Biology subjects

Farag, M.

Publications and source records attributed to Farag, M..

13 recordsLinked to original sources

bioRxiv2024

Hyperexcitability precedes CA3 hippocampal neurodegeneration in a dox-regulatable TDP-43 mouse model of ALS-FTD.

Neuronal hyperexcitability is a hallmark of amyotrophic lateral sclerosis (ALS) but its relationship with the TDP-43 aggregates that comprise the predominant pathology in over 90% of ALS cases remains unclear. Emerging evidence in tissue and slice culture models indicate that TDP-43 pathology induces neuronal hyperexcitability suggesting it may be responsible for the excitotoxicity long believed to be a major driver of ALS neuron death. Here, we characterized hyperexcitability and neurodegeneration in the hippocampus of doxycycline-regulatable rNLS8 mice (NEFH-tTA x tetO-hTDP-43{Delta}NLS), followed by treatment with AAV encoded DREADDs and anti-seizure medications to measure the effect on behavioral function and neurodegeneration. We found that approximately half of the CA3 neurons in the dorsal hippocampus are lost between 4 and 6 weeks after TDP-43{Delta}NLS induction. Neurodegeneration was preceded by selective hyperexcitability in the mossy fiber - CA3 circuit, leading us to hypothesize that glutamate excitotoxicity may be a significant contributor to neurodegeneration in this model. Interestingly, hippocampal injection of AAV encoded inhibitory DREADDs (hM4Di) and daily activation with CNO ligand rescued anxiety deficits on elevated zero maze (EZM) but did not reduce neurodegeneration. Therapeutic doses of the anti-seizure medications, valproic acid and levetiracetam, did not improve behavior or prevent neurodegeneration. These results highlight the complexity of TDP-43 - induced alterations to neuronal excitability and suggest that whereas targeting hyperexcitability can meliorate some behavioral deficits, it may not be sufficient to halt or slow neurodegeneration in TDP-43-related proteinopathies. Significance StatementCytoplasmic aggregates of TAR DNA Binding Protein 43 (TDP-43) are the predominant pathology in over 90% of Amyotrophic lateral sclerosis (ALS) and the majority of frontotemporal lobar degeneration (FTLD-TDP) cases. Understanding how TDP-43 pathology promotes neurodegeneration may lead to therapeutic strategies to slow disease progression in humans. Recent reports in mouse and cell culture models suggest loss-of-normal TDP-43 function may drive neuronal hyperexcitability, a key physiological hallmark of ALS and possible contributor to neurodegeneration. In this study, we identified region-specific hyperexcitability that precedes neurodegeneration in the inducible rNLS8 TDP-43 mouse model. Suppressing hyperexcitability with chemogenetics improved behavioral function but did not reduce hippocampal neuron loss. Anti-seizure medications had no beneficial effects suggesting directly targeting hyperexcitability may not be therapeutically effective.

neuroscience↗

bioRxiv2024

Differential interactions determine anisotropies at interfaces of RNA-based biomolecular condensates

Biomolecular condensates form via macromolecular phase separation, giving rise to coexisting phases delineated by interfaces. Here, we characterize the structures of interfaces formed by phase separation driven by heterotypic interactions in ternary mixtures of two types of RNA molecules and polyethylene glycol. We find that purine-rich RNAs are scaffolds that drive phase separation via strong heterotypic interactions. Conversely, pyrimidine-rich RNA molecules are defined by weaker heterotypic interactions. They function as adsorbents that accumulate at and wet the interfaces of coexisting phases formed by phase separation of scaffolds. Our computations predict that scaffolds and adsorbents have different non-random orientational preferences at interfaces. We tested these predictions using single-molecule super resolution imaging that tracks the motions of fluorogenic probes that are bound to RNA molecules. Motions parallel to the interface were found to be faster than motions perpendicular to the interface. These findings support previous predictions regarding anisotropies of motions at interfaces.

biophysics↗

bioRxiv2024

Biomolecular condensates can function as inherent catalysts

We report the discovery that chemical reactions can be catalyzed by condensates formed by intrinsically disordered proteins (IDPs). The proteins themselves lack any catalytic activities. Catalytic functions of condensates emerge as a consequence of sequence-dependent mesoscale electrochemical microenvironments created by phase separation. Stimulated Raman spectroscopy suggests that the catalytic behaviors of condensates are attributable to the spatial variations of water activities across condensate interiors and interfaces. We show that condensates are capable of catalyzing diverse cellularly relevant hydrolysis reactions. Through sequence design, the electrochemical properties of condensates can be programmed to exert control over catalytic behaviors. Incorporation of synthetic condensates into live cells alters transcription profiles and enables the activation of gene circuits that depend on products of hydrolysis reactions catalyzed by condensates. Our discovery of suggests that condensates, depending on their composition-dependent electrochemical properties, can be "Condenzymes", which contribute unexpected emergent chemical functions in cells.

