A protein-DNA surface hydrogel mechanically protects the cell nucleus

Presenting author:

MPI CBG, Molecular cell biology, Pfotenhauerstr. 108, 01307 Dresden [DE], adakkatt@mpi-cbg.de

Author(s):
Ramesh Adakkattil

The nuclear envelope (NE) safeguards the genome from mechanical stress during processes such as migration, division, and compression. However, how it buffers forces at the scale of individual DNA molecules remains poorly understood. Here, we combine biophysical, theoretical, and cell biological approaches to demonstrate that a multivalent protein–DNA co-condensate, comprising the NE protein LEM2 and the DNA-binding protein BAF, protects DNA from forces exceeding its melting force, directly enhancing DNA’s mechanical resilience. We show that DNA–BAF–LEM2 assembly generates forces that stiffen DNA, providing resistance to mechanical stress through an unconventional stiffening mechanism. We identify the intrinsically disordered region (IDR) of LEM2 to be essential for this force-mediated reinforcement on DNA. At the nuclear membrane inside cells, these elements combine to form an elastic surface hydrogel that protects chromatin, visible as a continuous amorphous layer around the chromatin surface in cryo-electron tomography. Disruption of this surface hydrogel leads to increased DNA damage and micronuclei formation upon nuclear deformation. Using a statistical mechanics framework, we link the molecular spring-stiffening behaviour to hydrogel-mediated nuclear shape stabilisation at the cellular level. Taken together, this work expands the functional repertoire of condensates, revealing a load-responsive nuclear surface hydrogel at the mesoscale that mitigates mechanical stress.

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Gangliosides and cholesterol, two major components of the membrane lipid rafts, as new regulatory partners for Stress Granules assembly.

Presenting author:

HiLIFE, University of Helsinki, Helsinki, Finland, , Haartmaninkatu 8, 00290 Helsinki [FI], anais.aulas@helsinki.fi

Author(s):
Anaïs Aulas, Coralie Di Scala

Condensates must be tightly regulated within the cell to prevent disease. While this regulation is often attributed to proteins or oligonucleotides, lipids (microdomains) in the plasma membrane have recently emerged as major potential regulators.

The impact of lipid dysregulation has often been overlooked, particularly in the context of condensate control. However, the literature suggests a correlation between lipid imbalance and the dysregulation of stress granule (SG) formation in the same diseases. SG are pro-survival ribonucleoprotein condensates linked to human diseases, from neurodegeneration to cancer.

At the plasma membrane, lipid rafts, mainly composed of gangliosides and cholesterol, act as organized platforms for signaling and trafficking. We hypothesized that the deregulation of these two types of lipids could interfere with the pro-survival properties of SG. We studied their action using two inhibitors, PPMP to inhibit gangliosides synthesis and MβCD to remove cholesterol from plasma membranes in two different cell lines: MDA-MB-231 (breast cancer) and SH-SY5Y (neuroblastoma). Interestingly, both inhibitors had a similar effect, suggesting a ubiquitous mechanism. They did not prevent SG formation but delayed it. Mechanistically, they act on SG formation by reducing the overall level of G3BP1 protein expression and stress-induced translation repression.

This result revealed that lipids could be potential new actionable targets for diseases involving SG.

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G3BP1: a tunable stress granule organizer in neurons?

Presenting author:

Universität Osnabrück, Department of Neurobiology, Barbarastrasse 11 , 49076 Osnabrück [DE], badrikoohi.mahshid@uni-osnabrueck.de

Author(s):
Mahshid Badri koohi, Roland Brandt, nataliya trushina, Daniel Villar Romero

G3BP1 (Ras-GTPase-activating protein SH3 domain-binding protein 1) is a critical component of stress granules, cytoplasmic aggregates of proteins and RNA that form in response to cellular stress. G3BP1 functions as a molecular switch, regulating the assembly and disassembly of stress granules through its RNA-binding activity and stress-sensing properties. This study investigates role of phosphorylation and acetylation as post-translational modifications (PTMs) of G3BP1 in the dynamics of neuronal stress granules under different stress factors.
Using phosphoproteomics, we identified four phosphorylation sites in G3BP1 neuronally differentiated cells exposed to arsenite or H₂O₂. To assess how PTMs affect stress granule dynamics in living cells, we performed FDAP experiments measuring G3BP1 distribution and mobility. PAGFP-tagged G3BP1 constructs were used, including wild-type and non-phosphorylatable mutants (S149A, S230–S232A, S253A). Additionally, we examined the role of acetylation using acetylation-mimic (K376Q) and non-acetylatable (K376R) mutants.
About 50% of exogenous PAGFP-G3BP1wt localized to arsenite-induced stress granules, with a time constant of ~120 s (t1/2 ~80 s). The non-phosphorylatable G3BP1(S230–S232A) mutant showed reduced dynamic exchange under both arsenite and H₂O₂ stress.In contrast, acetylation at K376 had no effect. These results suggest that G3BP1 phosphorylation regulates its dynamic behavior, with specific sites playing a selective modulatory role.

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Tracking initiation of paraspeckle assembly in real-time

Presenting author:

EMBL Heidelberg, Molecular Systems Biology Unit, Meyerhofstrasse 1, 69117 Heidelberg [DE], kyungmin.baeg@embl.de

Author(s):
Kyungmin Baeg, Lorenz Worf, Gaia Valeria Jark, Olivier Duss

Paraspeckles are nuclear membraneless organelles assembled co-transcriptionally on the long non-coding RNA NEAT1_2. Although in vivo studies suggest a multistep assembly mechanism, the current model lacks mechanistic and dynamic understanding of how NEAT1_2 transcription and interactions with paraspeckle proteins (PSPs) drive assembly and condensate formation. Here, we developed an in vitro system to dissect paraspeckle formation by recombinantly expressing all 7 essential full-length PSPs (NONO, SFPQ, RBM14, DAZAP1, FUS, HNRNPH3, TDP43) and visualizing their co-transcriptional assembly using multi-color single-molecule fluorescence microscopy. We first show that several PSPs can co-localize into the same condensate. Using biochemical and single-molecule FRET assays, we detect transient but specific binding of several PSPs to RNA. To address the co-transcriptional aspect of paraspeckle assembly, we established a co-transcriptional PSP binding assay at the single-molecule level and in real-time. Using our system, we observed that PSPs not only bind to nascent RNAs gradually, but also that preformed PSP micro-condensates bind to transcribing NEAT1_2 RNA. We find that the NEAT1 RNA region, RNA length, and PSP composition affect droplet recruitment to the nascent RNA during active transcription. Our system provides first mechanistic insight into how NEAT1_2 transcription seeds paraspeckle assembly and sheds light on broader transcription-regulated phase separation mechanisms.

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A Nuclear Actin Network Facilitates Transcriptional Activation by Transporting Specific Genes to Transcriptional Condensates

Presenting author:

Karlsruhe Insitute of Technology (KIT), IBCS-BIP, Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen [DE], yuzhi.bao@kit.edu

Author(s):
Yuzhi Bao, Elly Lohrer, Roshan Prizak, Svenja Ulferts, Marcel Sobucki, Robert Grosse, Lennart Hilbert, Lennart Hilbert

Stem cells and early embryonic cells show prominent transcriptional clusters, which are visited for a subclass of genes undergoing strong transcriptional induction. In zebrafish embryos, these visits require long-range movement of genes within 10 minutes or less.Here, we reveal a network of nuclear actin bundles that is essential for this rapid movement combined with selective transcription control in pluripotent zebrafish embryos. Specifically, acute chemical perturbation of the actin meshwork led to downregulation of transcription. The connection of actin and transcription was further supported by the microscopy observation of a nucleus-spanning actin filament bundle network that is coated with a film of recruited RNA polymerase II and anchored at prominent transcriptional clusters that are also enriched in nuclear actin monomers. Combining nucleus-targeted overexpression of wild type as well as polymerization-deficient actin with gene-specific labeling, we find that gene-condensate visit behavior and transcription levels are affected by perturbation of the nuclear actin network. In particular,for both perturbations, genes with high visit frequency visit cluster less often and become less transcribed, whereas genes with low visit frequency visit more often and become more frequently transcribed.Our work reveals pluripotency-specific nuclear actin bundle network enabling selective and rapid transport of a subset of embryonic genes to prominent transcriptional condensates.

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Quantification of Protein Folding Stability in the Cytoplasm and Stress Granules by Confocal Fast Relaxation Imaging

Presenting author:

, , Universitätsstraße 150, 44801 Bochum [DE], mailin.becker@ruhr-uni-bochum.de

Author(s):
Mailin Becker, Nirnay Samanta, Erik Tsvetaev, Miká Vollet, Sara Ribeiro, Simon Ebbinghaus

Biomolecular condensates, such as stress granules (SGs) are essential for cellular organization and regulation. Yet their dysregulation is associated with neurodegenerative diseases. Within these SGs, misfolded proteins are transiently sequestered. An example is the protein Superoxide Dismutase 1 (SOD1), which is particular relevant due to its aggregation in amytrophic lateral sclerosis (ALS). However, it is still unknown how SGs affect SOD1 stability and aggregation. Therefore, we developed confocal Fast Relaxation Imaging (cFReI). Using an infra-red laser to induce temperature jumps, we can monitor unfolding and aggregation relaxation kinetics of SOD1 simultaneously within SGs and the adjacent cytoplasm region of living cells. Our results show that SGs association does not destabilize SOD1. In most cells there was even a small stability increase inside the SGs in comparison to the cytoplasm. These findings suggest that SGs act primarily as passive reservoirs for misfolded proteins rather than as sites that promote their destabilization or aggregation. This offers important new insight into the role of SGs in protein homeostasis and disease progression.

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Hosts, clients, and competitors: the liquid-liquid phase separation story of two mRNA degradation pathways

Presenting author:

Institute of Physics, Polish Academy of Sciences, Laboratory of Biological Physics, aleja Lotników 32/46 , 02-668 Warsaw [PL], bialy@ifpan.edu.pl

Author(s):
Michal Kacper Bialobrzewski, Maja Kaja Cieplak-Rotowska, Zuzanna Staszalek, Marc Fabian, Nahum Sonenberg, Michal Dadlez, Anna Niedzwiecka

GW182 is a fuzzy, intrinsically disordered protein that plays a key role in degrading mRNA during post-transcriptional microRNA-mediated gene silencing. Its N-terminal Ago-binding domain (ABD) interacts with the Argonaute (Ago) protein, which is a core component of the miRNA-induced silencing complex (miRISC), while the C-terminal silencing domain (SD) recruits the CCR4-NOT deadenylase complex to targeted transcripts. CCR4-NOT also functions in a different silencing mechanism controlled by tristetraprolin (TTP), another intrinsically disordered RNA-binding protein that targets AU-rich elements in the 3′ untranslated regions of cytokine mRNAs. Although GW182 and TTP engage CCR4-NOT to degrade mRNA through distinct mechanisms, their overlap raises the question of whether the pathways converge or compete.

To explore this, we conducted biophysical studies showing that the GW182 SD is capable of driving liquid-liquid phase separation (LLPS). Phase diagrams reveal temperature-sensitive LLPS behaviour dependent on π–π interactions between tryptophan residues. Moreover, our results show that the GW182 SD forms multiprotein condensates with the CNOT1 subunit of CCR4-NOT, suggesting a host–client interaction. Notably, the presence of TTP as a third component disrupts condensate formation. These findings indicate that GW182 SD and TTP directly compete for binding to the same CNOT1 region, revealing potential molecular cross-talk between the two post-transcriptional silencing pathways.

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Describing NusG-mediated in vitro condensation: implications for bacterial transcription

Presenting author:

Bayreuth University, Biochemistry IV, Universitätsstraße 30, 95447 Bayreuth [DE], tamara.bosnjakovic@uni-bayreuth.de

Author(s):
Tamara Bosnjakovic, Benjamin Lau, Kristian Schweimer, Julia Mahamid, Olivier Duss, Janosch Hennig

The functional importance of biological condensates in bacterial cells has emerged in recent years as a novel approach to understanding the spatial and temporal organization of these unicellular organisms. It has been shown that conserved transcription factors are potentially partitioning in liquid-like condensates in vivo. Nevertheless, the molecular grammar behind this phenomenon remains unknown. Therefore, we employ a combination of in vitro phase separation assays and structural biology methods to dissect the molecular grammar of NusG condensate formation. Using an in vitro phase-separation assay in combination with fluorescent microscopy, we inspected the behavior of NusG upon addition of its native RNA target (rrnG). NusG-rrnG interactions are sufficient for in vitro induction of NusG condensate formation. Using NMR titration experiments, we were able to pinpoint the residues essential for RNA binding of NusG and condensate formation. Mutants based on these insights enabled us to test a potentially novel function of NusG and the correlation of RNA binding and condensate formation. Moreover, we are establishing E.coli NusG knock-out strain to test the effects of RNA-binding mutants in vivo and, for the first time, observe the formation of transcription-dependent membrane-less compartments in live bacteria.

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Liquid-Liquid Phase Separation as a Regulatory Principle of Gephyrin Organization: From Synaptic Architecture to Metabolic Integration

Presenting author:

Uniklinik Köln und Universität zu Köln , Chemistry and Biochemistry, Zülpicher Str. 47a, 50674 Cologne [DE], emanuel.bruckisch@uni-koeln.de

Author(s):
Emanuel H. W. Bruckisch, Arthur Macha, Konrad Benting, Nele Burdina, Imke von Stülpnagel, Monika Gunkel, Theresa Gehling, Filip Liebsch, Elmar Behrmann, Günter Schwarz

The postsynaptic scaffold protein gephyrin is essential for organizing glycine and GABA type A receptors at inhibitory synapses. Gephyrin is a multi-domain protein: its G-domain trimerizes, its E-domain dimerizes, and the central C-domain is unstructured, flexible and targeted by post-translational modifications. Recent studies show that gephyrin undergoes liquid-liquid phase separation (LLPS), forming dynamic condensates that support inhibitory postsynaptic density formation. Using cryo-EM, we found that receptor-loop binding aides the E-domain to form filaments via an interface between subdomain II regions of adjacent dimers promoting LLPS and receptor clustering. Structure-guided mutagenesis identified key residues whose disruption abolished filament formation, LLPS and receptor clustering in cells. These findings provide the first structural basis for gephyrin LLPS and suggest that filaments may form the core architecture of inhibitory postsynaptic condensates. This interface is also affected by mutations linked to epileptic encephalopathy, highlighting its physiological relevance. Gephyrin is derived from basic metabolism, as outside the brain it functions in molybdenum cofactor biosynthesis catalyzing an ATP-dependent metal insertion reaction. We will present latest results on how different nucleotide-based metabolites impact gephyrin LLPS, filament assembly and receptor clustering providing novel links between basic metabolism, redox signaling and synapse formation.

