Research

Research Summary

My research focuses on revealing mysteries about how cells are regulated at the very single molecular scale. To address this outstanding question, I focus on three exciting areas: liquid-liquid phase separation (LLPS), single-molecule imaging, and super-resolution optical microscopy. 

LLPS is a phenomenon that soluble proteins separate from aqueous protoplasm upon physical and chemical cues. It has been found that astonishingly many cellular processes have implications with LLPS, including amyloid formation, chromatin organization, temperature sensing, only to name a few. As a biophysicist, I can’t help but notice that there are huge inconsistencies between biological understanding and physical, quantitative understanding in enzyme reaction rates, cellular response times, etc. LLPS can explain these mysteries in cell biology.

Cells are regulated at different levels. I aim to understand how they are regulated by using single-molecule imaging. In particular, I am observing single RNA molecules in living cells to reveal spatiotemporal dynamics of regulatory RNAs such as miRNA and lncRNA. One of the questions I am recently answering is how lncRNA transcribed from nucleus regulate chloroplast function via anterograde signaling.

To study cellular mysteries at the molecular scale, it is necessary to image living cells at very high spatiotemporal resolution, necessitating super-resolution optical microscopy. I develop super-resolution microscopes and illuminate cellular processes with state-of-the-art techniques. I am solving the problems and pushing the limits of stimulated emission depletion (STED) microscopy, lattice light sheet microscopy (LLSM), etc.

Liquid-liquid phase separation; Biomolecular condensates

Liquid-liquid phase separation (LLPS) is an emerging picture that explains various biomolecular condensates in cells. What recent studies reveal is astonishing. Unbelievably many cellular processes are being found to be related to LLPS, including amyloid formation in neurodegenerative diseases, chromatin organization, mRNA translation, only to name a few.

In our lab, we seek to understand the molecular underpinnings of an emerging LLPS phenomena by using biophysical technologies and unveil condensates' role in different cellular contexts. Specifically, our lab is interested in studying LLPS taking place in mammalian cells and in plants.

Ultimately, we aim to find or invent a new orthogonal 'knob' based on the principles of LLPS for cellular modulation at high temporal resolution. We believe that in the future this novel 'knob' can be used to treat disease which is not druggable using conventional mechanisms. 

Super-resolution optical microscopy (STED, PALM/STORM)

Optical microscopy has been an enormously successful method in biology to visualize tissues, cells and 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. 

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 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. 

See Nobel Prize in Chemistry 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. 

Single-molecule biophysics: smFRET, SiMPull, smFISH

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 fluorescence in situ hybridization (smFISH), 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. 

smFRET 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. 

SiMPull 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 cell/tissue/liquid diagnosis.

smFISH, combined with novel amplification methods, enables observation of single mRNAs and regulatory RNAs (such as long non-coding RNAs and micro RNAs). Therefore, one can study individual behaviors of RNAs and their heterogeneity by using smFISH.

Our lab aims to take advantage of single-molecule biophysical techniques including smFRET, SiMPull, smFISH to study diverse complex living systems such as regulatory RNA - target RNA interaction, LLPS, and protein ubiquitination and degradation. Also, we aim to develop state-of-the-art single-molecule methods.

Imaging translation dynamics

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. 

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.