biophysics↗

bioRxiv2024

Metastable condensates suppress conversion to amyloid fibrils

Stress granules form via co-condensation of RNA-binding proteins containing prion-like low complexity domains (PLCDs) with RNA molecules. Homotypic interactions among PLCDs can drive amyloid fibril formation that is enhanced by ALS-associated mutations. We report that condensation-versus fibril-driving homotypic interactions are separable for A1-LCD, the PLCD of hnRNPA1. Separable interactions lead to thermodynamically metastable condensates and globally stable fibrils. Interiors of condensates suppress fibril formation whereas interfaces have the opposite effect. ALS-associated mutations enhance the stability of fibrils and weaken condensate metastability, thus enhancing the rate of fibril formation. We designed mutations to enhance A1-LCD condensate metastability and discovered that stress granule disassembly in cells can be restored even when the designed variants carry ALS-causing mutations. Therefore, fibril formation can be suppressed by condensate interiors that function as sinks. Condensate sink potentials are influenced by their metastability, which is tunable through separable interactions even among minority components of stress granules.

biophysics↗

bioRxiv2023

Sequence-encoded grammars determine material properties and physical aging of protein condensates

Biomolecular condensates are viscoelastic materials. Here, we report results from investigations into molecular-scale determinants of sequence-encoded and age-dependent viscoelasticity of condensates formed by prion-like low-complexity domains (PLCDs). The terminally viscous forms of PLCD condensates are Maxwell fluids. Measured viscoelastic moduli of these condensates are reproducible using a Rouse-Zimm model that accounts for the network-like organization engendered by reversible physical crosslinks among PLCDs in the dense phase. Measurements and computations show that the strengths of aromatic inter-sticker interactions determine the sequence-specific amplitudes of elastic and viscous moduli as well as the timescales over which elastic properties dominate. PLCD condensates also undergo physical aging on sequence-specific timescales. This is driven by mutations to spacer residues that weaken the metastability of terminally viscous phases. The aging of PLCD condensates is accompanied by disorder-to-order transitions, leading to the formation of non-fibrillar, beta-sheet-containing, semi-crystalline, terminally elastic, Kelvin-Voigt solids. Our results suggest that sequence grammars, which refer to the identities of stickers versus spacers in PLCDs, have evolved to afford control over the metastabilities of terminally viscous fluid phases of condensates. This selection can, in some cases, render barriers for conversion from metastable fluids to globally stable solids to be insurmountable on functionally relevant timescales.

biophysics↗

bioRxiv2023

FIREBALL: A tool to fit protein phase diagrams based on mean-field theories for polymer solutions

Biomolecular condensates form via phase transitions of condensate-specific biomacromolecules. Intrinsically disordered regions (IDRs) featuring the appropriate sequence grammar can contribute homotypic and heterotypic interactions to the driving forces for phase separation of multivalent proteins. At this juncture, experiments and computations have matured to the point where the concentrations of coexisting dense and dilute phases can be quantified for individual IDRs in complex milieus both in vitro and in vivo. For a macromolecule such as a disordered protein in a solvent, the locus of points that connects concentrations of the two coexisting phases defines a phase boundary or binodal. Often, only a few points along the binodal, especially in the dense phase, are accessible for measurement. In such cases and for quantitative and comparative analysis of parameters that describe the driving forces for phase separation, it is useful to fit measured or computed binodals to well-known mean-field free energies for polymer solutions. Unfortunately, the non-linearity of the underlying free energy functions makes it challenging to put mean-field theories into practice. Here, we present FIREBALL, a suite of computational tools designed to enable efficient construction, analysis, and fitting to experimental or computed data of binodals. We show that depending on the theory being used, one can also extract information regarding coil-to-globule transitions of individual macromolecules. Here, we emphasize the ease-of-use and utility of FIREBALL using examples based on data for two different IDRs. Statement of SignificanceMacromolecular phase separation drives the assembly of membraneless bodies known as biomolecular condensates. Measurements and computer simulations can now be brought to bear to quantify how the concentrations of macromolecules in coexisting dilute and dense phases vary with changes to solution conditions. These mappings can be fit to analytical expressions for free energies of solution to extract information regarding parameters that enable comparative assessments of the balance of macromolecule-solvent interactions across different systems. However, the underlying free energies are non-linear and fitting them to actual data is non-trivial. To enable comparative numerical analyses, we introduce FIREBALL, a user-friendly suite of computational tools that allows one to generate, analyze, and fit phase diagrams and coil-to-globule transitions using well-known theories.