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Multimodal binding of collybistin controls gephyrin filament formation in synaptic condensates

Presenting author:

Institute of Biochemistry, University of Cologne, Department of Chemistry and Biochemistry, Zülpicher Str. 47, 50674 Cologne [DE], nburdin1@uni-koeln.de

Author(s):
Nele Burdina, Filip Liebsch, Arthur Macha, Monika Gunkel, Joaquín Lucas Ortuño Gil, Pia Frommelt, Irina Rais, Fabian Basler, Simon Pöpsel, Elmar Behrmann, Guenter Schwarz

Gephyrin clusters glycine and GABA type A receptors at inhibitory postsynapses through the oligomerization of its G- and E-domains. Recently, we uncovered the formation of gephyrin E-domain–dependent filaments, which drive synaptic clustering via liquid-liquid phase separation. However, regulatory mechanisms controlling filament assembly at postsynaptic sites remained elusive. Using single-particle cryo-electron microscopy we revealed that collybistin, a key gephyrin-interaction partner at GABAergic synapses, controls gephyrin filament formation in a lipid-dependent manner: Collybistin alone inhibited filament formation while plasma-membrane phosphoinositides promoted the assembly of stable gephyrin-collybistin complexes that underwent filament assembly. Within these complexes, collybistin binds at distinct positions, with different stoichiometries and conformations, either promoting or restricting filament assembly and thereby tuning the phase separation properties of gephyrin. Additionally, disruption of the gephyrin-collybistin complex formation upon phosphorylation of gephyrin at Ser325 impaired collybistin-mediated phase separation and postsynaptic clustering at GABAergic synapses. Collectively, these findings highlight the critical role of gephyrin E-domain dimerization-dependent filaments for postsynaptic gephyrin condensate formation and demonstrate its tight regulation by collybistin, driving specific oligomerization at phosphoinositide-enriched GABAergic synapses.

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Glycogen phase separation drives macromolecular rearrangement and asymmetric division in Escherichia coli

Presenting author:

HHMI/Stanford University, ChEM-H, Biology, 290 Jane Stanford Way, 94305 Stanford [US], sjcd15@stanford.edu

Author(s):
Silvia J. Canas-Duarte, Yashna Thappeta, Haozhen Wang, Till Kallem, Alessio Fragasso, Yingjie Xiang, William Gray, Cheyenne Lee, Lynette Cegelski, Christine Jacobs-Wagner

Despite decades of study into E. coli, little is known about the processes that allow it to traverse the transition between exponential growth and the nutriend limited stationary phase. Using quantitative microscopy we found significant changes in the cytoplasmic localization of all major intracellular macromolecules in this ‘transition’. We identified the accumulation of glycogen as the driver of the observed macromolecular rearrangements and the resultant onset of asymmetric divisions in the transition phase. Glycogen was recently shown to undergo LLPS in eukaryotic cells and in vitro, but its accumulation in bacteria has long been described as the formation of granules. Thus, we set to explore the likelihood and consequences of glycogen forming phase-separated condensates in the cytoplasm of E. coli. We found that glycogen undergoes phase transitions in conditions resembling the bacterial cytoplasm. In vivo, our data suggests that glycogen accumulation alters the organization of intracellular components, likely by forming condensates capable of selectively excluding macromolecules. AFM measures in live cells indicate that glycogen condensates are “soft” in nature, compared to the significantly “hard” inclusion bodies measured under the same conditions. Finally, we found that glycogen phase separation likely results in cytoplasmic space compensation, which would allow cells to accumulate this glucose polymer in large amounts without affecting macromolecular homeostasis

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Dissecting the molecular architecture and splicing-linked functions of nuclear speckles

Presenting author:

Biozentrum, University of Basel, , Spitalstrasse 41, 4056 Basel [DE], kerstin.doerner@unibas.ch

Author(s):
Kerstin Dörner, Nicole Beuret, Jonas Bürki, Seraphine Lüscher, Lea Stadelmann, Mirjam Uhland, Giulia Basile, Daan Overwijn, Maria Hondele

Nuclear speckles (NS) are prominent biomolecular condensates that serve as regulatory hubs for multiple steps of gene expression, including transcription and splicing. Recent studies show that NS localize near highly transcribed genes, and likely enhance their splicing. However, the molecular mechanisms governing mRNA recruitment to NS and its impact on mRNA processing remain largely unknown.

 

Splicing inhibition leads to enlarged NS and accumulation of polyadenylated mRNPs. To better understand which mRNPs are recruited to NS, we screened mutants of all splicing- and NS-associated DEAD/DExH-box ATPases (DDX/DHXs), essential enzymes driving the splicing cycle, in HeLa cells and identified several that induced striking changes in NS morphology. We isolated the associated mRNP ‘cargo’ and identified seven candidate proteins likely involved in recruiting mRNPs to NS by mass spectrometry.

Using super-resolution (SIM/STED) microscopy, we found that NS are not homogeneous condensates. Instead, their core components SON and SRRM2 form dynamic meshwork-like scaffolds that undergo significant remodeling upon transcription or splicing inhibition. Depletion of the candidate recruiters resulted in smaller, more spherical speckles, suggesting that NS ultrastructure is highly dynamic and shaped by mRNP recruitment.

Together, our findings provide mechanistic insights into mRNP recruitment to NS and how NS morphology is coupled to mRNP splicing and maturation.

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Seek and You Shall Find: Exploring Protein Condensate Formation by Time-Resolved Small Angle X-ray Scattering and Molecular Simulations

Presenting author:

University of Copenahgen, Pharmacy, Universitetsparken 2, 2100 Copenhagen [DK], vito.fodera@sund.ku.dk

Author(s):
Samuel Lenton, Marco Polimeni, Fátima Herranz Trillo, Tobias Winckler-Carlsen, Ann Terry, Annette Eva Langkilde, Vito Foderà

Liquid–liquid phase separation (LLPS) is recognized as a critical early event in the formation of protein aggregates and amyloid fibrils, which are hallmarks of several neurodegenerative disorders. However, current studies predominantly focus on time scales where LLPS is already established (seconds to hours), leaving the initial molecular events and transient protein states largely unexplored. This gap stems from the lack of techniques capable of capturing early condensation dynamics in solution with sub-second temporal resolution.

In this study, we employ time-resolved small-angle X-ray scattering in conjunction with coarse-grained molecular simulations to investigate the rapid phase separation dynamics of bovine serum albumin (BSA) in the presence of polyethylene glycol (PEG) and potassium chloride (KCl). Utilizing a microfluidic platform with precisely controlled flow rates, we monitor the BSA–PEG–KCl mixing process and the nucleation of the dense phase in real time. We demonstrate that LLPS propensity increases with salt concentration, and through simulation-guided analysis of the scattering profiles, we quantify excluded volume effects and characterize the evolving interaction potential between BSA molecules.

This integrative approach provides a generalizable framework for probing early-stage phase behavior in protein systems and offers a powerful tool for elucidating the molecular determinants of pathological aggregation in disease-relevant proteins.

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Programmable DNA Protonuclei Reveal Hidden Determinants of Protein Phase Separation in Nuclear-Mimetic Environments

Presenting author:

JGU Mainz, Chemistry, Duesbergweg 10-14, 55128 Mainz [DE], johann.fritzen@uni-mainz.de

Author(s):
Johann Fritzen, Avik Samanta, Nele Kuhr, Erin Sternburg, Dorothee Dormann, Andreas Walther

Understanding how nucleic acid sequence and environment shape biomolecular condensate formation is key to decoding nuclear organization and disease-linked protein aggregation. Here, we present DNA-based protonuclei (PN), a fully synthetic, tunable platform that mimics essential nuclear features including crowding ([DNA]=5-13 g/L), sequence complexity, and viscoelasticity. Using the ALS-implicated protein FUS as a model, we demonstrate that phase separation (PS) behavior is critically dependent on loading ratio LR, revealing tunable binodal PS (LR=0.31-0.37) and spinodal PS (LR=0.61-0.72). Surprisingly, conventional affinity assays (e.g. EMSA) fail to predict condensate behavior in the PN environment, revealing a striking disconnect between binary binding and functional partitioning, with similar affinity sequences showing a 20-fold difference in partitioning. We further show that nucleic acid sequence, identity (RNA/DNA) or physical crosslinking of the DNA core modulate condensate morphology, retention, and the pathological liquid-to-solid transition, suggesting mechanical microenvironment as an underappreciated regulator of PS. Our results introduce PN as a versatile testbed bridging test tube simplicity and cellular complexity, enabling deeper insight into nucleic acid-protein interplay within crowded, confined compartments. This approach lays a foundation for programmable nuclear mimics in studying condensate biology and screening therapeutic modulators of phase behavior.

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Determinants of oxygen partitioning into biomolecular condensates

Presenting author:

Aarhus University, Department of molecular biology and genetics, universitetbyen 81, 8000 Aarhus [DK], au711045@uni.au.dk

Author(s):
Ankush Garg, Christopher Brasnett, Siewert Marrink, Klaus Koren, magnus Kjaergaard

Biomolecular condensates form through the self-assembly of proteins and nucleic acids to create dynamic compartments in cells. By concentrating specific molecules, condensates establish distinct microenvironments that regulate biochemical reactions in time and space. Macromolecules and metabolites partition into condensates depending on their interactions with the macromolecular constituents; however, the partitioning of gases has not been explored. We investigated oxygen partitioning into condensates formed by intrinsically disordered repeat proteins with systematic sequence variations using microelectrodes and phosphorescence lifetime imaging microscopy (PLIM). Unlike other hydrophobic metabolites, oxygen is partially excluded from the condensate with partitioning constants more strongly modulated by changes in protein length than hydrophobicity. For repeat proteins, the dense phase protein concentration drops with chain length, resulting in a looser condensate. We found that oxygen partitioning is anti-correlated with dense phase protein concentration. Several mechanisms could explain such an anti-correlation, including excluded volume or salting out effects. MD simulations suggest that oxygen does not form strong and specific interactions with the scaffold and is dynamic on the nanosecond timescale. Different biomolecular condensates maintained distinct oxygen concentrations, suggesting a possible mechanism for regulating oxygen-dependent reactions in cells

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Ubiquitin-dependent phase separation behavior of UPS shuttle factor RAD23B

Presenting author:

Institute of Molecular Biology (IMB), 55128 Mainz, AG Luck, Ackermannweg 4, 55128 Mainz [DE], J.Geist@imb-mainz.de

Author(s):
Johanna Lena Geist

Multiple ubiquitin shuttle factors like UBQLN2, p62 and RAD23A/B have been shown to phase separate either upon self-oligomerization or upon binding of (poly)ubiquitin. They all share a similar domain architecture, consisting of a N-terminal UBL (ubiquitin-like) domain, as well as of a one or multiple C-terminal UBA (ubiquitin-associated) domains. Phase separation of all three shuttle factors is mediated by ubiquitin binding, with RAD23B being known to phase separate in the presence of preferably K48-linked ubiquitin chains. The binding of RAD23B to ubiquitin is thereby mediated through at least one of its two UBA domains interacting with ubiquitin. Even though the interaction of UBA domains with ubiquitin is not new to the field, it however remains elusive what mechanism might drive the selective induction of phase separation of RAD23B upon polyubiquitin binding and how factors like length and architecture of ubiquitin chains might regulate the process. Therefore, we designed mutations in RAD23B that should affect its propensity to phase separate in the presence of ubiquitin by either enhancing the availability of or impairing the binding interface. We compare condensation of either purified wt and mutant RAD23B employing differential interference contrast and fluorescence microscopy. After identification of relevant mutants, we will investigate their effect on in cell condensation in unchallenged U-2 OS cells as well as under different stress conditions.

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Interplay of Thermodynamics, Configurations, and Counter Ions in Intrinsically Disordered Polyelectrolyte Complexation

Presenting author:

University of Zurich,, Department of Biochemistry,, Winterthurerstrasse 190, 8057 Zurich [CH], soundharar@gmail.com

Author(s):
Soundhara Rajan Gopi, Miloš Ivanović, Artemi Bendandi, Aritra Chowdhury, Valentin Von Roten, Paweł Łukijańczuk, Ruijing Zhu, Robert Best, Benjamin Schuler

The organization of the eukaryotic genome within the nucleus involves a complex interplay between densely packed, highly charged DNA, and dynamic nuclear proteins. Among these, intrinsically disordered proteins (IDPs), characterized by their lack of a fixed 3D structure and high charge density, play critical roles. Recent studies have highlighted that the release of counter-ions during polyelectrolyte complexation contributes to a thermodynamically stable yet dynamic protein complex and reflects the very strong dependence of K_D on salt concentration. In this study, we integrate single-molecule Förster Resonance Energy Transfer, all-atom molecular dynamics (MD), and coarse-grained MD simulations to elucidate the thermodynamics of polyelectrolyte complexation. We developed and validated a novel framework to estimate counter-ion condensation by coupling residue-level coarse-grained MD simulations with the Poisson-Boltzmann theory. Validation against all-atom MD simulations of charged peptide libraries, incorporating explicit solvent and ion models, demonstrates the accuracy of this approach. Our coarse-grained simulations, enhanced with optimized force-field parameters, successfully reproduce the conformational ensembles and dissociation constants of the H1-ProTα complex as a function of salt concentration. By dissecting the molecular interactions and thermodynamic forces, we advance our understanding of disordered polyelectrolyte assemblies prevalent in biological systems.

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Designing tuneable biomolecular condensates to mimic protein degradation pathways

Presenting author:

University of Cambridge, Pharmacology, Tennis Court Road, CB2 1QR Cambridge [GB], nalh2@cam.ac.uk

Author(s):
Nora Haanaes

The essential role of biomolecular condensates in organising cellular biochemistry is becoming increasingly clear. Being membraneless and entropy-driven through phase separation, biomolecular condensates can facilitate processes transiently and without requiring energy-expenditure from the cell. Our group is leveraging the unique chemistry of biomolecular condensates and their natural role in autophagic degradation for therapeutic purposes. We have established synthetic consensus tetratricopeptide repeat protein (CTPR) condensates that enable the grafting of short linear motifs (SLiMs) to facilitate specific binding to target and autophagy-related proteins. We are developing an iterative pipeline of in silico, in vitro and in cellulo experiments to guide the further design of CTPR condensates. Using molecular dynamics simulations and emerging AI tools, we are evolving the CTPR condensate sequences, and measuring how rational changes impact condensate properties. Importantly, we are building complexity into our in silico and in vitro experiments to better translate our structure-function relationships to the cellular environment. By directly targeting the autophagy pathway, we hope to use the CTPR-condensates for proximity-induced degradation of disease-related proteins.

