Abstract Biomolecular condensates formed via macromolecular phase separation of proteins and nucleic acids control a myriad of essential cellular processes, whereas abnormal phase transitions into solid-like aggregates are associated with a range of fatal neurodegenerative diseases. Here, we present a unique case to demonstrate that two neuronal proteins, TDP-43 and tau, undergo heterotypic phase separation via domain-specific interactions regulated by an autoinhibitory conformational switch. Using single-molecule FRET (Förster resonance energy transfer), in combination with multi-color high-resolution imaging, fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, single-droplet fluorescence anisotropy imaging, homoFRET microscopy, fluorescence lifetime-FRET imaging, vibrational Raman spectroscopy, and electron microscopy, we unmask the interplay of molecular drivers and dissect the sequence of events associated with the formation of TDP-43:tau co-condensates that undergo liquid-to-solid phase transitions into cytotoxic amyloids. Our cellular studies show that a disease-associated cytosolic fragment of TDP-43 recruits tau into oxidative stress-induced cytoplasmic granules, eliciting cellular toxicity. Our findings provide mechanistic underpinnings of co-condensation-mediated aberrant phase transitions associated with exacerbated neuropathological outcomes.

Hendra virus (HeV) is a biosafety level 4 human pathogen belonging to the Henipavirus genus within the Paramyxoviridae family. In HeV, the phosphoprotein-encoding gene also drives the synthesis of the V and W proteins that are two major players in the host innate immune response evasion. These three proteins share a common intrinsically disordered N-terminal domain (NTD) and have distinct C-terminal domains. We recently reported the ability of a short region (i.e., PNT3), located within the shared NTD, to form fibrils. We subsequently identified a PNT3 motif (EYYY) critically involved in fibrillation and deciphered the contribution of each tyrosine to the process. Herein, we combined mutational studies with various biochemical and biophysical approaches to further investigate the molecular mechanisms underlying PNT3 fibrillation. The results show that (i) lysine residues play a critical role in driving fibrillation, (ii) hydrophobic residues affect the nucleation step, and (iii) charge distribution strongly affects the fibrillation propensities. Vibrational Raman spectroscopy data further validated the role of lysine residues in promoting fibrillation and enabled documenting the formation of cross-β amyloid structures. Altogether, these results illuminate the molecular mechanisms involved in fibril formation and pave the way towards the rational design of inhibitors.

Physical properties of biomolecular condensates formed via phase separation of proteins and nucleic acids are associated with cell physiology and disease. Condensate properties can be regulated by several cellular factors including post-translational modifications. Here, we introduce an application of intermolecular energy migration via homo-FRET (F & ouml;rster resonance energy transfer), a nanometric proximity ruler, to study the modulation in short- and long-range protein-protein interactions leading to the changes in the physical properties of condensates of fluorescently-tagged FUS (Fused in Sarcoma) that is associated with the formation of cytoplasmic and nuclear membraneless organelles. We show that homoFRET captures modulations in condensate properties of FUS by RNA, ATP, and post-translational arginine methylation. We also extend the homoFRET methodology to study the in-situ formation of cytoplasmic stress granules in mammalian cells. Our studies highlight the broad applicability of homoFRET as a potent generic tool for studying intracellular phase transitions involved in function and disease. The properties of biomolecular condensates can be regulated by multiple factors, including intermolecular dynamics. Here, the authors use fluorescence anisotropy-based homoFRET imaging to monitor the intermolecular interactions and supramolecular packing that underlie the modulation of biomolecular condensate properties.

Biomolecular condensates formed via phase separation of intrinsically disordered proteins/regions (IDPs/IDRs) and nucleic acids are associated with cell physiology and disease. Water makes up for similar to 60-70% of the condensate volume and is thought to influence the complex interplay of chain-chain and chain-solvent interactions, modulating the mesoscale properties of condensates. The behavior of water in condensates and the key roles of protein hydration water in driving the phase separation remain elusive. Here, we employ single-droplet vibrational Raman spectroscopy to illuminate the structural redistribution within protein hydration water during the phase separation of neuronal IDPs. Our Raman measurements reveal the changes in the water hydrogen bonding network during homotypic and heterotypic phase separation governed by various molecular drivers. Such single-droplet water Raman measurements offer a potent generic tool to unmask the intriguing interplay of sequence-encoded chain-chain and chain-solvent interactions governing macromolecular phase separation into membraneless organelles, synthetic condensates, and protocells.

Prion-like self-perpetuating conformational conversion of proteins into amyloid aggregates is associated with both trans-missible neurodegenerative diseases and non-Mendelian inher-itance. The cellular energy currency ATP is known to indirectly regulate the formation, dissolution, or transmission of amyloid-like aggregates by providing energy to the molecular chaperones that maintain protein homeostasis. In this work, we demonstrate that ATP molecules, independent of any chaperones, modulate the formation and dissolution of amyloids from a yeast prion domain (NM domain of Saccharomyces cerevisiae Sup35) and restricts autocatalytic amplification by controlling the amount of fragmentable and seeding-competent aggregates. ATP, at (high) physiological concentrations in the presence of Mg2+, kinetically accelerates NM aggregation. Interestingly, ATP also promotes phase separation-mediated aggregation of a human protein harboring a yeast prion-like domain. We also show that ATP disaggregates preformed NM fibrils in a dose-independent manner. Our results indicate that ATP-mediated disaggrega-tion, unlike the disaggregation by the disaggregase Hsp104, yields no oligomers that are considered one of the critical species for amyloid transmission. Furthermore, high concentrations of ATP delimited the number of seeds by giving rise to compact ATP-bound NM fibrils that exhibited nominal fragmentation by either free ATP or Hsp104 disaggregase to generate lower mo-lecular weight amyloids. In addition, (low) pathologically rele-vant ATP concentrations restricted autocatalytic amplification by forming structurally distinct amyloids that are found seeding inefficient because of their reduced beta-content. Our results pro-vide key mechanistic underpinnings of concentration -dependent chemical chaperoning by ATP against prion-like transmissions of amyloids.