biophysics↗

bioRxiv2023

Phase Separation in Mixtures of Prion-Like Low Complexity Domains is Driven by the Interplay of Homotypic and Heterotypic Interactions

Prion-like low-complexity domains (PLCDs) are involved in the formation and regulation of distinct biomolecular condensates that form via coupled associative and segregative phase transitions. We previously deciphered how evolutionarily conserved sequence features drive phase separation of PLCDs through homotypic interactions. However, condensates typically encompass a diverse mixture of proteins with PLCDs. Here, we combine simulations and experiments to study mixtures of PLCDs from two RNA binding proteins namely, hnRNPA1 and FUS. We find that 1:1 mixtures of the A1-LCD and FUS-LCD undergo phase separation more readily than either of the PLCDs on their own. The enhanced driving forces for phase separation of mixtures of A1-LCD and FUS-LCD arise partly from complementary electrostatic interactions between the two proteins. This complex coacervation-like mechanism adds to complementary interactions among aromatic residues. Further, tie line analysis shows that stoichiometric ratios of different components and their sequence-encoded interactions jointly contribute to the driving forces for condensate formation. These results highlight how expression levels might be tuned to regulate the driving forces for condensate formation in vivo. Simulations also show that the organization of PLCDs within condensates deviates from expectations based on random mixture models. Instead, spatial organization within condensates will reflect the relative strengths of homotypic versus heterotypic interactions. We also uncover rules for how interaction strengths and sequence lengths modulate conformational preferences of molecules at interfaces of condensates formed by mixtures of proteins. Overall, our findings emphasize the network-like organization of molecules within multicomponent condensates, and the distinctive, composition-specific conformational features of condensate interfaces. Significance StatementBiomolecular condensates are mixtures of different protein and nucleic acid molecules that organize biochemical reactions in cells. Much of what we know about how condensates form comes from studies of phase transitions of individual components of condensates. Here, we report results from studies of phase transitions of mixtures of archetypal protein domains that feature in distinct condensates. Our investigations, aided by a blend of computations and experiments, show that the phase transitions of mixtures are governed by a complex interplay of homotypic and heterotypic interactions. The results point to how expression levels of different protein components can be tuned in cells to modulate internal structures, compositions, and interfaces of condensates, thus affording distinct ways to control the functions of condensates.

biophysics↗

bioRxiv2023

Single fluorogen imaging reveals spatial inhomogeneities within biomolecular condensates

Biomolecular condensates are viscoelastic materials. Simulations predict that fluid-like condensations are defined by spatially inhomogeneous organization of the underlying molecules. Here, we test these predictions using single-fluorogen tracking and super-resolution imaging. Specifically, we leverage the localization and orientational preferences of freely diffusing fluorogens and the solvatochromic effect whereby specific fluorogens are turned on in response to condensate microenvironments. We deployed three different fluorogens to probe the microenvironments and molecular organization of different protein-based condensates. The spatiotemporal resolution and environmental sensitivity afforded by single-fluorogen imaging shows that the internal environments of condensates are more hydrophobic than coexisting dilute phases. Molecules within condensates are organized in a spatially inhomogeneous manner, and this gives rise to slow-moving nanoscale molecular clusters that coexist with fast-moving molecules. Fluorogens that localize preferentially to the interface help us map their distinct features. Our findings provide a structural and dynamical basis for the viscoelasticity of condensates.

biophysics↗

bioRxiv2023

Adsorption of RNA to interfaces of biomolecular condensates enables wetting transitions

Biomolecular condensates form via spontaneous and driven phase transitions of multivalent proteins and nucleic acids. These macromolecules can be organized in spatially inhomogeneous ways that lead to multiple coexisting dense phases with distinct macromolecular interfaces. While considerable attention has focused on the physical driving forces that give rise to phase separation from bulk solutions, the interactions that underlie adsorption driven wetting transitions remain unclear. Here, we report that pyrimidine-rich RNAs function as adsorbents that enable cascades of wetting transitions that include partial and complete wetting of condensates formed by purine-rich RNAs. Computations show that macromolecules that are scaffolds of condensates are oriented perpendicular to condensate interfaces whereas adsorbents are oriented parallel to interfaces. Our results yield heuristics for the design of synthetic materials that can be based on RNA-rich condensates featuring bespoke interfaces and distinct local microenvironments created by the interplay between scaffolds versus adsorbents.