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Spatial organization of translation and translational repression in two phases of germ granules

Presenting author:

Institut de Génétique Humaine UMR9002 - CNRS, , 141 rue de la Cardonille, 34396 Montpellier [FR], ali.haidar@igh.cnrs.fr

Author(s):
Ali Haidar, Anne Ramat, Celine Garret, Martine Simonelig

RNA-Protein(RNP) condensates are hubs for post-transcriptional regulation. Several RNP condensates are not homogeneous, but rather composed of several immiscible phases. However, how these different phases are linked to their biological functions remains unclear. Germ granules are RNP condensates essential for germ cell fate, and mRNA localization and translational regulation. They represent an outstanding model to study the relationships between the organization and functions of RNP condensates. Using STED super-resolution microscopy, we showed that Drosophila germ granules have a biphasic organization of a shell and a core, with their main protein components enriched in the shell. We set up single-molecule imaging, including the Suntag approach to visualize translation taking place, and found that translation occurs in the shell and immediate periphery of the granule, but not in the core. Additionally, we revealed a correlation between mRNA translational status and their position; Translating mRNAs are enriched in the shell, whereas repressed mRNAs accumulate in the core. mRNA orientation and compaction within granules also depend on their translation status; the 5'end of translated mRNAs are oriented towards the surface and adopt a less compacted conformation than repressed mRNAs. Finally, we found that altering germ granule structure severely affects mRNA translation level. These findings reveal the importance of RNA granule architecture in organizing different functions.

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Recruitment of CAG Repeat RNAs relevant for Huntington's Disease into nuclear speckles occurs at both physiological and pathological repeat length and is governed by ATP

Presenting author:

Ruhr- Universitaet Bochum, Chair of Biophysical Chemistry, Universitaetsstr. 150, 44801 Bochum [DE], alexander.hautke@rub.de

Author(s):
Alexander Hautke, Arthur Voronin, Fathia Idiris, Anton Riel, Felix Lindner, Amandine Lelièvre-Büttner, Jikang Zhu, Bettina Appel, Edoardo Fatti, Karsten Weiß, Sabine Müller, Alexander Schug, Simon Ebbinghaus

CAG repeat RNAs and their folding into hairpins play an important role in Huntington's Disease (HD). Further, they are sequence-specifically recruited into nuclear speckles and sequester transcription and translation factors. This behavior intensifies as the number of CAG repeats increases.

Here, we study the impacts of macromolecular crowding, chemical interactions and hydration on the localization, folding stability and liquid-liquid phase separation of these RNAs. Key techniques for our study are Fast Relaxation Imaging, Fluorescence Recovery after Photobleaching and colocalization experiments.

We show that a (CAG)20 hairpin is largely destabilized inside cells compared to dilute buffer. Further, we report that CAG repeat RNAs are recruited into nuclear speckles at physiological and pathological repeat length and that folding stability remains unchanged compared to the nucleoplasm. Finally, both hairpin folding stability and mobility inside nuclear speckles are strongly affected by ATP due to preferential interactions between adenosine and RNA nucleobases in the unfolded state of the RNA. This finding is especially outstanding since declining cellular ATP levels are a frequently occurring condition in HD neurons. In recent far-infrared experiments, we investigated this pivotal role of ATP.

In summary, this study provides new insights into CAG repeat RNA biophysics and how it may impact HD pathology. Further, it suggests an important role of ATP for cellular RNA homeostasis.

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The Stress Response Choreography: RNA and Chromatin Orchestrate Nucleolar Reorganization

Presenting author:

EMBL Heidelberg, Cell Biology and Biophysics, Meyerhofstraße 1, 69117 Heidelberg [DE], yuki.hayashi@embl.de

Author(s):
Yuki Hayashi, Carlo Bevilacqua, Mateusz Brzezinski, Sapun Parekh, Jasper Michels, Robert Prevedel, Sara Cuylen-Häring

The nucleolus, a key biological condensate within the nucleus, is crucial for ribosome biogenesis and is organized into multiple nested subcompartments. Cellular stress, such as DNA damage, suppresses RNA polymerase I (Pol I) transcription, triggering profound nucleolar reorganization characterized by the relocation of inner subcompartments to the nucleolar surface, forming structures known as nucleolar caps. Despite the importance of the multi-layered architecture in facilitating ribosome synthesis, the molecular mechanisms that maintain its structural integrity and govern its reorganization under stress remain poorly understood.

Using quantitative live-cell imaging and micromanipulation, we reveal that nucleolar cap formation upon Pol I inhibition occurs via a two-step mechanism: first, RNA loss–mediated fusion of inner subcompartments; followed by chromatin compaction–dependent relocation to the nucleolar surface. These findings suggest a fundamental principle of nucleolar architecture: RNA acts as a physical barrier maintaining internal organization, while chromatin provides the mechanical force that enables dynamic, large-scale remodeling essential for cellular stress responses and genome regulation.

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Molecular dynamics simulation reveals the mechanism of polySUMO-mediated phase separation

Presenting author:

Leibniz Institute on Aging - Fritz Lipman Institute, Core Facility Imaging, Beutenberg Str. 11, 07745 Jena [DE], peter.hemmerich@leibniz-fli.de

Author(s):
Peter Hemmerich, Tobias Ulbricht, Peter Dittrich, Titus Franzmann, Simon Alberti

PML nuclear bodies (PML NBs) function as dynamic nuclear hubs that regulate genome maintenance, DNA repair, transcription, protein modification, and apoptosis. Liquid–liquid phase separation (LLPS) of the polySUMOylated PML scaffold and SUMO interacting motifs (SIMs) of partner clients are currently believed to be a major driving force for the assembly of PML NBs. Yet the precise mechanism(s) of LLPS at PML NBs is not fully understood. Here we present an in silico model of PML NB assembly. Guided by predictions of model simulations we identify a novel class of polySUMO-containing nuclear condensates, which we have coined polySUMO nanobodies (SNBs). In vitro, polySUMO chains form condensates at low nanomolar concentrations, indicating that multi-valent self-interaction is sufficient for polySUMO phase separation. Since (i) LLPS inhibitors fail to disassemble PML NBs, (ii) exchange rates of PML are extremely slow and (iii) PML does not move within the NB scaffold, we suggest that PML NBs are not liquid condensates. However, polySUMO assemblies may provide nano-sized volumes with LLPS properties in the shell of PML NBs as well as in SNBs. Our observation of polySUMO phase separation in the absence of SIM-containing binding partners adds an interesting new layer of complexity to our understanding of SUMO dynamics.

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TBK1 Induces the Formation of Optineurin Filaments that Condensate with Polyubiquitin and LC3 for Cargo Sequestration

Presenting author:

Ruhr Univeristy of Bochum, Medicine, Universitätsstraße 150, 44801 Bochum [DE], maria.herrera@rub.de

Author(s):
Maria Georgina Herrera, Lena Kühn, Lisa Jungbluth, Verian Bader, Laura Krause, David Kartte, Elias Adriaenssens, Sascha Martens, Jörg Tatzelt, Carsten Sachse, Konstanze Winklhofer

Optineurin is an autophagy receptor which plays an important role in the selective degradation of mitochondria, protein aggregates, and intracellular pathogens. It recognizes ubiquitylated cargo by its UBAN (ubiquitin-binding in ABIN and NEMO) domain and recruits the autophagic machinery through its LIR (LC3-interacting region) domain. Phosphorylation of Optineurin by TBK1 (Tank-binding kinase 1) increases the binding of Optineurin to both ubiquitin chains and LC3. Optineurin has been reported to form foci at ubiquitylated cargo, but the underlying mechanism and how these foci are linked to selective autophagy has remained largely unknown. This study shows that the phosphorylation of Optineurin by TBK1 induces the formation of filaments that phase separate upon binding to linear polyubiquitin. LC3 anchored to unilamellar vesicles co-partitions into Optineurin/polyubiquitin condensates, resulting in the local deformation of the vesicle membrane. These data suggest that the condensation of filamentous Optineurin with ubiquitylated cargo promotes the nucleation of cargo and its subsequent alignment with LC3-positive nascent autophagosomes.

__________

Establishment of a bioinformatics pipeline to characterise the phase separation of proteins in plant cells

Presenting author:

University of Hamburg, Department of Molecular Plant Physiology, Ohnhorststraße 18, 22609 Hamburg [DE], oliver.herzog@uni-hamburg.de

Author(s):
Oliver Herzog, Stefan Hoth, Magdalena Weingartner

Biomolecular condensation by liquid-liquid phase separation (LLPS) has significantly improved our understanding of protein compartmentalisation and stress responses. In plants, LLPS plays a crucial role in thermal adaptation: upon heat stress, mRNAs, RNA-binding proteins, and translation factors are sequestered into stress granules (SGs), adjusting global translation dynamics. Natural variations in SG components influence their LLPS ability and thereby affect resilience to temperature shifts. To investigate how isoforms encode temperature-responsive behaviour, we developed a systematic, quantitative framework.

We focused on two eukaryotic Elongation Factor 1B (eEF1B) isoforms with distinct temperature-dependent condensation and classified SG formation using a light microscopy-based bioinformatic approach. Fluorescently tagged isoforms were studied in roots and leaves of stably transformed plants and in Arabidopsis thaliana and Physcomitrium patens protoplasts after transient expression. Confocal images were processed via Nikon’s NIS Elements with its integrated deep-learning AI, complemented by a open-source pipeline using Fiji, CellProfiler’s granularity tool, and custom scripts.

From this, we identified three LLPS patterns: (1) diffuse, (2) semi-condensed, and (3) SG-like condensates, based on granularity, object size, intensity, and spatial clustering. The workflow enables reliable automated and comparative in vivo profiling of protein phase separation in plant cells.

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Stem cell-specific transcriptional condensates form on genomic scaffold regions that are lost upon differentiation

Presenting author:

Karlsruhe Insitute of Technology (KIT), Institute of Biological and Chemical Systems, Hermann-von-Helmholtz-Olatz 1, 76344 Eggenstein-Leopoldshafen [DE], lennart.hilbert@kit.edu

Author(s):
Tim Klingberg, Irina Wachter, Agnieszka Pancholi, Yomna Gohar, Priya Kumar, Matthias Akyel, Ana Miguel Fernandes, Yuzhi Bao, Alica Schmidt-Heydt, Alicia Günthel, Marcel Sobucki, Elisa Kämmer, Süheyla Eroğlu-Kayıkçı, Sylvia Erhardt, Carmelo Ferrai, Vasily Zaburdaev, Lennart Hilbert

Stem cells exhibit exceptionally prominent transcriptional clusters, which dissolve with progressing differentiation. Even though these clusters are assigned central roles in embryonic gene regulation, their formation and loss during differentiation remain poorly understood. Here, we reveal that prominent, stem cell-specific transcriptional condensates emerge and disperse in a conserved sequence across mouse embryonic stem cells, fruit fly spermatogonia, and zebrafish embryos. Using imaging, epigenetic profiling, and lattice simulations, we show that these clusters form via surface condensation on H3K27ac-marked super-enhancer regions, which act as genomic scaffolds. Upon differentiation, partial loss of these active epigenetic marks leads to dispersal of the prominent clusters. Our polymer-based simulations explain this process as a conserved trajectory through a three-dimensional state space, governed by surface condensation principles that extend beyond canonical liquid-liquid phase separation. This work provides a biophysical mechanism for the dynamic organization of stem cell-specific transcriptional hubs and demonstrates evolutionary conservation in intact organisms. By uncovering a conserved biophysical mechanism for transcriptional organization in development, our work illustrates how polymer properties can shape cell identity and fate.

__________

Profiling phase-separated condensates induced by therapeutic antisense oligonucleotides

Presenting author:

Eberhard Karls Universität Tübingen, Interfaculty Institute of Biochemistry, Auf der Morgenstelle 15, 72076 Tübingen [DE], daniel.hofacker@uni-tuebingen.de

Author(s):
Daniel Hofacker, Stefanie Gackstatter, Thorsten Stafforst

Chemically modified therapeutic antisense oligonucleotides (ASOs) can induce or alter the formation and composition of phase-separated condensates, e.g. paraspeckles, nucleoli, or stress granules. They interact with multiple proteins and heavily influence the fate of their interaction partners. These effects range from protein mislocalization to the degradation of essential protein components.

Recently, we developed an assay (isASO-ID) to discover the interaction partners of ASOs in a cellular environment at physiologically relevant concentrations (Hofacker et al. 2024). This assay allows for the first time to directly connect ASO-protein interactions with pharmacological properties like efficacy, their appearance in condensates, and adverse effects including toxicity connected to this.

In an unpublished work, we utilized isASO-ID to take insight into the molecular basis of toxicity in the context of RNA Editing with ADAR-recruiting ASOs. These display a novel class of therapeutic ASOs and recently reached the first clinical trials. However, in comparison to other ASO classes, their toxicity profile is under-studied. We discovered that they do not share the same (unspecific) protein binding pattern with other ASO classes. Instead, we discovered previously undescribed interactors. These enzymatic components were inhibited upon ASO binding with respect to the applied concentration and the chemical modifications. Our findings pave the way towards a safer ASO design in the future.

__________

A high-throughput approach for systematic characterization of protein condensates

Presenting author:

Hochschule Darmstadt, FB Chemie und Biotechnologie, Stephanstraße 7, 64295 Darmstadt [DE], sarah.hofmann@h-da.de

Author(s):
Sarah Hofmann

The systematic evaluation of the liquid-liquid phase separation (LLPS) of proteins is a valuable approach to observe their propensity to form protein condensates and to understand the conditions under which these condensates form. Factors like protein concentrations, certain amino acid sequences and environmental conditions all contribute to phase separation. To enable a high-throughput and standardized approach, we expose proteins to a fixed set of solutions that cover different environmental conditions including pH, ionic strength, charge and crowding agent, by using an automated liquid handling system. For efficient analysis by a microscopy-based screening assay, an automated method is developed using the microscope’s built-in software algorithm, which is trained to detect protein condensates. Our focus is on sirtuins, which are a specific family of lysine deacetylases that require NAD+ as cofactor for their enzymatic activity. They are involved in various cellular processes including metabolism, aging, stress response and gene expression, with indications that they play a role in the formation or are a component of some cellular protein condensates e.g. in PML nuclear bodies. We have detected condensate formation for several sirtuins under specific environmental conditions and by correlating structural changes with condensate formation, we can gain insights into the molecular mechanisms driving condensate formation as a biological process.

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Frosty Foci - The E. coli DEAD-box ATPase CsdA forms nucleoid-associated foci at cold temperature

Presenting author:

University of Basel, Biozentrum, Spitalstrasse 41, 4056 Basel [CH], maria.hondele@unibas.ch

Author(s):
Michelle Jennifer Gut, Ruta Prakapaité, Alexia Loynton-Ferrand, Alexander Schmidt, Maria Hondele

Subcellular organization is essential for maintaining cellular homeostasis. Unlike eukaryotic cells, bacteria lack membranebound organelles and instead rely on alternative structures, including biomolecular condensates.

