Biology subjects

Bremer, A.

Publications and source records attributed to Bremer, A..

12 recordsLinked to original sources

bioRxiv2026

Multivalent Tau-Fyn interactions cooperatively arrest postsynaptic density condensate dynamics

Tau-Fyn signaling at postsynaptic sites is implicated in synaptic dysfunction and neuronal hyperexcitability in Alzheimers disease and related tauopathies, yet the molecular mechanisms by which Tau-Fyn interactions influence postsynaptic signaling assemblies remain poorly understood. Here, we reconstitute biomolecular condensates and membrane-associated assemblies that mimic the postsynaptic density (PSD) to investigate how Tau and Fyn regulate PSD organization and dynamics. We show that Tau and Fyn co-partition into PSD condensates and N-methyl D-aspartate receptor subtype 2B (NR2B)-associated PSD clusters, with both proteins preferentially enriching in the condensed phase relative to core PSD scaffold proteins. Enrichment of Fyn within PSD condensates is recapitulated by an SH3-SH2 fragment lacking the kinase domain, indicating that the kinase domain is dispensable for condensate partitioning. Fluorescence recovery after photobleaching reveals that Tau markedly reduces Fyn mobility and, together with Fyn, cooperatively drives a pronounced dynamic arrest of PSD condensate components, including the central scaffold protein PSD-95. Disruption of a major Tau-Fyn interaction hotspot by P216A/P219A mutations in Tau nearly completely restores the mobility of both Fyn and PSD-95, demonstrating that cooperative Tau-Fyn interactions are required for dynamic arrest. NMR spectroscopy identifies a multivalent interaction network between the Tau proline-rich region and the Fyn SH3 domain involving multiple binding motifs, with residues P216 and P219 acting as major interaction determinants. A 1.4 [A] crystal structure of the Fyn SH3 domain bound to a Tau peptide reveals a ternary 2:1 Fyn:Tau assembly, providing structural support for multivalent Tau- Fyn engagement. Together, our findings establish a condensate-based mechanism by which multivalent Tau-Fyn interactions cooperatively regulate PSD condensate dynamics and provide a molecular framework linking Tau-Fyn signaling to synaptic dysfunction in tauopathies.

biochemistry↗

bioRxiv2025

Viscoelasticity and interface properties of multi-component condensates govern protein sequestration and suppression of amyloid formation

Stress granules (SGs) are multi-component biomolecular condensates widely implicated as sites of protein aggregation by virtue of the high concentrations of amyloidogenic RNA-binding proteins they contain. This model, in which SGs are viewed as crucibles for amyloid formation, has not been rigorously tested. Here, we employed twelve multi-component protein-nucleic acid condensate systems as SG-mimics with diverse physicochemical features. Utilizing three fibril-forming proteins, hnRNPA1, Tau, and FUS, which are concentrated more than 50-fold in condensates, we report that multi-component biomolecular condensates robustly suppress, rather than promote, amyloid formation. Multiscale experimental analyses, including quantitative kinetic measurements, rheology, and microscopy, combined with computational modelling, reveal that condensates serve as sinks for soluble protein, and fibrils form in the dilute phase, although interfaces can promote nucleation. Three key physicochemical properties of condensates govern suppression of fibril formation: First, condensate-mediated sequestration lowers the concentration of fibril-forming proteins in the dilute phase. Second, condensate viscoelasticity constrains efflux-driven fibril growth in the dilute phase. And third, dilution of fibril-forming proteins at condensate interfaces mitigates fibril nucleation. The sink potential of SG-mimics is recapitulated in G3BP1-RNA condensates and SGs reconstituted in mammalian cell lysate, suggesting that SGs may have evolved to suppress stress-induced protein aggregation.

biophysics↗

bioRxiv2024

Reconciling competing models on the roles of condensates and soluble complexes in transcription factor function

Phase separation explains the exquisite spatial and temporal regulation of many biological processes, but the role of transcription factor-mediated condensates in gene regulation is contentious, requiring head-to-head comparison of competing models. Here, we focused on the prototypical yeast transcription factor Gcn4 and assessed two models for gene transcription activation, i.e., mediated via soluble complexes or transcriptional condensates. Both models rely on the ability of transcription factors and coactivators to engage in multivalent interactions. Unexpectedly, we found that propensity to form homotypic Gcn4 condensates does not correlate well with transcriptional activity. Contrary to prevailing models, binding to DNA suppresses Gcn4 phase separation. Notably, the ability of Gcn4 to form soluble complexes with coactivator subunit Med15 closely mirrored the propensity to recruit Med15 into condensates, indicating that these properties are intertwined and cautioning against interpretation of mutational data without head-to-head comparisons. However, Gcn4 variants with the highest affinity for Med15 do not function as well as expected and instead have activities that reflect their abilities to phase separate with Med15. These variants therefore indeed form cellular condensates, and those attenuate activity. Our results show that transcription factors can function as soluble complexes as well as condensates, reconciling two seemingly opposing models, and have implications for other phase-separating systems. Highlights- Homotypic phase separation propensities of Gcn4 variants do not predict in vivo activities well. - DNA binding leads to solubilization of GCN4 condensates, which is reversed by Med15 association. - The abilities to co-phase separate and form soluble complexes with Med15 are highly intertwined. - Variants with high affinities for Med15 form condensates that attenuate function.

