Research

Research Summary 

The human body consists of roughly 30 trillion cells, and a single eukaryotic cell can host up to several billion protein molecules to perform their functions. These protein molecules, in combination with several nucleic acids, are involved in an array of biochemical processes within cells, such as immune protection, cell cycle regulation and differentiation, cellular trafficking, catalyzing biochemical reactions, and so on. Phase separation of biomolecules into liquid-like supramolecular assemblies has newly emerged as a critical organizing principle within living cells. The intrinsically disordered group of proteins (IDPs/IDRs) comprising low-complexity (LC), prion-like domains have been identified as the major drivers of cellular phase separation leading to the formation of membrane-less organelles (MLOs) enriched in proteins and nucleic acids. In addition to performing diverse physiological functions, these non-canonical MLOs are also known to undergo aberrant phase transitions and aggregation, resulting in a range of fatal neurodegenerative diseases. Hence, obtaining insights into critical molecular events rendering the formation of these pathological assemblies has gained significant importance. A plethora of ensemble tools have been employed to delineate the biophysical principles of phase separation and maturation. However, a detailed understanding of the fundamental molecular drivers governing phase separation and liquid-to-solid transitions remains elusive. See below for detailed sections. 

I. Single-molecule FRET dissects crucial molecular events during biological phase separation in a droplet-by-droplet manner

This work revolves around the prion-like intrinsically disordered low complexity region of Fused in Sarcoma (FUS-LC), a protein associated with the pathology of ALS and FTLD. FUS-LC serves as an archetypal Intrinsically Disordered Protein (IDP) characterized by a low-complexity amino acid sequence, enriched in serine, tyrosine, glycine, and glutamine residues, playing a crucial role in driving the phase transition of FUS. Employing a highly sensitive technique, single-molecule Förster Resonance Energy Transfer (sm-FRET), along with ultrafast fluorescence spectroscopy, fluorescence correlation spectroscopy in conjunction with vibrational Raman spectroscopy, we investigate the protein chain in both monomeric and droplet phases. Our analysis captures the alterations in the polypeptide chain during phase separation. The results unveil significant conformational diversity and the presence of distinct subpopulations within the FUS-LC polypeptide chain in both dispersed and condensed phases, accompanied by unwinding of the polypeptide chain upon phase separation. The introduction of a disease-associated glycine-to-glutamate (G156E) mutation further expands the FUS-LC polypeptide chain in the condensed phase. We propose that this unwinding leads to enhanced multivalency, facilitating a transition from intra-molecular to intermolecular contacts, potentially accelerating the aggregation of the mutant FUS-LC. This conformational unwinding, resulting in the formation of a dense protein network, may represent a general phenomenon in the phase transition process.


II. Single-droplet surface-enhanced Raman scattering illuminates the conformational fingerprint within phase-separated biomolecular compartments of Fused in Sarcoma (FUS)

 

We also demonstrate the vibration Raman spectroscopy utilizing plasmonic silver nanoparticles for single-droplet surface-enhanced Raman scattering (SERS). This approach allows us to uncover the internal dynamics of protein droplets, capturing their conformational diversity and distribution on a droplet-by-droplet basis. These precise measurements enable a detailed examination of the mesoscopic condensed phase, revealing the molecular factors influencing the fascinating biophysics of condensates. The exceptional sensitivity of our measurements provides unprecedented insight into the crucial interactions, conformational variations, and structural arrangements within the condensed phase of the well-known RNA-binding protein, FUS, in a single-droplet fashion. Our results indicate that the structured C-terminal RNA-binding domain of FUS experiences partial unwinding in the presence of RNA, leading to increased disorder in the polypeptide chain. This disorder promotes both homotypic (FUS-FUS) and heterotypic (FUS-RNA) interactions within the condensed phase. The highly sensitive single-droplet vibrational methodology serves as a powerful tool for unraveling the fundamental molecular drivers behind biological phase transitions in various biomolecular condensates relevant to physiology and disease. By employing different surface functionalities and alternative metals as SERS substrates, this ultra-sensitive approach can be applied to investigate a broad range of biomolecular condensates.

III. Energy migration via homoFRET captures the dynamic architecture of in vitro and in situ formed phase-separated biomolecular compartments of Fused in Sarcoma (FUS)

 

Aberrant maturation of dynamic, phase-separated liquid-like assemblies into irreversible gel-like or solid-like aggregates is implicated in a broad spectrum of fatal neurodegenerative diseases. These assemblies differ in the interior material properties, including diffusivity and viscoelasticity determined by the nature of intermolecular interactions. In order to dissect the alterations in the internal material characteristics of these biomolecular condensates, influenced by various factors such as sequence composition, truncations, mutations, post-translational modifications, and nucleic acid and biomolecule stoichiometry, new investigative tools are indispensable. Here, we utilize homo-Förster Resonance Energy Transfer (homoFRET) as a molecular proximity ruler to investigate intermolecular energy migration, shedding light on molecular packing at the nanometric scale within biomolecular condensates. The homoFRET efficiency, estimated from the decrease in fluorescence anisotropy due to rapid depolarization, serves as an indicator of molecular packing influencing the material properties of biomolecular condensates. Employing single-droplet anisotropy imaging, we document spatially-resolved homoFRET efficiencies in condensates formed by fluorescent protein-tagged Fused in Sarcoma (FUS). Through single-droplet picosecond time-resolved anisotropy measurements, we discern various energy migration events within the intricate network of polypeptide chains in FUS condensates. Our homoFRET investigations also capture the modulation of material properties by RNA, ATP, and post-translational modification. Additionally, utilizing mammalian cell lines with stably expressed FUS, we explore nuclear FUS and stress granule formation induced by oxidative stress in the cytoplasm. Our findings highlight that spatially-resolved homoFRET methodology serves as a powerful tool for investigating intracellular phase transitions in both cell physiology and disease.

Undergraduate Research Summary

Eukaryotic transcription is a highly regulated process which requires several proteins at its various stages of initiation, elongation and termination. Initially, pre-initiation and initiation were thought to be the only stages at which transcription was regulated. However, studies in the past decade or so have shown that transcription elongation is equally important for control of gene expression. ELL (Eleven Nineteen Lysine Rich in Leukemia) family of proteins have been shown to suppress transient pausing of the RNA Polymerase II during transcriptional elongation in vitro, whereas the EAF (ELL Associated Factor) family members are known to physically interact and regulate the transcriptional elongation function of ELL. It is well established that many transcription factors interact with the multi-subunit mediator complex to control gene expression. In this study, we decided to test if the S. pombe orhologs of ELL and EAF can interact with the various subunits of the mediator complex including Med19, Med7, Med8, Med31 and Cdk8, as well as Cdk9, which is a part of elongation factor P-TEFb. Towards this goal, coding sequences of each of these mediator subunits were cloned in both the DNA binding and Activation domain vectors. Subsequently, their auto-activation potential and ability to interact with ELL and EAF was checked using the yeast two hybrid assay.


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