In eukaryotes, DEAD-box ATPases (DDXs) play crucial roles in many steps of RNA processing and have emerged as important regulators of condensate formation. Intriguingly, we found that three of the five E. coli DDXs form condensates in vitro, suggesting that bacteria also employ DDX-mediated condensation for cellular organization.

Of the E. coli DDXs, the ribosome biogenesis factor CsdA showed the highest condensation propensity and is the only DDX essential for growth at cold temperatures. Truncation studies indicated that CsdA condensation correlates with ATPase activity and influences growth at low temperatures.

Quantitative mass spectrometry showed that cold temperature significantly induces CsdA expression, raising its cellular concentration above its in vitro condensation threshold. Intriguingly, super-resolution microscopy revealed that endogenous CsdA forms prominent clusters in close proximity to the nucleoid, and that condensation-deficient mutants fail to form these foci.

Our data suggest that at low temperatures, CsdA clusters promote ribosome biogenesis. This function is reminisent of the eukaryotic nucleolus, a hierarchically organized condensate containing multiple DDXs, hinting at an evolutionarily conserved mechanism for ribosomal RNA processing.

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PAX3::FOXO1 phase separation leads to sticky situations

Presenting author:

St. Jude Children's Research Hospital, Molecular Oncology, 262 Danny Thomas Place MS 354, 38103 Memphis [US], jack.hopkins@stjude.org

Author(s):
Jack Hopkins

The PAX3::FOXO1 (P3F) oncofusion transcription factor is the malignant hallmark of fusion-positive alveolar rhabdomyosarcoma (FP-RMS). While the N-terminal DNA-binding domains of PAX3 alter chromatin accessibility, the FOXO1 C-terminal intrinsically disordered transactivation domain simultaneously recruits transcriptional machinery to drive gene expression and impedes effective therapeutic targeting. Consequently, the survival rate for patients with FP-RMS has remained at 30% for almost 50 years. However, novel strategies targeting intrinsically disordered transcription factors via their aberrant phase separation are being developed as our understanding of condensates improves. This project explores whether P3F condensates are essential for FP-RMS rhabdomyosarcomagenesis. We identified the amino acids responsible for generating P3F condensates in FP-RMS cell lines using machine learning algorithms developed by the Kriwacki lab. Blocking P3F phase separation through mutation of these key residues altered target gene expression and subsequently terminated P3F-mediated tumorigenesis. Furthermore, maintaining non-mutated P3F in the dilute phase prevented tumor growth in a mouse xenograft model of FP-RMS. These results revealed that phase separation is an essential component of P3F-mediated transformation, and that targeting the biophysical properties or biomolecular components within P3F condensates may be a viable therapeutic strategy for FP-RMS treatment.

__________

Combined TDP-43-CTD and FUS -LC Phase Separation: from Liquid State to Fibril State

Presenting author:

, , Mittlere Bleiche12A, 55116 Mainz [DE], hosseinie@mpip-mainz.mpg.de

Author(s):
Elnaz Hosseini, Pablo G. Argudo, Jasper J. Michels, Sapun H. Parekh

Understanding the phase separation phenomenon is key to unravelling this transition from soluble proteins to pathological aggregates. TAR DNA binding protein 43 (TDP-43) and Fused-In-Sarcoma (FUS) protein share several structural (in reverse order), and functional similarities and they are co-localized within various subcellular compartments, most notably in stress granules. However, their co-phase separation into liquid droplets is until now unknown. Here, we investigate the phase behavior and maturation dynamics of their disordered, prion-like domains: the low-complexity domain of FUS (FUS-LC) and C-terminal domain of TDP43 (TDP43-CTD). We found that at conditions where each protein individually does not phase separate, they can co-phase separate when mixed. Studies using fluorescence recovery after photobleaching (FRAP) show that 1 hour after droplet formation the condensates are dynamic, with FUS-LC recovering faster than TDP-43-CTD. However, after 24 hours, the proteins are less mobile. After four days, amyloid-like clusters form in the solution; data from coherent Raman microscopy shows that the secondary structure of proteins within the droplets and clusters becomes more β-sheet-rich with time. Measurements using fluorescence lifetime microscopy indicate molecular proximity of FUSC-LC and TDP43-CTD in droplets that increases with cluster formation, showing how these proteins mature into solid-like fibrils.

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The NLS region of TDP-43 is crucial for its cellular localization and phase separation behavior

Presenting author:

JGU Mainz-Biocenter/IMB, imP, Hanns-Dieter-Hüsch Weg 15/17, 55128 Mainz [DE], shutten@uni-mainz.de

Author(s):
Saskia Hutten, Xiaofei Ping, Nele Kuhr, Lukas Stelzl, Dorothee Dormann

Cytosolic inclusions of the RBP TDP-43 are a pathological hallmark in ALS and FTD. Yet, the underlying mechanisms causing cytoplasmic mislocalisation/ aggregation of TDP-43 are barely understood.

TDP-43 contains two disordered regions, a long C-terminal low complexity domain (LCD), which strongly contributes to its phase separation/aggregation behavior, and a short, N-terminal region comprising the nuclear localization signal (NLS), whose role in TDP-43’s phase separation/aggregation is so far unknown.

We characterized the phase separation of TDP-43 variants carrying mutations in either basic, acidic, polar or aliphatic residues in the NLS. Alanine substitutions of basic but not of any other residues suppress condensate/aggregate formation, as well as cluster formation at subsaturated concentrations. Our data are supported by molecular dynamics simulations, identifying basic residues in the NLS form inter-chain contacts with the C-terminal LCD during condensation.

TDP-43 carrying basic-to-alanine substitutions in the NLS (NLSmut) also shows reduced recruitment into stress granules, despite being able to bind RNA. Live-cell imaging of cells stably expressing WT or NLSmut GFP-TDP-43 demonstrates strongly impaired recruitment of TDP-43 NLSmut into nuclear stress bodies.

Our data suggest that basic residues in the NLS region of TDP-43 are crucially involved in TDP-43 self-interactions, and thereby contribute to its condensation and localization in membrane-less organelles.

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Investigation of the condensation behavior of plant RS splicing regulators

Presenting author:

Johannes Gutenberg - Universität Mainz, Institute for Molecular Physiology, Hanns-Dieter-Hüsch-Weg 17, 55128 Mainz [DE], a.huebenthal@uni-mainz.de

Author(s):
Anna Hübenthal, Frederik Kölpin, Stephan Hobe, Yannick Witzky, Friederike Schmid, Andreas Wachter

During early seedling development of plants, light exposure triggers a switch to photomorphogenesis, which is accompanied by rapid alternative splicing (AS) responses affecting numerous genes. Preceding studies have identified members of the RS subfamily of serine/arginine-rich (SR) proteins as regulators of light-dependent AS. Previous research showed that the four Arabidopsis thaliana RS proteins (RS31a, RS31, RS40, and RS41) localize in nuclear speckles upon illumination and form condensates in vitro. However, the mechanisms of these processes and their role in AS are poorly understood.
We examine complex forming properties of recombinantly expressed RS proteins by turbidity assays and microscopic imaging. These experiments are complemented by coarse-grained simulations of the RS proteins’ phase behavior in collaboration with the Schmid group. Our current findings indicate a distinct condensation behavior of RS31a, RS31, and RS41 in vitro, resulting in different turbidity and condensate sizes. Further studies with chimeric proteins composed of various sections from different RS proteins will help determine the specific features that influence protein complex formation. Moreover, analysis of in vivo localization and splicing regulation of wild type and mutant RS proteins will provide insights into the molecular determinants and biological implications of RS condensate formation for AS in light-dependent seedling morphogenesis.

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A Multi-Scale View of Multicomponent IDP Condensates from Simulation and Experiment

Presenting author:

University of Zurich, Biochemistry, Winterthurerstrasse 190, 8057 Zurich [CH], m.ivanovic@bioc.uzh.ch

Author(s):
Miloš Ivanović, Nicola Galvanetto, Aritra Chowdhury, Robert Best, Benjamin Schuler

Condensates assembled from IDPs govern myriad cellular processes, but the principles linking molecular motions to emergent material properties remain poorly understood. Combining single-molecule FRET and multi-million–atom explicit-solvent simulations, we show that condensates of oppositely charged IDPs retain sub-microsecond chain reconfiguration and pico- to nanosecond side-chain exchange despite high viscosity [1]. Extending this experiment–simulation approach [2, 3] to multicomponent condensates spanning diverse sequences and salt concentrations, we identify striking correlations between viscosity, translational diffusion, and chain dynamics [4]. Simulations recapitulate these trends and reveal that sequence- and salt-dependent inter-residue contact lifetimes govern the effective friction linking molecular and macroscopic scales, in line with both polymer-physics models and independent measurements. Rapid exchange of charged interaction partners at high residue densities may represent a general mechanism by which molecules avoid dynamic arrest in the highly charged environment of the cell nucleus. Finally, we developed and validated a tailored strategy to refine force-field parameters for condensates and benchmark complementary routes, providing a toolkit for predictive simulations.

[1] Galvanetto*, Ivanović*, et al., Nature (2023)
[2] Nettels et al., Nat. Rev. Phys. (2024)
[3] Ivanović & Best, Curr. Opin. Struct. Biol. (2025)
[4] Galvanetto*, Ivanović*, et al., PNAS (2025)

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Towards characterization of pathological liquid-to-solid phase transitions of TDP-43 by in situ correlative cryogenic light and cryo-electron microscopy

Presenting author:

EMBL, Molecular Systems Biology, Meyerhofstrasse 1, 69117 Heidelberg [DE], reeba.jacob@embl.de

Author(s):
Reeba Susan Jacob, Jik Nijssen, Xiao Yan, Steffen Klein, Simon Alberti, Anthony Hyman, Julia Mahamid

TDP-43 is an essential RNA-binding protein implicated in several neurodegenerative diseases, including ALS and FTLD. Recent work (Yan et al., 2025) shows that pathological aggregation of TDP-43 requires two key events: its concentration within stress granules (SG) and exposure to oxidative conditions. These synergistically induce intra-condensate demixing of TDP-43, to form a TDP-43-enriched phase on the surface of SGs, that promotes the development of aggregates with characteristics of pathological TDP-43 inclusions. Here, we employ in situ cryogenic electron tomography (cryo-ET) to elucidate the structural transitions involved in TDP-43 aggregation at molecular resolution and provide insights into the role of the SG environment in this process. To this end, we have established a correlative light and electron microscopy (CLEM)-based workflow (Klumpe at al. 2021, Zhang et al. 2023) to characterize the intra-condensate demixed TDP-43 within SGs of human cells. To precisely localize GFP-tagged TDP-43 molecules in the crowded cellular environment visualized by cryo-ET, we are optimizing our recently developed genetically-encoded multimeric (GEM) tags that can bind to GFP upon ligand induction (Fung et al., 2022). Thus, by leveraging advanced in situ cryo-EM approaches, we aim to uncover the nanometer-scale structures, spatial organization, and cellular interactions of TDP-43 aggregates, offering insight into the mechanisms driving its pathological aggregation.

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Tracing the birth and evolution of a biomolecular condensate

Presenting author:

Physics of Life Cluster of Excellence & BIOTEC, TU Dresden, , Tatzberg 47/49, 01307 Dresden [DE], marcus.jahnel@tu-dresden.de

Author(s):
Marcus Jahnel

Earth. 715 million years ago. The Cryogenian period. Four ice ages freeze the planet for eons. Closed ice sheets repeatedly cover oceans and continents. The cold is unbearable, and life is almost wiped out. Proteins that mitigate cold stress, particularly RNA chaperones, experience extreme and prolonged evolutionary pressure. What is the result? Only a few organisms survive in a handful of places. However, the world of slimes that existed before this severe environmental stress will soon explode into all the complex body plans that we see around us today. But how did life survive the cold? Did condensates play a role?

Our team has traced the evolution of cold-shock proteins - RNA-binding proteins that still help protists combat the cold but which were co-opted by mammals to facilitate brain development during embryonic growth. Interestingly, this protein family shows a gradual expansion of intrinsically disordered regions (IDRs) that correlates with organismal complexity. We find that flanking IDRs not only enable the formation of condensates, but also fine-tune the binding affinity to regulatory RNA regions. High-resolution, single-molecule experiments involving targeted long non-coding RNAs highlight the influence of IDR-mediated protein-protein interactions on the folding process of complex regulatory RNAs. Together, our findings illustrate how this biomolecular condensate may have emerged in response to prolonged stress and evolved to enable recovery and growth.

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The RNA helicase eIF4A1 regulates chromatin decondensation during mitotic exit

Presenting author:

RWTH Aachen University, Medical School, Institute of Biochemistry and Molecular Cell Biology, Pauwelsstraße 30, 52074 Aachen [DE], rjuehlen@ukaachen.de

Author(s):
Ramona Jühlen, Sabine Wiesmann, Wolfram Antonin

Chromatin undergoes structural changes during cell division. It massively condenses at the beginning of mitosis to enable the segregation of the genome. At the end of mitosis, chromosomes decondense to re-establish their interphase chromatin structure. This process is indispensable for reinitiating transcription and perpetuation of genetic information.

We identified the DEAD-box RNA helicase eIF4A1 as a chromatin decondensation factor. eIF4A1 is known as a translation initiation factor within the eIF4F complex during interphase. Here, it catalyzes the ATP-dependent unwinding of RNA duplexes.

Live-cell imaging of the chromatin revealed that reducing eIF4A1/2 levels in cells slows down chromatin decondensation. Conversely, increasing eIF4A1/2 concentration on mitotic chromosomes accelerates their decondensation.

Mitotic chromosomes are covered by a liquid-like layer of RNAs and proteins, collectively known as perichromatin. Down-regulation of eIF4A1/2 reduces the RNA signal on mitotic chromatin. Moreover, proteins associated with the perichromatin are partially displaced in cytoplasmic foci. We propose that eIF4A1 balances the RNA content of the perichromatin and, thereby, controls chromatin transition from individualized chromosomes to a single nuclear chromatin mass during mitotic exit. To understand chromatin decondensation in more detail, we focus on other RNA helicases as potential chromatin decondensation factors, which seem to play critical but different roles from eIF4A1.

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The folding pathway of SOD1 under cell stress

Presenting author:

Ruhr University Bochum, Faculty of Chemistry and Biochemistry, Biophysical Chemistry, Universitätsstraße 150, 44801 Bochum [DE], linda.sistemich@rub.de

Author(s):
Linda Kartaschew, Sara da Silva Ribeiro, Lukas Johannknecht, Evgeniia Nikitina, Simon Ebbinghaus

Liquid-liquid phase separation (LLPS) drives the formation of stress granules (SGs), which are dynamic condensates critical for cellular stress responses. SG formation has been observed under heat stress which also leads to the unfolding of proteins. Using superoxide dismutase 1 (SOD1), a protein linked to amyotrophic lateral sclerosis (ALS), we investigate how different folding states partition between the cytoplasm and SGs, and how these environments reshape unfolding and aggregation pathways.

