Biology subjects

Arora, L.

Publications and source records attributed to Arora, L..

4 recordsLinked to original sources

bioRxiv2024

Chaperone-mediated heterotypic phase separation prevents the amyloid formation of the pathological Y145Stop variant of the prion protein

Biomolecular condensates formed via phase separation of proteins and nucleic acids are crucial for the spatiotemporal regulation of a diverse array of essential cellular functions and the maintenance of cellular homeostasis. However, aberrant liquid-to-solid phase transitions of such condensates are associated with several fatal human diseases. Such dynamic membraneless compartments can contain a range of molecular chaperones that can regulate the phase behavior of proteins involved in the formation of these biological condensates. Here, we show that a heat shock protein 40 (Hsp40), Ydj1, exhibits a holdase activity by potentiating the phase separation of a disease-associated stop codon mutant of the prion protein (Y145Stop) either by recruitment into Y145Stop condensates or via Y145Stop-Ydj1 two-component heterotypic phase separation that prevents the conformational conversion of Y145Stop into amyloid fibrils. Utilizing site-directed mutagenesis, multicolor fluorescence imaging, single-droplet steady-state and picosecond time-resolved fluorescence anisotropy, fluorescence recovery after photobleaching, and fluorescence correlation spectroscopy, we delineate the complex network of interactions that govern the heterotypic phase separation of Y145Stop and Ydj1. We also show that the properties of such heterotypic condensates can further be tuned by RNA that promotes the formation of multicomponent multiphasic protein-RNA condensates. Our vibrational Raman spectroscopy results in conjunction with atomic force microscopy imaging reveal that Ydj1 effectively redirects the self-assembly of Y145Stop towards a dynamically-arrested non-amyloidogenic pathway, preventing the formation of typical amyloid fibrils. Our findings underscore the importance of chaperone-mediated heterotypic phase separation in regulating aberrant phase transitions and amyloid formation associated with a wide range of deadly neurodegenerative diseases.

biophysics↗

bioRxiv2024

Intermolecular Energy Migration via HomoFRET Captures the Modulation in the Material Property of Phase-Separated Biomolecular Condensates

Biomolecular condensation via phase separation of proteins and nucleic acids has emerged as a crucial mechanism underlying the spatiotemporal organization of cellular components into functional membraneless organelles. However, aberrant maturation of these dynamic, liquid-like assemblies into irreversible gel-like or solid-like aggregates is associated with a wide range of fatal neurodegenerative diseases. New tools are essential to dissect the changes in the internal material properties of these biomolecular condensates that are often modulated by a wide range of factors involving the sequence composition, truncations, mutations, post-translational modifications, and the stoichiometry of nucleic acids and other biomolecules. Here, we employ homo-Forster Resonance Energy Transfer (homoFRET) as a proximity ruler to study intermolecular energy migration that illuminates the molecular packing in the nanometric length-scale within biomolecular condensates. We used the homoFRET efficiency, measured by a loss in the fluorescence anisotropy due to rapid depolarization, as a readout of the molecular packing giving rise to material properties of biomolecular condensates. Using single-droplet anisotropy imaging, we recorded spatially-resolved homoFRET efficiencies of condensates formed by fluorescent protein-tagged Fused in Sarcoma (FUS). By performing single-droplet picosecond time-resolved anisotropy measurements, we were able to discern various energy migration events within the dense network of polypeptide chains in FUS condensates. Our homoFRET studies also captured the modulation of material properties by RNA, ATP, and post-translational modification. Additionally, we utilized mammalian cell lines stably expressing FUS to study nuclear FUS and oxidative stress-induced stress granule formation in the cytoplasm. Our studies demonstrate that spatially-resolved homoFRET methodology offers a potent tool for studying intracellular phase transitions in cell physiology and disease.

biophysics↗

bioRxiv2023

Single-Molecule FRET Illuminates Structural Subpopulations and Dissects Crucial Molecular Events During Phase Separation of a Prion-Like Low Complexity Domain

Biomolecular condensates formed via phase separation of proteins and nucleic acids are thought to be associated with a wide range of cellular functions and dysfunctions. We dissect critical molecular events associated with phase separation of an intrinsically disordered prion-like low-complexity domain of Fused in Sarcoma by performing single-molecule studies that permit us to access the wealth of molecular information that is skewed in conventional ensemble experiments. Our single-molecule FRET experiments reveal the coexistence of two conformationally distinct subpopulations in the monomeric form. Single-droplet single-molecule FRET studies coupled with fluorescence correlation spectroscopy, picosecond time-resolved fluorescence anisotropy, and vibrational Raman spectroscopy indicate that structural unwinding switches intramolecular interactions into intermolecular contacts allowing the formation of a dynamic network within condensates. A disease-related mutation introduces enhanced structural plasticity engendering greater interchain interactions that can accelerate pathological aggregation. Our findings provide key mechanistic underpinnings of sequence-encoded dynamically-controlled structural unzipping resulting in biological phase separation.

biophysics↗

bioRxiv2021

Spatiotemporal Modulations in Heterotypic Condensates of Prion and α-Synuclein Control Phase Transitions and Amyloid Conversion

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) of proteins and nucleic acids are thought to govern critical cellular functions. These multicomponent assemblies provide dynamic hubs for competitive homotypic and heterotypic interactions. Here, we demonstrate that the complex coacervation between the prion protein (PrP) and -synuclein (-Syn) within a narrow stoichiometry regime results in the formation of highly dynamic liquid droplets. Domain-specific electrostatic interactions between the positively charged intrinsically disordered N-terminal segment of PrP and the negatively charged C-terminal domain of -Syn drive the formation of these highly tunable, reversible, thermo-responsive condensates. Picosecond time-resolved measurements revealed the existence of relatively ordered electrostatic nanoclusters that are stable on the nanosecond timescale and can undergo breaking-and-making on a much slower timescale giving rise to the liquid-like behavior on the second timescale and mesoscopic length-scale. The addition of RNA to these preformed coacervates yields multiphasic, anisotropic, vesicle-like, hollow condensates. LLPS promotes liquid-to-solid maturation of -Syn-PrP condensates resulting in the rapid conversion into heterotypic amyloids. Our results suggest that synergistic interactions between PrP and -Syn in liquid condensates can offer mechanistic underpinnings of their physiological role and overlapping neuropathological features.

biophysics↗