biophysics↗

bioRxiv2024

Biomolecular condensates are characterized by interphase electric potentials

Biomolecular condensates form via processes that combine phase separation and reversible associations of multivalent macromolecules. Condensates can be two- or multi-phase systems defined by coexisting dense and dilute phases. Here, we show that solution ions can partition asymmetrically across coexisting phases defined by condensates formed by intrinsically disordered proteins or homopolymeric RNA molecules. Our findings were enabled by direct measurements of the activities of cations and anions within coexisting phases of protein and RNA condensates. Asymmetries in ion partitioning between coexisting phases vary with protein sequence, condensate type, salt concentration, and ion type. The Donnan equilibrium set up by asymmetrical partitioning of solution ions generates interphase electric potentials known as Donnan and Nernst potentials. Our measurements show that the interphase potentials of condensates are of the same order of magnitude as membrane potentials of membrane-bound organelles. Interphase potentials quantify the degree to which microenvironments of coexisting phases are different from one another. Importantly, and based on condensate-specific interphase electric potentials, which are membrane-like potentials of membraneless bodies, we reason that condensates are mesoscale capacitors that store charge. Interphase potentials lead to electric double layers at condensate interfaces. This helps explain recent observations of condensate interfaces being electrochemically active.

biophysics↗

bioRxiv2023

Design of intrinsically disordered protein variants with diverse structural properties

Intrinsically disordered proteins (IDPs) perform a wide range of functions in biology, suggesting that the ability to design IDPs could help expand the repertoire of proteins with novel functions. Designing IDPs with specific structural or functional properties has, however, been diffcult, in part because determining accurate conformational ensembles of IDPs generally requires a combination of computational modelling and experiments. Motivated by recent advancements in effcient physics-based models for simulations of IDPs, we have developed a general algorithm for designing IDPs with specific structural properties. We demonstrate the power of the algorithm by generating variants of naturally occurring IDPs with different levels of compaction and that vary more than 100 fold in their propensity to undergo phase separation, even while keeping a fixed amino acid composition. We experimentally tested designs of variants of the low-complexity domain of hnRNPA1 and find high accuracy in our computational predictions, both in terms of single-chain compaction and propensity to undergo phase separation. We analyze the sequence features that determine changes in compaction and propensity to phase separate and find an overall good agreement with previous findings for naturally occurring sequences. Our general, physics-based method enables the design of disordered sequences with specified conformational properties. Our algorithm thus expands the toolbox for protein design to include also the most flexible proteins and will enable the design of proteins whose functions exploit the many properties afforded by protein disorder.

biophysics↗

bioRxiv2023

Crowder titrations enable the quantification of driving forces for macromolecular phase separation

Macromolecular solubility is an important contributor to the driving forces for phase separation. Formally, the driving forces in a binary mixture comprising a macromolecule dissolved in a solvent can be quantified in terms of the saturation concentration, which is the threshold macromolecular concentration above which the mixture separates into coexisting dense and dilute phases. Additionally, the second virial coefficient, which measures the effective strength of solvent-mediated intermolecular interactions provides direct assessments of solvent quality. The sign and magnitude of second virial coefficients will be governed by a combination of solution conditions and the nature of the macromolecule of interest. Here, we show, using a combination of theory, simulation, and in vitro experiments, that titrations of crowders, providing they are true depletants, can be used to extract the intrinsic driving forces for macromolecular phase separation. This refers to saturation concentrations in the absence of crowders and the second virial coefficients that quantify the magnitude of the incompatibility between macromolecules and the solvent. Our results show how the depletion-mediated attractions afforded by crowders can be leveraged to obtain comparative assessments of macromolecule-specific, intrinsic driving forces for phase separation. SIGNIFICANCEPhase separation has emerged as a process of significant relevance to sorting macromolecules into distinct compartments, thereby enabling spatial and temporal control over cellular matter. Considerable effort is being invested into uncovering the driving forces that enable the separation of macromolecular solutions into coexisting phases. At its heart, this process is governed by the balance of macromolecule-solvent, inter-macromolecule, and solvent-solvent interactions. We show that the driving forces for phase separation, including the coefficients that measure interaction strengths between macromolecules, can be extracted by titrating the concentrations of crowders that enable macromolecules to phase separate at lower concentrations. Our work paves the way to leverage specific categories of measurements for quantitative characterizations of driving forces for phase separation.

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

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

A complete digital karyotype of the B-cell leukemia REH cell line resolved by long-read sequencing

The B-cell acute lymphoblastic leukemia (ALL) cell line REH, with the t(12;21) ETV6-RUNX1 translocation, is known to have a complex karyotype defined by a series of large-scale chromosomal rearrangements. Taken from a 15-year-old at relapse, the cell line offers a practical model for the study of high-risk pediatric B-ALL patients. In recent years, short-read DNA and RNA sequencing have emerged as a complement to analog karyotyping techniques in the resolution of structural variants in an oncological context. However, it is challenging to create a comprehensive digital karyotype of a genome with these techniques alone. Here, we explore the integration of long-read PacBio and Oxford Nanopore whole genome sequencing (WGS), IsoSeq RNA-sequencing, and short-read sequencing to create a detailed digital karyotype of the REH cell line. WGS refined the breakpoints of known aberrations and clarified the molecular traits of disrupted ALL-associated genes BTG1 and TBL1XR1, as well as the glucocorticoid receptor NR3C1. Several previously underreported structural variants were also uncovered, including deletions affecting the ALL-associated genes VPREB1 and NFATC1. Meanwhile, transcriptome sequencing identified seven fusion genes within the genomic breakpoints. Together, our extensive whole-genome investigation makes high-quality open-source data available to the leukemia genomics community. KEY POINTSO_LIA complete digital karyotype of the REH cell line was produced with short- and long-read DNA and RNA sequencing technologies. C_LIO_LIThe study enabled precise identification of structural variants, and the fusion genes expressed as the result of these variants. C_LI

cancer biology↗

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↗