In previous studies, we investigated a truncated version of SOD1 (SOD1_bar), showing that destabilized SOD1 mutants with higher hydrophobicity and flexibility exhibit enhanced partitioning to SGs [1]. Here we present results studying monomeric full length SOD1, by confocal microscopy and Fast Relaxation Imaging (FReI). We detect an intermediate state in the unfolding and aggregation pathway that could specifically engage with molecular chaperones and is also prone to aggregation under stress conditions. We also show that Hsp70 chaperones promote faster folding and prevent aggregation.

The comparison of the folding and aggregation pathways of different variants of SOD1 will allow to decipher the role of SGs in hosting and processing amyloidogenic proteins.

[1] N. Samanta, S. S. Ribeiro, M. Becker et al., Sequestration of Proteins in Stress Granules Relies on the In-Cell but Not the In Vitro Folding Stability, JACS 2021 143 (47), 19909-19918, DOI:10.1021/jacs.1c09589

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Characterisation of Neurogenin-3 and its Propensity to Form Condensates

Presenting author:

Institute of Molecular Biology (IMB), 55128 Mainz, , Ackermannweg 4, 55128 Mainz [DE], r.khatter@imb-mainz.de

Author(s):
Radhika Khatter

Neurogenin-3 (NGN3) is a master regulating transcription factor of the basic Helix Loop Helix
family that is both necessary and sufficient for the endocrine lineage commitment in the pancreas.
Through a tightly regulated cascade of gene expression events, NGN3 drives pancreatic progen-
itors to endocrine fate. While its regulatory pathways and transcriptional candidates are well char-
acterized, little is known about its intrinsic biophysical and biochemical properties. Here, we in-
vestigate the molecular features of recombinant NGN3 in vitro, with a focus on its DNA binding
ability and sequence specificity both, qualitatively and quantitatively. Using Electrophoretic Mobil-
ity Shift Assay (EMSA) and streptavidin-biotin pulldown assay, we assess NGN3’s interaction with
short and extended DNA motifs.

We also find that NGN3 exhibits condensate formation behavior under defined conditions. We
characterize this emergent property using Right Angle Light Scattering (RALS) and fluorescence
microscopy across a range of different protein concentrations, salt, pH, and crowding environ-
ments. Interestingly, condensate formation is also enhanced in the presence of DNA, suggesting
DNA-driven condensation.

Furthermore, NGN3 contains intrinsically disordered regions (IDRs), which are frequently asso-
ciated with biomolecular condensate formation and dynamic interactions. Domain-deletion con-
structs targeting these IDRs and clinically reported NGN3 mutants have been generated to iden-
tify sequence determinants essential for DNA binding and condensate formation. These mutants
will be tested systematically.
Collectively, our characterisation of NGN3 aims to reveal how its intrinsic molecular properties
underpin its essential role in driving pancreatic endocrine lineage specification, thereby deepen-
ing our understanding of the mechanisms underlying endocrine cell fate decisions.

References:
[1] Gradwohl et al., Proc. Natl. Acad. Sci. USA, 97, 1607-1611 (2000).
[2] Huang et al., Nature Communications, 9, 4273 (2018).
[3] Banani et al., Nature Reviews Molecular Cell Biology, 18, 285-298 (2017).
[4] Alberti et al., Cell, 176, 419-434 (2019).

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Biomolecular condensation in bacterial rRNA transcription

Presenting author:

EMBL Heidelberg, MSB, Meyerhofstraße 1, 69117 Heidelberg [DE], benjamin.lau@embl.de

Author(s):
Benjamin Lau, Anastasiia Chaban, Kyungmin Baeg, Janosch Hennig, Julia Mahamid, Olivier Duss

Bacterial cells lack membrane-delimited specialized compartments; yet, essential cellular processes rely on subcellular compartmentalization. Recently, it has been shown that the formation of biomolecular condensates through liquid-liquid phase separation plays a crucial role in many key processes within bacterial cells, including ribosomal RNA (rRNA) transcription. In E. coli, RNA polymerases are organized into clusters in vivo, which include nascent rRNA and the rRNA transcription anti-termination complex (rrnTAC).

Here, we show that the rrnTAC proteins together form a biomolecular condensate under near-physiological conditions in vitro. Furthermore, we demonstrate the co-condensation of a minimal rDNA transcription elongation complex within the rrnTAC condensate, mimicking the environment of bacterial rRNA transcription under condensate conditions. We show that the ribosomal protein S4, as part of the rrnTAC, is essential for condensate formation, suggesting that transcriptional condensation is linked with ribosome biogenesis. To monitor these dynamic processes, we are currently developing in vitro single-molecule Fluorescence Microscopy (smFM) approaches to measure co-localization, protein copy numbers, and the rate of transcription initiation at single rDNA templates, providing a powerful tool to monitor condensate formation and function in real-time.

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The molecular basis of natural variation in RNA phase transitions

Presenting author:

Institute of biology Valrose, , 28 avenue Valrose, 06100 Nice [FR], amelie.le-parc@univ-cotedazur.fr

Author(s):
Amelie Le Parc, Asma Sandjak, Karine Jacquet, Arnaud Hubstenberger, Christian Braendle

RNA granules are condensates composed of RNA-binding proteins and translationally repressed RNAs. Although widespread across organisms, their functions remain poorly understood. We study how environmental stress affects RNA granule formation in the oogenic germline of the nematode Caenorhabditis elegans across natural strains. Using a CRISPR-Cas9-engineered strain expressing fluorescent reporters for P-bodies (PUF-5) and stress granules (GTBP-1), we observed consistent granule formation under heat (32°C), cold (6°C), and osmotic stress (500 mM NaCl). Using these experimental settings, we detected significant quantitative variation in granule number and size across a global panel of 12 natural strains. Next, we will perform quantitative trait locus (QTL) linkage mapping to identify polymorphisms contributing to such natural variation, providing insights into the molecular mechanisms of RNA granule formation. In a complementary objective, we aim to decipher the impact of RNA granule formation on oocyte viability. While the three stresses have only weak effects on oocyte viability, preventing P-body formation by RNAi significantly reduced viability at extreme temperatures, indicating that RNA granule formation likely contributes to protecting C. elegans oocytes under thermal stress. We are now extending these experiments to the 12 natural strains to test for correlations between granule formation and oocyte viability across stressful environments.

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β-importin TNPO1 regulates phase separation of ALS-associated proteins by remodeling the dilute phase

Presenting author:

University of Pennsylvania Perelman School of Medicine, Biochemistry and Biophysics, 422 Curie Boulevard, 19104 Philadelphia [US], miriam.linsenmeier@pennmedicine.upenn.edu

Author(s):
Miriam Linsenmeier, Min Kyung Shinn, Thomas Mumford, Lukasz Bugaj, Rohit Pappu, James Shorter

Proteins encompassing RNA recognition motifs (RRMs) and intrinsically disordered prion-like domains (PrLDs) undergo phase separation, forming micron-scale dense phases that coexist with dilute phases above protein-specific saturation concentrations. These proteins also form a hierarchy of other assemblies, including nanoscale clusters in subsaturated solutions and amyloid-like structures. Thus, cells face the challenge of regulating the totality of concentration-dependent assembly processes to ensure the formation of physiologically functional species while avoiding those that drive pathological outcomes.

Here, we demonstrate that β-importin Transportin 1 (TNPO1) regulates clustering and phase separation of ALS-linked proteins FUS, hnRNPA1, and hnRNPA2. It does so by preferentially binding to and remodeling distributions of clusters in subsaturated solutions, thereby weakening driving forces for phase separation through polyphasic linkage effects. Specific high-affinity 1:1 interactions renormalize the pool of self-association-competent cargo molecules. Through this mode-of-action, TNPO1 universally regulates self-assembly, independent of the protein-specific phase separation pathways. Importantly, TNPO1-mediated chaperoning of the ALS-linked FUS-P525L variant is impaired due to weakened interaction with this variant. These findings provide a mechanistic understanding of importin-mediated chaperoning and highlight how disease mutations impair these regulatory mechanisms.

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Competition between stoichiometric protein-RNA clustering and phase separation drives reentrant phase behavior of hnRNPA1

Presenting author:

ETH Zurich, Department of Chemistry and Applied Biosciences, Vladimir Prelog Weg 1, 8093 Zurich [CH], katarzyna.makasewicz@chem.ethz.ch

Author(s):
Katarzyna Makasewicz, Chiara Morelli, Lenka Faltova, Paolo Arosio

Phase separation of RNA-binding proteins (RBPs) is modulated by RNA, including promotion and suppression of phase separation at low and high RNA concentration, respectively. Previous efforts to understand molecular mechanisms behind this phenomenon focused on model systems of positively-charged peptides and oligonucleotides, where suppression of phase separation at high RNA concentration was shown to be due to over-screening of peptide macromolecular charges by RNA binding. However, the molecular origin of reentrant phase separation of full length RBPs was not studied before. In this work, we show that suppression of phase separation of hnRNPA1 at high RNA concentration is driven by competition with stoichiometric protein-RNA co-assembly - a fundamentally different mechanism compared to model peptides. We show that this phenomenon is mediated by the multi-domain architecture of hnRNPA1 and modulated by the strength of protein-RNA interactions. Our results shed light on the behavior of hnRNPA1 in the nucleus and cytoplasm, where both the concentrations and identities of available RNA molecules modulate protein phase behavior. By showing that cluster formation and phase separation are in competition and how it can be modulated, we provide a framework through which the behavior of hnRNPA1 in the cell can be understood in a comprehensive way, which can be advantageous when trying to better understand both its physiological as well as pathological functions.

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Topological confinement by a membrane anchor suppresses liquid-liquid phase separation into protein aggregates: Implications for prion diseases

Presenting author:

Ruhr Universität Bochum, Biochemistry of Neurodegenerative Diseases, MA 2/131, Universitätsstrase 150, 44801 Bochum [DE], Fatemeh.Mamashli@ruhr-uni-bochum.de

Author(s):
Kalpshree Gogte, Fatemeh Mamashli, Maria Georgina Herrera, Simon Kriegler, Verian Bader, Janine Kamps, Prerna Grover, Roland Winter, Konstanze F. Winklhofer, Jörg Tatzelt

Liquid–liquid phase separation (LLPS) of proteins linked to neurodegenerative diseases has been implicated in the initiation of neurotoxic protein aggregation. This study provides compelling evidence that posttranslational modification of the prion protein (PrP) with a glycosylphosphatidylinositol (GPI) anchor plays a crucial role in regulating phase separation. Evidence from human inherited prion diseases and neurodegeneration in transgenic mice points to a critical role of the C-terminal glycosylphosphatidylinositol (GPI) anchor in prion protein (PrP) function. Specifically, the absence of this anchor promotes the formation of neurotoxic and infectious PrP species. Using complementary in vitro and in vivo models, we demonstrate that membrane attachment suppresses LLPS and spontaneous aggregation of PrP. Once detached from the membrane, PrP adopts a misfolded, detergent-insoluble conformation. These findings offer novel insight into how membrane-induced topological confinement modulates phase behavior and shed light on the molecular mechanisms underlying prion disease pathogenesis.

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A label-free method for measuring the composition of multi-component biomolecular condensates

Presenting author:

Leibniz Institut für Polymerforschung Dresden e.V., Polymer Biomaterials Science, Hohe Straße 6, D-01069 Dresden [DE], mccall@ipfdd.de

Author(s):
Patrick M. McCall, Kyoohyun Kim, Anna Shevchenko, Martine Ruer-Gruß, Jan Peychl, Jochen Guck, Andrej Shevchenko, Anthony A. Hyman, Jan Brugués

Many sub-cellular compartments are biomolecular condensates made of multiple components, often including several distinct proteins and nucleic acids. However, current tools to measure condensate composition are limited and cannot capture this complexity quantitatively, as they either require fluorescent labels, which can perturb composition, or can distinguish only 1-2 components. Here, we describe a label-free method based on quantitative phase imaging and Analysis of Tie-lines and Refractive Index (ATRI) to measure the composition of reconstituted condensates with multiple components. We first validate the method empirically in binary mixtures, revealing sequence-encoded density variation and complex aging dynamics for condensates composed of full-length proteins. We then use ATRI to simultaneously resolve the concentrations of five macromolecular solutes in multi-component condensates containing RNA and constructs of multiple RNA-binding proteins. Our measurements reveal an un-expected decoupling of density and composition, highlighting the need to determine molecular stoichiometry in multi-component condensates. We foresee this approach enabling the study of compositional regulation of condensate properties and function.

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Towards an evolutionary model of phase separation–driven ribosome biogenesis

Presenting author:

Max-Planck-Institute for Molecular Genetics, , Ihnestr. 63, 14195 Berlin [DE], katrina.meyer@molgen.mpg.de

Author(s):
Katrina Meyer, Deana Haxhiraj, Marcel Wittmund, Kiersten Ruff, René Buschow, Nadine Brombacher, Guillermo Pérez-Hernández, Melissa Bothe, Edda Schulz, Denes Hnisz, Rohit Pappu, Rainer Nikolay, Matthew Kraushar

In eukaryotes, 80 ribosomal proteins (RPs) must be efficiently targeted to the nucleolus, where they assemble with four rRNAs to form ribosomes. The nucleolus is a prominent biomolecular condensate, characterized by a multilayered liquid-like architecture. Yet, the molecular features that guide RPs to the nucleolus and their evolution remain poorly understood. To address this, from bacteria to humans,
i) we developed a high-throughput screen for subcellular peptide localization. Our method relies on pooled oligonucleotide synthesis, lentiviral delivery, and stable cell line generation to express tiled peptide libraries. This strategy reliably recapitulates known nucleolar localization signals, aligns with computational predictions, and identifies novel nucleolar-targeting sequences in RPs that had not been previously annotated. The data can be mapped back to ribosomal structures, offering insights into how localization motifs relate to ribosome architecture.
ii) we systematically investigated the condensation behavior of RPs in vitro. We developed a method to purify the full set of RPs en masse circumventing the need for individual expression and purification. Using these preparations, we screened buffer conditions (pH, salt concentration, temperature) to determine how environmental parameters influence condensate formation. Our results indicate subunit-specific properties of RPs as suggested by our initial bioinformatic analyses.

