The Investigation of Biophysical Properties of Biomolecular Condensates and Aggregates in Live Cells
Open Access
- Author:
- Hsiung, Chia Heng
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology (PHD)
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- September 29, 2021
- Committee Members:
- Lorraine Santy, Major Field Member
Xin Zhang, Chair & Dissertation Advisor
Moriah Szpara, Major Field Member
Ken Keiler, Professor in Charge/Director of Graduate Studies
Lauren Zarzar, Outside Field Member
Emily Weinert, Outside Unit Member - Keywords:
- Biomolecular Condensates
Liquid-Liquid Phase Separation
Polarity
Viscosity
AggTag - Abstract:
- The work presented in this dissertation details the efforts in applying a novel class of environmental sensitive fluorophores in live cells to directly detect the physicochemical properties of intracellular biomolecular condensates and protein aggregates through microscopies. Ultimately, the objectives of such efforts are fulfilled through examining different types of membraneless biostructures including RNA-binding protein condensates, protein aggregates derived from different stresses, and RNA granules. This dissertation is organized into three parts: Introduction and Literature reviews (Chapter 2), Experimental Data (Chapter 3-5), and Conclusion and Future Direction (Chapter 6). Chapter 2 reviews the literature of current fields related to the research presented herein: biomolecular condensates and liquid-liquid phase separation (LLPS), regulations of biomolecular condensates, RNA-binding proteins (RBPs) in these condensates and the factors effects RBPs’ LLPS, structure and function of TDP43, and the multi-step protein aggregation process and its detection as well as the efforts in the Zhang group to detect the multi-step protein aggregation process in live cells. Focusing on author’s research work, Chapter 3 is titled Biophysical properties of anisosomes formed by TDP43 RNA-binding defective mutants in live cell. It describes the experimental efforts in addressing whether the N-terminal domain and RNA-binding domain of TDP43 influence the ability of the protein to undergo LLPS and to form granules under stress condition in live cell. The investigation of the physicochemical properties of anisosomes formed by RNA-binding deficient TDP43 mutants and its location relative to the existing nuclear bodies. Together, the work presented in chapter 3 demonstrated the importance of N-terminal domain influencing TDP43’s ability to undergo LLPS and to form stress granules, and RNA-binding deficient TDP43 mutants go through rapid LLPS form structure called anisosomes. The environmental sensitive fluorophores successfully detected and reported the polarity and viscosity of the anisosomes through fluorescence lifetime microscopy (FLIM). The fourth Chapter is titled: Single-atom replacement as a general approach towards solvatochromic probes to detect protein aggregation in live cells. It describes the single-atom replacement synthetical design of acridone-based fluorophores which enhances the polarity sensing ability of the fluorophore for the utilization in biological application. The fluorophore (SO2X)’s ability to shift in emission wavelength in responds to polarity changes makes it an ideal candidate for live cell imaging. Data presented in this Chapter also demonstrated the practical utilization of SO2X in live cell imaging and new physicochemical insight reported regarding to protein aggregates derived from different origin through lambda scan in confocal microscopy application. Finally, Chapter 5 is titled: Biophysical properties of RNA granules inside the cell. It describes the effort in developing a new experimental platform for detecting the physicochemical properties of RNA granules that utilizes environmental sensitive fluorophores in live cell imaging. RNA granules (or RNA foci), a type of ribonucleoproteins (RNPs) whose assembly is mostly driven by RNA-RNA interactions. It started to receive more appreciation within this decade. While studies have been conducted, however, the underlying mechanism in terms of how RNA-RNA interaction drives and stabilizes the formation of RNA foci is still unclear. The work presented in this Chapter, demonstrated the establishment of a new platform for direct detection of the polarity and viscosity of the RNA foci.