Biology subjects

Avni, A.

Publications and source records attributed to Avni, A..

8 recordsLinked to original sources

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↗

bioRxiv2022

ATP modulates self-perpetuating conformational conversion generating structurally distinct yeast prion amyloids that limit autocatalytic amplification

Prion-like self-perpetuating conformational conversion of proteins into amyloid aggregates is associated with both transmissible neurodegenerative diseases and non-Mendelian inheritance. Here, we demonstrate that ATP modulates 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 dose independently disaggregates preformed NM fibrils. Furthermore, high concentrations of ATP delimited the number of seeds by generating compact, ATP-bound NM fibrils that exhibited nominal fragmentation by either free ATP or Hsp104 disaggregase. Additionally, (low) pathological ATP concentrations restricted autocatalytic amplification by forming structurally distinct seeding-inefficient amyloids. Our results provide mechanistic underpinnings of concentration-dependent chemical chaperoning by ATP against prion-like transmissions.

biochemistry↗

bioRxiv2022

Heterotypic electrostatic interactions control complex phase separation of tau and prion into multiphasic condensates and co-aggregates

Biomolecular condensates formed via phase separation of proteins and nucleic acids are thought to perform a wide range of critical cellular functions by maintaining spatiotemporal regulation and organizing intracellular biochemistry. However, aberrant phase transitions are implicated in a multitude of human diseases. Here, we demonstrate that two neuronal proteins namely, tau and prion undergo complex coacervation driven by domain-specific electrostatic interactions to yield highly dynamic, mesoscopic liquid-like droplets. The acidic N-terminal segment of tau interacts electrostatically with the polybasic N-terminal intrinsically disordered segment of the prion protein (PrP). We employed a unique combination of time-resolved tools that encompass several orders of magnitude of timescales ranging from nanoseconds to seconds. These studies unveil an intriguing orchestra of molecular events associated with the formation of heterotypic condensates comprising ephemeral, domain-specific, short-range electrostatic nanoclusters. Our results reveal that these heterotypic condensates can be tuned by RNA in a stoichiometry-dependent manner resulting in reversible, multiphasic, immiscible, ternary condensates of different morphologies ranging from core-shell to nested droplets. This ternary system exhibits a typical three-regime phase behavior reminiscent of other membraneless organelles including nucleolar condensates. We also show that upon aging, tau-PrP droplets gradually convert into solid-like co-assemblies by sequestration of persistent intermolecular interactions. Our vibrational Raman spectroscopic data in conjunction with atomic force microscopy and multi-color fluorescence imaging results reveal the presence of amorphous and amyloid-like co-aggregates upon maturation. Our findings provide mechanistic underpinnings of overlapping neuropathology involving tau and PrP and highlight a broader role of complex phase transitions in physiology and disease.

biophysics↗

bioRxiv2022

TOR mediates cytokinin-driven development and defense cues

Plants constantly perceive and process environmental signals and balance between the energetic demands of growth and defense. Growth arrest upon pathogen attack was previously suggested to result from a redirection of the plants metabolic resources towards the activation of plant defense. The energy sensor Target of Rapamycin (TOR) kinase is a conserved master coordinator of growth and development in all eukaryotes. Although TOR is positioned at the interface between development and defense, little is known about the mechanisms in which TOR may potentially regulate the relationship between these two modalities. The plant hormones cytokinin (CK) and gibberellin (GA) execute various aspects of plant development and defense. The ratio between CK and GA was reported to determine the outcome of developmental programs. Here, investigating the interplay between TOR-mediated development and TOR-mediated defense in tomato, we found that TOR silencing resulted in rescue of several different aberrant developmental phenotypes, demonstrating that TOR is required for the execution of developmental cues. In parallel, TOR inhibition enhanced immunity in genotypes with a low CK/GA ratio but not in genotypes with a high CK/GA ratio. TOR-inhibition mediated disease resistance was found to depend on developmental status, and was abolished in strongly morphogenetic leaves, while being strongest in mature, differentiated leaves. CK repressed TOR activity, suggesting that CK-mediated immunity may rely on TOR downregulation. At the same time, TOR activity was promoted by GA, and TOR silencing reduced GA sensitivity, indicating that GA signaling requires normal TOR activity. Our results demonstrate that TOR likely acts in concert with CK and GA signaling, executing signaling cues in both defense and development. Thus, differential regulation of TOR or TOR-mediated processes could regulate the required outcome of development-defense prioritization.

plant biology↗

bioRxiv2022

Single-Droplet Surface-Enhanced Raman Scattering Decodes the Molecular Language of Liquid-Liquid Phase Separation

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.