An intense research activity reveals that living cells contain (noncanonical) membraneless organelles that are formed via phase separation of intrinsically disordered proteins/regions (IDPs/IDRs) with nucleic acids and other biomolecules. These biomolecular condensates are involved in a myriad of critical cellular functions and neurodegenerative diseases. Unmasking the role of intrinsic disorder and conformational heterogeneity of IDPs/IDRs in promoting promiscuous and ephemeral interactions resulting in a liquid-like behavior of these condensates is crucial to understanding the molecular drivers of phase separation. While a host of existing microscopic and spectroscopic tools exist for studying phase separation, most of these methodologies are inadequate in illuminating the conformational heterogeneity and distribution within individual droplets. To overcome these limitations, we developed and adapted a novel and highly-sensitive methodology that combines the capabilities of vibrational spectroscopy and optical microscopy that can illuminate the unique molecular details of the polypeptide chains within the mesoscopic liquid condensed phase at the single-droplet resolution. Often low Raman scattering cross-section of proteins makes the recording of vibrational signatures under physiological conditions in aqueous solutions extremely challenging. In this direction, we utilize surface-engineered, plasmonic metal nanostructures giving rise to high electromagnetic/chemical enhancement of Raman signals even at extremely low analyte concentrations that can increase Raman scattering cross-section by several orders of magnitude, allowing single-molecule detection and characterization even at a much lower laser power. Our ultra-sensitive single-droplet SERS methodology captures the exquisite details involving conformational disorder, heterogeneity, and distribution within FUS condensates. Additionally, this method can capture some key structural details within the droplets formed in the presence of varying RNA-protein ratios and highlight RNA-mediated partial unwinding of the ordered domains in the C-terminal RBD that increases polypeptide chain disorder that can promote both homotypic and heterotypic interactions within the condensed phase.

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 permitting 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.

This chapter summarizes various experimental protocols that are currently being used to characterize liquid–liquid phase separation. The basic characterizations involve turbidity and microscopic assays and the procedure to construct phase diagrams. The in silico studies and in vitro structural investigations involving nuclear magnetic resonance, fluorescence, and Raman help unmask the key molecular interactions within the condensed phase. The rheological studies are essential to quantify the mesoscale material properties. Overall, this chapter will be useful for the practitioners of the field as well as for beginners.

Our laboratory at the Indian Institute of Science Education and Research (IISER) Mohali is involved in studying the intriguing conformational. characteristics of intrinsically disordered proteins (IDPs) that do not fold up into a well-defined 3D structure and exist as rapidly fluctuating conformational ensemble. Our research over the past decade or so has dealt with the IDPs and partially unfolded proteins and their conversion into amyloid aggregates that are associated with deadly neurodegenerative diseases such as Alzheimer’s, Parkinson’s, Huntington’s, and prion diseases. We were particularly interested in characterizing the monomeric and oligomeric precursors of amyloid fibrils. This work has been summarized in an invited Feature Article. The current article primarily deals with the newer research direction of our lab in liquid- liquid phase separation of IDPs. Here we summarize our recent exciting discoveries in phase separation of range of disease-related IDPs. Below we provide a background of the emerging field of biomolecular condensates and describe our published results.

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.

Eukaryotic cells contain a host of non-canonical membrane-less organelles that are formed via liquid-liquid phase separation (LLPS) of intrinsically disordered proteins/regions (IDPs/IDRs) along with nucleic acids and other biomolecules. These biomolecular condensates are involved in a myriad of critical cellular functions and neurodegenerative diseases. Unmasking the role of intrinsic disorder and conformational heterogeneity of IDPs/IDRs in promoting promiscuous and ephemeral interactions resulting in liquid-like behavior of these condensates is crucial to understand the molecular drivers of LLPS.

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) are involved in a myriad of critical cellular functions and debilitating neurodegenerative diseases. Elucidating the role of intrinsic disorder and conformational heterogeneity of intrinsically disordered proteins/regions (IDPs/IDRs) in these phase-separated membrane-less organelles is crucial to understanding the mechanism of formation and regulation of biomolecular condensates. Here we introduce a unique single-droplet surface-enhanced Raman scattering (SERS) methodology that utilizes surface-engineered, plasmonic, metal nanoparticles to unveil the inner workings of mesoscopic liquid droplets of Fused in Sarcoma (FUS) in the absence and presence of RNA. These highly sensitive measurements offer unprecedented sensitivity to capture the crucial interactions, conformational heterogeneity, and structural distributions within the condensed phase in a droplet-by-droplet manner. Such an ultra-sensitive single-droplet vibrational methodology can serve as a potent tool to decipher the key molecular drivers of biological phase transitions of a wide range of biomolecular condensates involved in physiology and disease.