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Regulation of protein biosynthesis: biophysical investigation of a repressive mRNP complex

Presenting author:

, , Friedrichstr. 25, 95444 Bayreuth [DE], julia.meyer@uni-bayreuth.de

Author(s):
Julia Meyer, Marco Payr, Andrea Lomoschitz, Kristian Schweimer, Karine Lapouge, Miroslav Krepl, Jiri Sponer, Janosch Hennig

Protein biosynthesis must be highly regulated since dysregulation can have severe consequences. To investigate the mechanism of translation regulation we use the model system Drosophila melanogaster. In female flies, translation repression of msl2 mRNA is essential for the survival of organism, since even traces of the protein Msl2 would lead to hypertranscription of both X chromosomes and death. This repressive mechanism depends on the RBP Sxl, that binds to the 5´UTR and 3´UTR. At the latter, two additional co-repressor RBPs, Unr and Hrp48 are needed. The current model postulates that the complex at the 3´UTR inhibits the recruitment of the 43S PIC to the 5´cap. Our research focuses on the assembly, structure, dynamics and function of this mRNP complex. Previous studies revealed that Sxl and Unr bind cooperatively to msl2. Since only little is known about the interaction between Hrp48 and msl2, NMR and ITC experiments combined with MD simulations and interaction studies of pointmutants were used for characterization. Additionally, we could expand our structrual knowledge about Sxl, and confirmed that it harbors a transient a-helix at the C-terminal end of the RRM domain that is stabilized upon msl2 binding and strengthens complex formation. Next steps will include in vitro experiments in full-length context to gain mechanistic understanding which we can combine with our structural details to get a full picture of the 3´UTR-mediated translation repression mechanism of msl2.

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Trailer Hitch coordinates P-body organization and facilitates transcript-specific mRNA regulation through a nuclear actin-mediated feedback loop

Presenting author:

, , 70 West 82nd St., 10024 New York [US], smilano@gradcenter.cuny.edu

Author(s):
Samantha Milano

Processing bodies (P-bodies) are dynamic, membraneless organelles that mediate mRNA storage, translational repression, and decay. While many of the protein and RNA components of P-bodies have been identified, how these components contribute to the emergent physical state and functions of these condensates remains poorly understood. Here, we identify the RNA-binding protein Trailer Hitch (Tral) as a key regulator of P-body composition and phase state during Drosophila melanogaster oogenesis. Loss of Tral alters P-body architecture, resulting in increased Cup and decreased Me31B levels. This compositional shift is driven by the aberrant release and degradation of twinstar mRNA from P-bodies, resulting in reduced nuclear actin levels which, in turn, trigger transcriptional reprogramming of P-body components. Through super-resolution microscopy, RNAi knockdowns, and chemical treatments, we show that Tral is essential for maintaining P-body physical properties, thus enabling transcript-specific mRNA partitioning. We demonstrate that selective mRNA retention in P-bodies is governed by a network of molecular interactions—including electrostatic forces, hydrophobic contacts, and protein:RNA binding—that are modulated, in-part, by Tral. Together, our findings position Tral as a central coordinator of P-body autoregulation, integrating transcript stability, nuclear actin dynamics, and condensate organization.

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Harnessing PML nuclear bodies to prevent protein aggregation

Presenting author:

Goethe University Frankfurt, Germany, Institute of Biochemistry II, Faculty of Medicine, Theodor-Stern-Kai 7, 60596 Frankfurt [DE], ste.mueller@em.uni-frankfurt.de

Author(s):
Stefan Müller

The ubiquitin proteasome system is a central pillar of cellular protein quality control (PQC) processes and SUMO-targeted ubiquitin ligases (StUbLs) contribute to the clearance of misfolded and damaged proteins in the nucleus. Within the nucleus PQC is spatially regulated and PML bodies are sites where SUMOylated proteins are targeted by StUbLs. The current view is that aberrant, e.g. damaged or misfolded, nuclear proteins are marked by SUMOylation and adressed to PML NBs. Proof-of-concept experiments now demonstrate that targeted recruitment of distinct misfolded proteins, such as TDP-43, to the StUbL machinery in PML NBs is a powerful strategy to limit the formation of pathogenic protein aggregates. We will outline how reprogramming of StUbL signaling can be exploited for cellular proteostasis.

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The interplay of nuclear speckles with SRSF3, ZC3H14, NXF1 license spliced mRNAs for mRNA export.

Presenting author:

Goethe Universität Frankfurt, Germany, RNA Regulation, Max-von-Laue-Str. 13, 60438 Frankfurt am Main [DE], mueller-mcnicoll@bio.uni-frankfurt.de

Author(s):
Michaela Müller-McNicoll, Harsh Oza

A key quality control step in gene expression is the coupling of pre-mRNA splicing to nuclear export, ensuring that only fully processed transcripts are translated. We have previously shown that the splicing factor SRSF3 connects alternative splicing to mRNA export. Here, we examine whether SRSF3 also links mis-splicing to export competence, contributing to splicing surveillance.

Using isoginkgetin, a mild spliceosome inhibitor, we observed widespread intron retention in >14,500 transcripts. These RNAs are polyadenylated but export-incompetent, accumulating in enlarged nuclear speckles alongside hyperphosphorylated SRSF3, while the speckle-resident RNA MALAT1 is displaced. This sequestration is reversible, suggesting temporary retention of mis-spliced transcripts.

Hyperphosphorylated SRSF3 remains bound to RNAs upon splicing inhibition but loses interaction with the export receptor Nxf1 and other adaptors. Here we identify Zc3h14 as a novel splicing-sensitive SRSF3 interactor. Zc3h14 and Nxf1 associate only with hypo-phosphorylated SRSF3 and properly spliced RNAs. iCLIP reveals that Zc3h14 binds alternative exons and 3′UTRs near SRSF3 binding sites, but this binding is lost upon splicing inhibition, while binding to poly(A) tails persists. Our data support a model wherein correct splicing enables SRSF3-dependent recruitment of Nxf1 and Zc3h14, marking mRNAs for export, while mis-spliced transcripts are sequestered in enlarged nuclear speckles likely through increased cohesion.

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Nucleosome sliding in chromatin condensates

Presenting author:

Felix Mülller-Planitz, , MTZ, Phys. Chemistry, Fetscherstraße 74, 01307 Dresden [DE], Felix.Mueller-Planitz@tu-dresden.de

Author(s):
Petra Vizjak, Dieter Kamp, Stigler Johannes, Felix Müller-Planitz

Chromatin folds into dense structures and can undergo phase separation in vitro and in vivo, forming condensates. It remains largely unexplored how chromatin enzymes fulfil their functions in compacted chromatin. The dense environment may prevent local accessibility and fluidity of chromatin creating profound challenges for enzymatic processes. We investigated these challenges using the ISWI remodeling ATPase, which translocates (‘slides’) nucleosomes along DNA. Surprisingly, condensate formation with chromatin fibers did not substantially affect nucleosome sliding rates in vitro. Notably, optical tweezer and FRAP data showed that ISWI remains immobile and stiffens chromatin unless the enzyme can advance through the ATP hydrolysis cycle. ATP hydrolysis therefore powers ISWI’s diffusion through dense condensates and prevents ISWI from affecting mesoscale mechanical properties of chromatin. Molecular dynamics simulations of a ‘monkey-bar’ model in which ISWI grabs onto neighboring nucleosomes, then withdraws from one before rebinding another in an ATP hydrolysis-dependent manner agree with our data. We speculate that ‘monkey-bar’ mechanisms could be shared with other chromatin factors and that changes in the stiffness of chromatin caused by mutations in remodeling enzymes could contribute to pathologies. 

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Rapid depletion and super-resolution microscopy reveal dual roles for SRSF5 in mediating the crosstalk between nuclear speckles and paraspeckles during cellular stress

Presenting author:

Goethe-Universität Frankfurt , , Biologicum Raum 1.223 | Max-von-Laue Str. 13, 60438 Frankfurt am Main [DE], ellen.okuda@biophys.mpg.de

Author(s):
Ellen Okuda, Mara Rudigier, Laurell Kessler, Benjamin Arnold, Mike Heileman, Kathi Zarnack, Michaela Müller-McNicoll

Nuclear speckles (NS) and paraspeckles (PS) are adjacent yet distinct nuclear condensates that undergo stress-induced reorganization. Here, we identify a dual role for the splicing factor SRSF5 in coordinating the crosstalk between both condensates. Super-resolution imaging shows that SRSF5, while enriched in NS, also overlaps with the shell of a subset of PS. SRSF5 binds purine-rich sequences at the 5'end of NEAT1_2 promoting its alignment to PS shells and the formation of large PS cluster during stress. We propose that SRSF5 binding occurs transiently during PS maturation and must later be removed from NEAT1_2 by nuclear helicases. Inhibition of this remodeling by Rocaglamide A, which locks helicases onto purine-rich RNA leads to the aberrant fusion of PS and NS —which can be partially rescued by acute SRSF5 depletion. Surprisingly, while short-term SRSF5 loss impairs PS formation, prolonged depletion activates a feedback loop involving intron retention and premature polyadenylation of TARDBP, reduction of TDP-43 levels and NEAT1_2 isoform switching, ultimately restoring PS clusters. Our findings reveal that SRSF5 serves both architectural and regulatory roles in PS biogenesis and that helicase-mediated remodeling is essential to maintain PS identity and function under stress. These insights uncover fundamental principles of nuclear body dynamics.

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Expression and self-assembly of heterologous bacterial microcompartments in cyanobacteria

Presenting author:

Friedrich-Schiller Universität Jena, Mathias Schleiden Institut, Philosophenweg 12, 07743 Jena [DE], carlos.panerio@uni-jena.de

Author(s):
Carlos Emmanuel Panerio, Julie A. Z. Zedler

Bacterial microcompartments (BMCs) are self-assembling, protein-based structures consisting of an enzymatic core encapsulated by various shell proteins. Specific interactions of these components facilitate the compartmentalization of enzymes and metabolites within the cytoplasm, ostensibly allowing BMCs to act as prokaryotic organelles. Depending on the encapsulated enzyme core, BMCs can be functionally categorized as catabolic, such as in carbon-concentrating carboxysomes, or anabolic, such as in diverse metabolosomes of bacteria. Confining reactions within BMCs can be highly beneficial by (1) increasing the proximity, concentration, and stability of enzymes and substrates, (2) limiting the presence of inhibitory substances (e.g. O2 in carboxysomes), and (3) confining highly reactive or toxic compounds to prevent deleterious effects (e.g. aldehyde intermediate in metabolosomes). Reprogramming BMCs and their cargos has the potential to apply the same benefits for heterologous reactions, allowing the generation of efficient miniature reactors within cell factories. However, engineering BMCs in cyanobacteria can be difficult due to incompatibilities with native carboxysome assembly, and thus heterologous BMC formation must be initially tested in model cyanobacterial species such as Synechococcus elongatus and Synechocystis sp. Here, we provide proof-of-concept for the expression and assembly of Pdu BMC from Citrobacter freundii in model cyanobacteria.

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Phosphorylation of hnRNPA1 modulates the balance between phase separation and aggregation

Presenting author:

ETH Zürich
, Department of Chemistry and Applied Biosciences, Vladimir-Prelog-Weg 1-5/10, 8093 Zürich [CH], marcell.papp@chem.ethz.ch

Author(s):
Marcell Papp, Lenka Faltová, Paolo Arosio

Various RNA-binding proteins can form amyloid aggregates linked to neurodegenerative disorders. In some cases, amyloids are associated with the formation of membraneless organelles, however, the molecular mechanisms governing the interplay between amyloid formation and liquid-liquid phase separation remain poorly understood. An illustrative example is the aggregation of the ALS-related protein hnRNPA1, which forms fibrils following phase separation. In this study, we identify a specific single-point phosphorylation mutant of hnRNPA1 that is capable to suppress aggregation without impacting condensate formation. By combining experimental data with coarse-grained molecular dynamics simulations, we demonstrate that phosphorylation at this residue perturbs interactions between the aggregation-prone region of hnRNPA1 and other segments of the protein that act as a molecular safeguard against aggregation. We validate our model by predicting the effect of other single-point phosphomimetic mutants, which either inhibit or accelerate aggregation. Overall, this work shows a molecular mechanism through which post-translational modifications can perturb the balance between protein phase separation and aggregation.

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Condensation in the trans-Golgi network drives insulin granule biogenesis

Presenting author:

Yale University, School of Medicine, Cell Biology and Internal Medicine, 333 Cedar St, 06510 New Haven, CT [US], anup.parchure@yale.edu

Author(s):
Anup Parchure

Secretory granules (SGs) are specialized organelles in pancreatic beta cells which store and secrete insulin in response to glucose stimulation. Defects in SG biogenesis are associated with type 2 diabetes. At the trans-Golgi network (TGN), the terminal sorting station in the endomembrane system, a key challenge is sorting proinsulin and other SG cargoes away from the constitutive secretory pathway, despite the absence of a known sorting receptor or consensus sorting motif. We find that condensation of Chromogranin proteins is essential for sorting proinsulin within the TGN lumen and initiating SG formation. These proteins act as “drivers” that recruit client proteins like proinsulin into condensates. Disruption of this condensation leads to proinsulin mis sorting, a hallmark of diabetes. We uncover mechanisms that spatially restrict condensation to the TGN, and not earlier in the secretory pathway involving both intrinsic sequence features and the secretory pathway’s pH gradient. A conserved disulfide-bonded omega loop in Chromogranins regulates condensate formation and size, thereby influencing insulin granule properties and quantal release. Together, our work uncovers a receptor-independent, conserved mechanism of protein sorting based on phase separation principles. This paradigm not only advances our understanding of insulin SG biogenesis but also offers broader implications for regulated secretion in other specialized cell types.

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RNAs untangled: Shedding light onto complex structures of long RNAs

Presenting author:

TU Dresden, Physics of Life excellence cluster, Arnoldstraße 18, 01307 Dresden [DE], lukas.pekarek1@tu-dresden.de

Author(s):
Lukas Pekarek, Andreas Hartmann, Fiona Anilkumar, Shashank Shekhar, Fathima Ferosh, Jovana Vasiljevic, Simon Doll, Manthan Raj, Michael Schlierf, Marcus Jahnel

RNA is an intriguing molecule. Despite its relatively simple composition, RNA's functional versatility underscores the crucial role of RNA structure. Proper folding enables distant segments of the RNA molecule to come into close proximity, facilitating essential biological functions. This is particularly critical for long RNAs such as mRNAs, rRNAs, and lncRNAs, which can span over 1000 nucleotides. The function of these RNAs depends critically on their structure and ability to cooperatively interact with RNA-binding proteins, which often contain intrinsically disordered regions prone to condensation. When their structure or function is compromised, the consequences for the cell can be severe.

This raises a fundamental challenge: how do living organisms ensure the robust and accurate folding of long regulatory RNAs? Do biological condensates help to shape RNA structures? And conversely, how do regulatory RNAs influence the formation of these condensates?

In this project, we aim to understand how long RNA molecules fold into their complex structures and what the role of RNA-binding proteins is. We took lncRNA HOTAIR and YBX1 RNA-binding protein as a case study to shed some light on this folding enigma. By employing methods like single-molecule optical tweezers, fluorescence correlation spectroscopy, or confocal microscopy, we want to understand the key aspects of the dynamic folding process of complex RNAs in the context of biomolecular condensates.