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↗

bioRxiv2021

An Intrinsically Disordered Pathological Variant of the Prion Protein Y145Stop Transforms into Self-Templating Amyloids via Liquid-Liquid Phase Separation

Biomolecular condensation via liquid-liquid phase separation of intrinsically disordered proteins/regions (IDPs/IDRs) along with other biomolecules is thought to govern critical cellular functions, whereas, aberrant phase transitions are associated with a range of deadly neurodegenerative diseases. Here we show, a naturally occurring pathological truncation variant of the prion protein (PrP) by a mutation of a tyrosine residue at 145 to a stop codon (Y145Stop) yielding a highly disordered N-terminal IDR that spontaneously phase-separates into liquid-like droplets. Phase separation of this N-terminal segment that is rich in positively charged and aromatic residues is promoted by the electrostatic screening and a multitude of other transient, intermolecular, noncovalent interactions. Single-droplet Raman measurements in conjunction with an array of bioinformatic, spectroscopic, microscopic, and mutagenesis studies revealed that the intrinsic disorder and dynamics are retained in the liquid-like condensates. Lower concentrations of RNA promote the phase transition of Y145Stop at low micromolar protein concentrations under physiological condition. Whereas, higher RNA to protein ratios inhibit condensation indicating the role of RNA in modulating the phase behavior of Y145Stop. Highly dynamic liquid-like droplets eventually transform into dynamically-arrested, ordered, {beta}-rich, amyloid-like aggregates via liquid-to-solid transition upon aging. These amyloid-like aggregates formed via phase separation display the self-templating characteristic and are capable of recruiting and autocatalytically converting monomeric Y145Stop into amyloid fibrils. In contrast to this disease-associated intrinsically disordered Y145 truncated variant, the wild-type full-length PrP exhibited a much lower propensity for phase separation and liquid-to-solid maturation into amyloid-like aggregates hinting at a potentially crucial, chaperone-like, protecting role of the globular C-terminal domain that remains largely conserved in vertebrate evolution. Such an intriguing interplay in the modulation of the protein phase behavior will have much broader implications in cell physiology and disease.

biophysics↗

bioRxiv2017

CRISPys: Optimal sgRNA design for editing multiple members of a gene family using the CRISPR system

The discovery and development of the CRISPR-Cas9 system in the past few years has made eukaryotic genome editing, and specifically gene knockout for reverse genetics, a simpler, efficient, and effective task. The system is directed to the genomic target site by a programmed single-guide RNA (sgRNA) that base-pairs with the DNA target, subsequently leading to site-specific double-strand breaks. However, many gene families in eukaryotic genomes exhibit partially overlapping functions and, thus, the knockout of one gene might be concealed by the function of the other. In such cases, the reduced specificity of the CRISPR-Cas9 system, which may lead to the cleavage of genomic sites that are not identical to the sgRNA, can be harnessed for the simultaneous knockout of multiple homologous genes. Here, we introduce CRISPys, an algorithm for the optimal design of sgRNAs that would potentially target multiple members of a given gene family. CRISPys first clusters all the potential targets in the input sequences into a hierarchical tree structure that specifies the similarity among them. Then, sgRNAs are proposed in the internal nodes of the tree by embedding mismatches where needed, such that the cleavage efficiencies of the induced targets are maximized. We suggest several approaches for designing the optimal individual sgRNA, and an approach that provides a set of sgRNAs that also accounts for the homologous relationships among gene-family members. We further show by in-silico examination over all gene families in the Solanum lycopersicum genome that our suggested approach outperforms simpler alignment-based techniques.\n\nGraphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY\n\nHighlightsO_LIMany genes in eukaryotic genomes exhibit partially overlapping functions. This imposes difficulties on reverse-genetics, as the knockout of one gene might be concealed by the function of the other.\nC_LIO_LIWe present CRISPys, a graph-based algorithm for the optimal design of CRISPR systems given a set of redundant genes.\nC_LIO_LICRISPys harnesses the lack of specificity of the CRISPR-Cas9 genome editing technique, providing researchers the ability to simultaneously mutate multiple genes.\nC_LIO_LIWe show that CRISPys outperforms existing approaches that are based on simple alignment of the input gene family.\nC_LI

bioinformatics↗