them in time and to achieve super-resolution. By breaking the diffraction-limit, super-resolution imaging has been a very powerful tool to investigate cellular dynamics at nanoscale. Nobel prize in Chemistry is awarded to this imaging technique in 2014.
In our lab, we seek to develop novel super-resolution imaging methods including both STED and PALM/STORM. We have developed a novel method called ‘background-free STED’ to overcome one of the major bottleneck of STED imaging: the background noise. Moreover, we are looking for various collaborative researches that can adopt super-resolved investigation of cellular processes. One example can be the focal adhesion processes, which needs to be spatially and temporarily decoded in nanoscale.
2. Translation imaging
Cells respond to environmental cues by controlling protein translation dynamically. Single-molecule fluorescence in situ hybridization (smFISH) is the method of choice to locate individual mRNAs in fixed cells. In the fixed sample, an array of fluorophore-tagged oligodeoxynucleotides is introduced to be specifically hybridized with an mRNA of interest. Simultaneous detection of multiple mRNAs has been demonstrated with barcoding mRNA with different colors (multiplexed smFISH).
Although smFISH offers wealth of information within fixed-cell sample, imaging mRNAs in live cells can give information on the regulation of gene expression by imaging mRNAs and translations in real time. Individual mRNA molecules can be visualized in live cells by probe hybridization or by engineering mRNA itself. Molecular beacons are probes that target endogenous transcript and reduces background by turning on fluorescence after target hybridization. By engineering mRNAs to incorporate, for example, MS2 stem loops and using MS2 coat protein (MCP), single-molecule tracking of mRNAs is possible.
Annual Review of Biophysics 2018
Also, it is possible to observe single mRNA translation events by visualizing nascent peptides produced by single mRNA tagged with MS2/PP7 systems. To visualize the translation site (TLS) the Suntag system, which allows single-molecule readout of translation by producing mature fluorescent proteins, are used. This SunTag can be multimerized to enhance the fluorescent signal. Also after the target encoding regin, the auxin-induced degron (AID) is fused to remove the peptide after being fully translated.
Our lab aims to study real-time translation events in live cells to understand how protein abundance is regulated upon different environmental changes. Also, we seek to study transcription initiation, transcription elongation and cotranscriptional processing with combination of the methods described above.
3. Single-molecule Biophysics: smFRET, SiMPull, single-molecule optogenetics
Conventional methods to study living systems does not reflect the heterogeneity nor does reveal molecular dynamics. Single-molecule biophysical techniques, including single-molecule fluorescence resonance energy transfer (smFRET), single-molecule pull-down (SiMPull), and single-molecule optogenetics, are very powerful tool to overcome these limitations. By studying individual single-molecules, genuine molecular dynamic details can be unveiled that are not averaged out by taking an ensemble measurement of multiple heterogeneous molecules.
Nature 2011, Nature Chemical Biology 2014
Single-molecule FRET is a technique that can measure the nanometric distance between two fluorophores by using resonance energy transfer that is very sensitive to (typically) 1-10 nm range. This distance range makes smFRET ideal for detecting conformational changes of a protein, RNA or DNA as well as studying receptor-ligand binding because the size of typical proteins fall into this scale. The dyes can be labeled to two (or more if multi-color FRET is implemented) specific loci within a protein or oligonucleotides or labeled to receptor and ligand depending on purposes of study.
Single-molecule pull-down is a single-molecule version of immunoprecipitation. By using surface immobilized antibodies, one can pull-down indivisual proteins or individual protein complexes to study the stoichiometry or interaction between proteins inside the cell. SiMPull has the potential to be used as a new method for cellular or tissue diagnosis.
By using a protein domain that exhibits light-induced conformation changes, such as LOV2, our lab plans to develop and utilize single-molecule optogenetics. It is possible to fuse LOV2 domain with helicase or myosin motors to control the activity. Also, light-induced conformation changes can be used to manipulate the phase property of certain proteins in vivo. Moreover, single-molecule optogenetics allows hiding/exposing active enhancer domains, making possible fast optogenetic regulation.
Our lab aims to develop state-of-the-art biophysical techniques including smFRET, SiMPull, single-molecule optogenetics, force spectroscopy and to apply them to study diverse complex living systems.
4. Liquid-liquid phase separation; Membrane-less granules
Liquid-liquid phase separation (LLPS) is an emerging picture that explains various cellular membrane-less granules. Recently, the study of LLPS and the role and pathology of membrane-less granules attract great attention.
Our research interests include biological processes happening inside and outside of cells and molecular mechanisms of neurodegenerative diseases. Recently, we seek to understand the molecular underpinnings of an emerging liquid-liquid phase separation (LLPS) phenomena and its role in cellular context and in neurodegenerative diseases. Ultimately, we aim to discover pathological mechanism of Alzheimer’s (AD) and Parkinson’s disease (PD) at the molecular level for contributing to a new effective drug or therapy.
- “Background-free STED nanoscopy by polarization switching” (unpublished)
- “Molecular defect of ALS mutant FUS-RNA interaction drives aberrant phase separation and cellular aggregation” (unpublished)
- Lee, J.-C., Park, K.-K., Zhao, T.-M., Kim, Y.-H., “Einstein-Podolsky-Rosen Entanglement of Narrow-Band Photons from Cold Atoms” Phys. Rev. Lett. 117, 250501 (2016).
- Lee, J.-C., Lim, H.-T., Hong, K.-H., Jeong YC, Kim, M. S., Kim, Y.-H., “Experimental demonstration of delayed-choice decoherence suppression,” Nature Commun. 5, 4522 (2014).
- *Kim, Y.-S., *Lee, J.-C., Kwon, O., Kim, Y.-H., “Protecting entanglement from decoherence using weak measurement and quantum measurement reversal,” Nature Phys. 8, 117 (2012).
- Lee, J.-C., Jeong YC, Kim, Y.-S., Kim, Y.-H., “Experimental demonstration of decoherence suppression via quantum measurement reversal,” Opt. Express 19, 16309 (2011).