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Deciphering Munc13 assembly and dynamics through mass spectrometry

Presenting author:

Johannes Gutenberg - Universität Mainz, Department of chemistry - biochemistry, Hanns-Dieter-Hüsch Weg 17, 55128 Mainz [DE], zahra.riazimand@uni-mainz.de

Author(s):
Zahra Riazimand, Carla Schmidt

Neurotransmission takes place at synapses and involves the precise transfer of information between neurons. This process relies on specialised membrane-less compartments in the pre-synaptic neuron: the synaptic vesicle reserve pool and the active zone. These compartments assemble from specific scaffold proteins and organise the synapse into functionally distinct domains. The active zone controls synaptic vesicle docking and priming. It includes key proteins, that have been found to form condensates and organise voltage-gated Ca²⁺-channels and synaptic vesicles. However, mechanistic and quantitative insights into their assembly and regulation remain limited.

Munc13 is an active zone scaffold protein and, due to its intrinsically disordered regions (IDRs) an interesting candidate for studying dynamic protein interactions. In this study, a variant of Munc13 including the C2A domain linked to an IDR was expressed in E. coli. After purification through an affinity tag, ion exchange and size exclusion chromatography, its identity was confirmed by mass spectrometry (MS). Currently, we are employing different techniques including microscopy and cross-linking MS to investigate condensate formation and protein interactions at residue level. Native MS maintains non-covalent interactions and, therefore, enables the characterisation of proteins and their complexes which will help us gaining insights into the oligomeric state of the protein and the stoichiometry of the assemblies.

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Differential conformational expansion of Nup98-HOXA9 oncoprotein in micro- and macrophases

Presenting author:

Johannes Gutenberg University Mainz, , Hanns-Dieter-Hüsch-Weg 17, 55128 Mainz [DE], hruan@uni-mainz.de

Author(s):
Hao Ruan, Rodrigo Dillenburg, Sina Wittmann, Martin Girard, Edward Lemke

A majority of eukaryotic transcription factors have a block copolymer architecture, with at least one block being a folded DNA interaction domain, and another block being highly enriched in intrinsic disorder. In this study, we focus on Nup98-HOXA9 (NHA9), a chimeric transcription factor implicated in leukemogenesis, in which two FG-repeat-rich IDRs derived from Nup98 get fused to the C-terminal part of transcription factor HOXA9. By integrating experiments and simulations, we examined the structural dynamics of NHA9's FG domain across assembly states. We found that the FG domain has different conformational compactness in the monomeric state, oligomeric, and densely packed condensate state. Notably, the oligomeric state exhibits micelle-like organisation, with the DNA-binding domain exposed at the periphery. While their architecture is non-random, their sizes depend on NHA9 concentration, consistent with non-core-shell spherical micelles. Molecular dynamics simulations support the expansion behaviour of NHA9’s FG domain as oligomeric assemblies grow in size and reveal micelle-like structural features in oligomeric assemblies. These findings offer molecular insight into the phase behaviour of NHA9 and highlight the dynamic conformational transitions of IDRs during condensate formation, with implications for understanding transcriptional regulation in cancer.

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Dissecting the Structural Dynamics of Fused in Sarcoma (FUS) ensembles in different Clusters and Phase-Separated Condensates

Presenting author:

Heinrich-Heine Universität Düsseldorf, Physical Chemsitry, Universitätsstr. 1, 40225 Düsseldorf [DE], Archita.Sarkar@hhu.de

Author(s):
Laura T. Vogel, Titus M. Franzmann, Oleg Opanasyuk, Suren Felekyan, Jan-Hendrik Budde, Tim Loibl, Lize-Mari van der Linden, Giacomo Bartolucci, Archita Sarkar, Christoph A. Weber, Ralf Kühnemuth, Simon Alberti, Claus A.M. Seidel

Significant attention has been given to neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Fronto-Temporal Dementia (FTD), whose increased pathogenesis is linked directly to the biomolecular condensates formed by the Intrinsically Disordered RNA-Binding Protein FUS (Fused in Sarcoma), which forms condensates. However, the conformational behavior and chain dynamics of individual FUS molecules remain poorly understood due to their structural heterogeneity and sensitivity to environmental conditions. Using in vitro biophysical studies based on multiparameter fluorescence detection (MFD), we find that FUS can be best described as a dynamic conformational ensemble. Polarization-resolved FCS (pFCS) measurements show a salt-induced compaction of the protein's hydrodynamic dimensions by approximately threefold. With increasing KCl concentration, the average shape of FUS changes from a prolate to an oblate, and the volume shrinks threefold with a concomitant reduction of the average exchange time from 200-400 µs. These conformational shifts may create a thermodynamically favorable landscape for self-interaction and phase separation, offering mechanistic insights into both functional condensate formation and pathological aggregation.

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Plant Organellar RNA-Editing - In a Condensed Phase ?

Presenting author:

Humbold-Universität zu Berlin, , Genter Str. 68, 13353 Berlin [DE], frederik.saulich@hu-berlin.de

Author(s):
Frederik Saulich

Plant organellar RNA-editing is the conversion of specific cytidines to uridines via deamination. While the exact role of RNA-editing in organellar gene regulation remains unknown, a variety of associated essential or supportive nuclear-encoded factors have been identified by forward and reverse geetic approaches. The current mode of action model for organellar RNA editing, suggests an editosome composed of different combinations of known RNA-editing factors. However, how these rather lowly expressed factors assemble into a complex in the crowded organellar environment remains unclear. Here the process of phase separation into a condensate, to concentrate the required factors, could be a potential explanation. One such factor is MORF8, described as a dually targeted scaffold protein affecting a multitude of mitochondrial and several plastid RNA-editing sites. MORF8 has a protein interaction domain called the morf-box and a long c-terminal intrinsically disordered region, which contains a predicted prion like domain (PLD). We hypothesize that this PLD is key for condensate formation of MORF8 and investigate its phase separation characteristics in vitro. We find that MORF8 forms solid-like condensates autonomously in a concentration-, PEG-, and pH-dependent manner. Further genetic complementation studies suggest a role of the PLD for multiple mitochondrial and few plastid RNA editing events.

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De novo design of peptides localizing at the interface of biomolecular condensates

Presenting author:

ETH Zurich, D-CHAB, Vladimir-Prelog-Weg 1, 8049 Zurich [CH], timo.schneider@chem.ethz.ch

Author(s):
Timo N. Schneider, Marcos Gil-Garcia, Marco A. Bühler, Lucas F. Santos, Lenka Faltova, Gonzalo Guillén-Gosálbez, Paolo Arosio

The interface of biomolecular condensates has been shown to play an important role in processes such as protein aggregation and biochemical reactions. Targeted modulation of these interfaces could, therefore, serve as an effective strategy for engineering condensates and modifying aberrant behaviors. However, the molecular grammar driving the preferential localization of molecules at condensate interfaces remains largely unknown. In this study, we developed a computational pipeline that combines coarse-grained simulations, machine learning, and mixed-integer linear programming to design peptides that selectively partition at the interfaces of specific condensate targets. Using this workflow, we designed and synthesized peptides that localize at the interfaces of condensates formed by the intrinsically disordered protein regions (IDRs) of hnRNPA1, LAF-1, and DDX4. These peptides exhibit surfactant-like architectures, with one tail incorporated into the condensate and the other excluded from the dense phase. The distinct peptide sequences highlight the importance of the net charge of the scaffold protein as a key physicochemical parameter for designing peptides with preferential interfacial localization. Overall, our pipeline represents a promising strategy for the rational design of interface-localizing peptides and the identification of the corresponding molecular grammar.

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Stress-dependent regulation of a liquid droplet component, Rbfox1

Presenting author:

Medizinische Hochschule Hannover (MHH), Institute for Cell Biochemistry, Carl-Neuberg-Straße 1, 30625 Hannover [DE], Schumann.Nils@mh-hannover.de

Author(s):
Nils Schumann

The RBFOX family of proteins is a family of RNA-binding proteins, first identified as splicing factors. A well-conserved RNA recognition motif (RRM) and multiple low-complexity domains (LCDs) characterize them.

Due to the conservation of these proteins across species, we have used Drosophila melanogaster as a model to study the functions of individual domains. To this end, we employed CRISPR/Cas9 to remove both the RRM domain and one LCD, which is predicted to form a coiled-coil (CC) structure, from Rbfox1. These mutant proteins were then studied for their ability to form RNP granules in-vivo during stress using the Drosophila ovary as a model system. Preliminary results from these experiments suggest that removing the RRM might be sufficient to alter the three-dimensional structure of granules, while removing the CC domain hinders Rbfox1 inclusion into RNPs. These results indicate that the CC domain may be required for efficient phase separation.

Additionally, the CC domain was found to have previously uncharacterized functions in facilitating protein-protein interactions during Notch signaling. Loss of the CC domain leads to dysregulation of the Notch pathway and results in phenotypic abnormalities in the ovarian stem cell niche. These findings highlight a novel regulatory mechanism through which Rbfox1 modulates Notch signaling.

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AGO1, a Key miRNA Protein, Helps Cells Grow by Acting in the Nucleolus

Presenting author:

Medizinische Hochschule Hannover (MHH), Institut für Zellbiochemie, Carl-Neuberg-Str. 1, 30625 Hannover [DE], Shcherbata.Halyna@mh-hannover.de

Author(s):
Halyna Shcherbata

Environmental changes trigger the formation of dynamic RNA-protein assemblies, or RNP granules, in both the cytoplasm and nucleus. These membrane-less compartments arise through liquid-liquid phase separation of RNA-binding proteins with intrinsically disordered regions, preceding major transcriptional reprogramming. Our lab previously showed that microRNAs (miRNAs) modulate stress responses and that miRNA biogenesis is highly stress-sensitive. Since Argonaute1 (AGO1)—the core component of the RNA-induced silencing complex (RISC)—is essential for miRNA function, we hypothesized that AGO1 behavior may change under stress. Using Drosophila S2 cells and oogenesis models, we found that AGO1 relocates under stress to cytoplasmic (stress granules, processing bodies) and nuclear (Cajal bodies, nucleolus) RNP granules. Notably, AGO1 consistently colocalizes with Fibrillarin in the nucleolus, suggesting a potential role in ribosome biogenesis. Small RNA sequencing revealed that AGO1 binds various small RNAs, especially C/D box snoRNAs, which guide rRNA modification. Fibrillarin, AGO1’s nucleolar partner, is a core component of the C/D box small nucleolar RNP (snoRNP) complex required for pre-rRNA cleavage and rRNA modification. Given that nucleolar size reflects rDNA transcription and protein synthesis capacity, our findings support a model where AGO1, together with Fibrillarin, regulates rRNA processing and nucleolar size, thereby controlling protein synthesis and cell growth.

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Biomimetic Minimal Peptide Coacervates for Biocatalysis

Presenting author:

Max-Planck Institute for Polymer Research, , MPIP, Ackermannweg 10, 55128 Mainz [DE], sis2@mpip-mainz.mpg.de

Author(s):
Tsvetomir Ivanov, Shutian Si, Katharina Landfester

Biomolecular condensates play a critical role in the transmission of cellular functions and the regulation of biological processes within cells. In this study, we have developed a minimalist design of dipeptide coacervates and photocatalytic monopeptide coacervates that are capable of performing bio-organic reactions in an aqueous medium.The dynamic self-assembling coacervate system demonstrates enhanced stability, catalytic performance, and hydrophobic characteristics. The hydrophobic microenvironment facilitated the efficient partitioning of hydrophobic species and the incorporation of a variety of substrates for organic chemical reactions, thereby enhancing their efficiency in compartmentalized aqueous environments.The presence of photocatalytic monomers in the coacervate structure enabled the execution of redox reactions, resulting in aldehyde formation, obviating the necessity for an external catalyst, such as enzymes or additional organic molecules. The partitioning of hydrophobic cargos resulted in an augmentation of the size of the coacervate droplets due to efficient loading. Upon initiation of chemical conversion by light, the size of the droplets returned to the initial state.The development of coacervates with intrinsic photocatalytic activity and a hydrophobic environment represents a novel approach to catalysis in the domain of bio-inspired materials. This advancement holds significant potential for applications in synthetic biology,organic chemistry,catalysis.

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Kinetic Analysis of Biomolecular Condensate Nucleation Using Droplet Microfluidics

Presenting author:

University of Basel, Biozentrum, Spitalstrasse 41, CH-4056 Basel [CH], matej.siketanc@unibas.ch

Author(s):
Matej Siketanc, Jonas Keller, Miriam Linsenmeier, Maria Hondele

Biomolecular condensates represent a dynamic and adaptive mode of cellular organization. Both in vitro and in-cell studies have shown that their behavior is highly sensitive to chemical conditions, rapidly responding to changes in pH, temperature, salt concentration, and protein levels. However, the kinetics of condensate formation, particularly during the initial nucleation phase, remain poorly understood due to the limited temporal resolution of conventional techniques [1].
Droplet-based microfluidics offer a powerful solution, enabling precise control over experimental conditions and real-time observation of liquid–liquid phase separation (LLPS) in picoliter- to nanoliter-scale compartments [2 & 3]. Recent developments show that this method can yield complete phase diagrams and kinetic parameters, bridging thermodynamic properties with condensate formation dynamics [4].
We combined a microfluidics platform with a dual-camera microscopy system, enabling high-temporal-resolution analysis of condensate kinetics across sub-second to minute timescales. This robust setup lays the foundation for future quantitative studies of condensate nucleation and growth under physiologically relevant conditions.

References:
[1] Alberti et al., 2019
[2] Linsenmeier et al., 2022
[3] Arter et al., 2022
[4] Villois et al., 2022

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Selective ion binding and uptake shape the microenvironment of biomolecular condensates

Presenting author:

Radboud University (IMM), Nijmegen, Physical Organic Chemistry, Heyendaalseweg 135, 6525 AJ Nijmegen [NL], iris.smokers@ru.nl

Author(s):
Iris Smokers, Mazdak Khajehpour, Evan Spruijt

Biomolecular condensates modulate various ion-dependent cellular processes and can regulate subcellular ion distributions by selective uptake of ions. To understand these processes it is essential to uncover the molecular grammar governing condensate-ion interactions. In this work, we use NMR spectroscopy of ions and model condensate components to quantify and spatially resolve selective ion binding to condensates and show that these interactions follow the “law of matching water affinities”, resulting in strong binding between proteins and chaotropic anions, and between nucleic acids and kosmotropic cations. Ion uptake into condensates directly follows binding affinities, resulting in selective uptake of strong-binding ions, but exclusion of weak-binding ions. Ion binding further shapes the condensate microenvironment: it remodels the phase diagram by effectively neutralizing charges and altering the condensate composition, it modulates condensate viscosity and can even flip their interface potential. Such changes can have profound effects on biochemical processes taking place inside condensates, as we show for RNA duplex formation. Our findings provide a new perspective on the role of condensate-ion interactions in cellular bio- and electrochemistry and the driving forces behind small molecule uptake into condensates and may aid design of condensate-targeting therapeutics.