biophysics↗

bioRxiv2022

Uncovering molecular grammars of intrinsically disordered regions that organize nucleolar fibrillar centers

The nucleolus is a multilayered structure. Each layer is thought to be a compositionally distinct phase, although how these phases form and interface with one another remains unclear. Using computational, proteomics, in vitro, and in vivo studies, we uncover distinct molecular grammars within intrinsically disordered regions (IDRs) of nucleolar proteins that localize to fibrillar centers (FCs) and dense fibrillar components (DFCs). FC and DFC proteins feature two distinct types of IDRs namely those with long tracts of acidic residues and those with blocks of lysines interspersed by acid-rich-regions. We find that phase separation driven by complex coacervation in mixtures of nucleolar proteins, with their distinctive IDRs, and ribosomal DNA and RNA molecules is sufficient to drive the formation of structural facsimiles of FCs and DFCs. One-Sentence SummaryFacsimiles of core nucleolar substructures were reconstituted via phase separation of key protein and nucleic acid mixtures.

biophysics↗

bioRxiv2022

Quantifying coexistence concentrations in multi-component phase-separating systems using analytical HPLC

Over the last decade, evidence has accumulated to suggest that numerous instances of cellular compartmentalization can be explained by the phenomenon of phase separation. This is a process by which a macromolecular solution separates spontaneously into dense and dilute coexisting phases. Semi-quantitative, in vitro approaches for measuring phase boundaries have proven very useful in determining some key features of biomolecular condensates, but these methods often lack the precision necessary for generating quantitative models. Therefore, there is a clear need for techniques that allow quantitation of coexisting dilute and dense phase concentrations of phase-separating biomolecules, especially in systems with more than one type of macromolecule. Here we report the design and deployment of analytical High-Performance Liquid Chromatography (HPLC) for in vitro separation and quantification of distinct biomolecules that allows us to measure dilute and dense phase concentrations needed to reconstruct coexistence curves in multicomponent mixtures. This approach is label-free, detects lower amounts of material than is accessible with classic UV-spectrophotometers, is applicable to a broad range of macromolecules of interest, is a semi-high-throughput technique, and if needed, the macromolecules can be recovered for further use. The approach promises to provide quantitative insights into the balance of homotypic and heterotypic interactions in multicomponent phase-separating systems.

biophysics↗

bioRxiv2022

Condensates of disordered proteins have small-world network structures and interfaces defined by expanded conformations

The formation of membraneless biomolecular condensates is driven by macromolecules with sticker-and-spacer architectures that undergo phase separation coupled to percolation (PSCP). Driving forces for PSCP are governed by the interplay between reversible inter-sticker crosslinks and solvation preferences of spacers. Here, we introduce molecular and mesoscale descriptions of structures within, outside, and at the interfaces of condensates that are formed by prion-like low complexity domains (PLCDs), which are exemplars of intrinsically disordered, linear multivalent proteins. Our studies are based on simulations that accurately describe sequence-specific phase behaviors of PLCDs. We find that networks of reversible, intermolecular, inter-sticker crosslinks organize PLCDs into small-world topologies within condensates. These topologies result from distinct conformational preferences within dense, dilute, and interfacial regions. Specifically, the degree of conformational expansion varies non-monotonically, being most expanded at the interface and most compact in the dilute phase with molecules preferring to be oriented perpendicular to condensate interfaces. This contrasts with dense and dilute phases where molecules are randomly oriented relative to one another. Our results demonstrate that even simple condensates, with only one type of macromolecule, feature inhomogeneous spatial organizations of molecules and interfacial features that likely prime them for being locations of biochemical activity.

biophysics↗

bioRxiv2021

Deciphering how naturally occurring sequence features impact the phase behaviors of disordered prion-like domains

Phase separation of intrinsically disordered prion-like low-complexity domains (PLCDs) derived from RNA-binding proteins enable the formation of biomolecular condensates in cells. PLCDs have distinct amino acid compositions, and here we decipher the physicochemical impact of conserved compositional biases on the driving forces for phase separation. We find that tyrosine residues make for stronger drivers of phase separation than phenylalanine. Depending on their sequence contexts, arginine residues enhance or weaken phase separation, whereas lysine residues weaken cohesive interactions within PLCDs. Increased net charge per residue (NCPR) weakens the driving forces for phase separation of PLCDs and this effect can be modeled quantitatively. The effects of NCPR also weaken known correlations between the dimensions of single chains in dilute solution and the driving forces for phase separation. We build on experimental data to develop a coarse-grained model for accurate simulations of phase separation that yield novel insights regarding PLCD phase behavior.

biophysics↗