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Spatial organization of protein quality control by SUMO-Ub networks

Presenting author:

Universitätsklinikum Frankfurt, IBCII, Sandhofstrasse 2-4, 60528 Frankfurt (Main) [DE], T.Stark@em.uni-frankfurt.de

Author(s):
Tabea Stark, Jan Keiten-Schmitz, Kristina Wagner, Stefan Müller

There is accumulating evidence that spatial organization of protein quality control in membrane-less organelles enables cells to cope with protein misfolding in distinct cellular compartments. This is exemplified by stress granules (SGs), promyelocytic leukemia (PML) nuclear bodies (NBs) and nucleoli that are critically involved in compartmentalizing proteostatic pathways in the cytosol and nucleus, respectively. Importantly, we recently found that the ubiquitin-like SUMO system regulates the interplay and crosstalk of cytosolic and nuclear protein quality control systems. We demonstrated that the nuclear SUMO-targeted ubiquitin ligase (StUbL) pathway which is associated with PML NBs contributes to proteotoxic stress resilience by regulating the dynamics of cytosolic SGs. In our current work, we explore how the SUMO system is intertwined with protein quality control in the nucleolus. In this context we will present data on SUMO-dependent nucleolar compartmentalization and clearance of defective ribosomal products (DRiPs). We further characterized how nucleophosmin 1 (NPM1), the principle organizer of nucleoli, contributes to balanced proteostasis.

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Partial demixing of RNA Polymerase II condensates in the nucleus

Presenting author:

, , , [0], lstelzl@uni-mainz.de

Author(s):
Lukas Stelzl, Arya Changiarath, Jan Padeken, Rosa Herrera Rodriguez, Jasper Michels

Phase separated condensates could spatio-temporally fine tune RNA polymerase II behavior during two key stages, transcription initiation and the elongation of the nascent RNA transcripts. However, it has remained unclear whether these two condensate would mix when present at the same time or would remain distinct chemical environments. To understand whether and how RNA Polymerase II condensates could modulate transcription regulation, we combined particle-based multi-scale simulations and experiments in the model organism C. elegans. Simulations and in vivo experiments describe a lower critical solution temperature (LCST) behavior of RNA Polymerase II, with condensates dissolving at lower temperatures whereas higher temperatures promote condensation. Importantly condensation correlates with an incremental transcriptional response to temperature but is largely uncoupled from the classical heat stress response. Importantly, we show in simulations how the degree of phosphorylation of the disordered CTD, which is characteristic for each step of transcription, controls demixing of CTD and pCTD. Depending on system composition, we observe full or partial engulfment of CTD by pCTD. Remarkably, we observe such full and partial engulfment of RNA polymerase II condensates by phosphorylated RNA polymerase II by super resolution microscopy of C. elegans embryos. Taken together our results suggest a role of partially demixed condensates in transcription initation and elongation.

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Transcription Factor Condensation on Chromatin

Presenting author:

Institute of Molecular Biology (IMB), 55128 Mainz, Wittmann group, Ackermannweg 4, 55128 Mainz [DE], l.thews@imb-mainz.de

Author(s):
Leonhard Thews

Pioneer transcription factors (PTFs) are critical regulators of cell fate decisions due to their
ability to bind condensed chromatin and initiate local decompaction. Recent studies (Ji et al.
Mol Cell, 2024) suggest that the phase separation of PTFs into condensates may be essential for
their chromatin-opening function, but the underlying biophysical mechanisms remain poorly
understood. This project aims to investigate how PTF condensates interact with chromatin and
influence nucleosome stability and chromatin accessibility. Using reconstituted chromatin
assembled from fluorescently labeled histone octamers and λ-DNA, nucleosome positioning
sequences (NPSs) or native enhancer sequences, we apply single-molecule assays - including
optical tweezers - to quantify PTF-induced chromatin remodeling. Preliminary force-extension
measurements reveal characteristic nucleosome unwrapping patterns in reconstituted λ- and
NPS-chromatin. Upcoming experiments will compare chromatin opening in the presence and
absence of PTF condensates to assess their potential to destabilize chromatin structure. To this
end, the PTFs Klf4, FoxA1, and Sox2 are currently being produced and tested for their phase
separation capabilities. Ultimately, a comparative analysis of wild-type, MBP-tagged, and
condensation-deficient PTF variants will elucidate the specific contribution of phase separation
to chromatin interaction.

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ADP-RIBOSYLATION OF FUS IN THE DNA DAMAGE REPAIR RESPONSE

Presenting author:

JGU Mainz, IMP, Hanns-Dieter-Hüsch weg 15-17, 55128 Mainz [DE], ftiryaki@uni-mainz.de

Author(s):
Fatmanur Tiryaki, Orsolya Leidecker, Ivan Matic, Dorothee Dormann

FUS is a DNA/RNA-binding protein genetically linked to ALS and FTD, forming insoluble protein aggregates in these disorders. In healthy cells, FUS primarily localizes to the nucleus, where it regulates DNA/RNA-related processes such as DNA damage repair (DDR), transcription, splicing, and mRNA transport. It can undergo liquid-liquid phase separation (LLPS) and localize to nuclear and cytosolic condensates, including DNA damage sites and stress granules. Proteomics and in vitro studies suggest FUS is ADP-ribosylated by PARP1, and that both proteins are important for DDR. However, it remains unclear whether and when FUS is ADP-ribosylated during DDR, and how this modification influences FUS phase separation and function. We show that FUS undergoes ADP-ribosylation in vitro, promoting the dissolution of FUS condensates. Using an in vitro system modeling DDR foci, we found PARP1 and FUS form immiscible condensates with dsDNA. Upon NAD⁺ addition, PARP1 becomes active, leading to fusion of PARP1/DNA and FUS droplets. Progressive ADP-ribosylation causes both condensates to dissolve. We also observed basal FUS ADP-ribosylation in cells, which increases upon oxidative stress in a PARP1-dependent manner. Our findings reveal how ADP-ribosylation regulates FUS dynamics and contributes to DNA repair foci resolution.

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mRNA condensation, translation and decay control in oogenesis

Presenting author:

Institute of biology Valrose, Sciences Nat., 28 avenue Valrose, 06100 Nice [FR], florian.valero@univ-cotedazur.fr

Author(s):
Florian Valero, Sarah Ouertani, Arnaud Hubstenberger

The fate of mRNAs is governed by two intricate and compartmentalized pathways: translation and decay. Translationally repressed mRNAs accumulate in condensates, whereas actively translated mRNAs remain dispersed. While the link between translation control and decay remains debated, the influence of condensation on this connection is even less understood. We investigated this relationship during oogenesis, a context in which

transcription is silenced and gene expression is regulated exclusively at the post-transcriptional level. In C. elegans, oocytes are arranged by maturation stage, providing a system to examine the spatiotemporal dynamics of mRNA control (Cardona et al., Cell 2023). Using single-molecule imaging of endogenous mRNAs, we quantified changes in mRNA copy numbers to infer half-lives and simultaneously measured the proportion of mRNAs in condensed versus soluble states. Our quantitative analysis of mRNAs with opposing expression profiles across the cell cycle supports a model in which active translation sensitizes mRNAs to RNAi-mediated decay. However, genetic manipulations that uncouple translational repression from condensation reveal that condensation does not enhance protection of repressed mRNAs from RNAi, but may instead safeguard them from an unidentified decay pathway. Altogether, without relying on pharmacological treatments nor artificial reporter constructs, we establish quantitative relationships among mRNA translation, condensation, and decay.

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Investigating the role of FUS/EWSR1::TFCP2 fusion proteins on condensate formation and transcriptional regulation

Presenting author:

German Cancer Research Center (DKFZ), Heidelberg, Germany. Faculty of Biosciences, Heidelberg University, Heidelberg, Germany., B066 - Chromatin Networks, Im Neuenheimer Feld 267, 69120 Heidelberg [DE], claire.vargas@dkfz-heidelberg.de

Author(s):
Claire Vargas, Sina Jasmin Wille, Stefan Fröhling, Claudia Scholl, Karsten Rippe

Fusion proteins (FPs) drive ~16.5% of cancers, especially in pediatric malignancies. The recently discovered FUS/EWSR1::TFCP2 fusions in aggressive rhabdomyosarcoma subtype combine intrinsically disordered regions from FUS and EWSR1 with the TFCP2 transcription factor, resulting in the overexpression of ALK and shortened TERT oncogenes (Schöpf et al., Nat. Comm. 2024). However, the functional impact of the assembly of TFCP2 fusions into micrometer-sized “onco-condensates” remains unknown. We conducted a spatial transcriptomics analysis of FUS/EWSR1::TFCP2 condensates on patient tissue sections and examined the formation of onco-condensates using immunostaining. This was complemented by studies on cell line models, where we observed heterogeneous assembly properties and evaluated the transcription activation capacity of FPs. Both FUS/EWSR1::TFCP2 fusions and TFCP2 demonstrated limited ability to activate transcription. This, along with the heterogeneous morphology and cellular localization of these assemblies, indicates a more complex oncogenic mechanism than simply enhancing the transcriptional activation potential of TFCP2. Our work highlights that combining spatial transcriptomics with visualization of onco-condensates offers a powerful approach to gain new insights into the mechanism by which aberrant assembly properties of fusion proteins drive tumorigenesis.

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Interaction proteomics display the spatio-temporal dynamics of mitochondrial RNA granules

Presenting author:

Institute of Molecular Systems Medicine, , University Hospital Building 75 - Theodor-Stern-Kai 7 , 60590 Frankfurt (Main) [DE], vignane@med.uni-frankfurt.de

Author(s):
Melinda Brunstein, Thibaut Vignane, Christian Münch

Over the past decade, protein phase separation has emerged as a cornerstone of biological processes and stress regulation. While nuclear and cytoplasmic condensates are primarily studied, those located within organelles are largely overlooked. Mitochondrial RNA granules (MRGs) are one such condensate, proposed to coordinate mitochondrial RNA metabolism, yet their composition, dynamics, and regulatory mechanisms remain poorly understood. Here, we used TurboID-based proximity labeling coupled with 20 known MRG-associated proteins, enabling in situ mapping of transient and low-affinity interactions within physiological context. This approach identified a high-confidence interaction network comprising over 1,700 protein-protein interactions. Our results suggest that MRGs are structured condensates enriched in proteins involved in RNA processing and translation. We further demonstrated that inhibition of mitochondrial transcription triggers MRG disassembly, while elevated double-stranded RNA levels—resulting from impaired RNA degradation—stabilize them. Together, our findings establish MRGs as dynamic condensates with temporally adaptable functions that respond to mitochondrial transcription and RNA turnover, thereby spatially coordinating transcription, RNA processing, and translation. This work provides a new framework to investigate MRGs’ roles in mitochondrial function, stress response and disease.

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Profiling Phase-Separated Condensates by Proximity Biotinylation

Presenting author:

Eberhard Karls Universität Tuebingen , Interfakultäres Institut für Biochemie (IFIB) Universität Tübingen Verfügungsgebäude (IFIZ) , Auf der Morgenstelle 15, 72076 Tübingen [DE], sarah.wasilewski@uni-tuebingen.de

Author(s):
Daniel Hofacker, Sarah Wasilewski, Thorsten Stafforst

Membrane-less organelles such as nucleoli, stress granules and nuclear speckles are dynamic biomolecular condensates that compartmentalize key cellular functions including ribosome biogenesis, mRNA regulation during stress and splicing regulation. Their formation and function is often orchestrated through RNA-protein interactions, many of which remain unexplored. Traditional approaches to map these interactions inside the cell typically rely on genetic manipulation, limiting their applicability.

We introduce a method that enables the in situ mapping of RNA-protein interactions without the need for genetic manipulation. The technique harnesses modified oligonucleotide probes complementary to an RNA-of-interest which allow the recruitment of a biotin ligase, enabling proximity-based biotinylation of nearby proteins. These proteins are then identified through mass spectrometry. The method is easily adaptable to new targets, it operates effectively with relatively low cell numbers and with its high sensitivity and low background, it is suitable even for slowly growing cell types.

With this technique we identified the interactome of specific lncRNAs that reside in phase-separated nuclear condensates, successfully distinguishing their unique protein landscapes. Ongoing work applies this strategy to dissect the molecular composition of newly identified speckles, highlighting its potential to advance our understanding of RNA biology and uncover previously hidden regulatory networks.

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Assessing the contribution of phase separation and HP1α in organizing heterochromatin domains

Presenting author:

, , Im Neuenheimer Feld 267, 69120 Heidelberg [DE], r.weinmann@dkfz-heidelberg.de

Author(s):
Robin Weinmann

Chromatin self-organizes into silenced and active subcompartments on the mesoscale of 0.1-1 µm. Liquid-liquid phase separation (LLPS) has been proposed as a mechanism driving this process, but it is frequently not clear if it occurs under endogenous conditions (Rippe 2022, CSH Perspect Biol). We addressed this question for pericentric repeat sequences, which can cluster into condensed heterochromatic foci termed chromocenters and for which an organization by LLPS of HP1a has been proposed, but our previous work found no supporting evidence in mouse fibroblasts (Erdel 2020, Mol Cell). To further dissect the role of HP1a, we developed DART (dCas9 Activator Recruitment Toolbox) and COBRA (Co-Binding Reporter Array). DART and super-resolution microscopy revealed that chromocenters are organized in subclusters which mostly disperse upon activation. HP1a localized in ~50 nm foci which were broadly distributed but enriched at chromatin. Charge changes from histone acetylation or increased RNA levels facilitated chromocenter decondensation, even in presence of HP1a and H3K9me3. HP1a localization and binding dynamics remained largely unaffected by chromocenter perturbation. Instead, COBRA experiments characterized HP1a as a direct transcriptional repressor when promoter-proximal. Our findings support a model in which chromocenters consist of repeat units that switch between active and repressed states through localized interactions, without contribution of HP1a phase separation.

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Visualizing the Interaction of genes with Transcriptional Condensates during Embryonic Lineage Specification

Presenting author:

Karlsruhe Insitute of Technology (KIT), Institute of Biological and Chemical Systems, Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen [DE], mona.wellhaeusser@kit.edu

Author(s):
Mona Wellhäusser, Irina Wachter, Maria Panarisi, Alicia Günthel, Lennart Hilbert

Stem cells exhibit prominent, long-lived transcriptional condensates that are implicated in long-range contacts between genes and regulatory elements contributing to embryonic development. While rapid changes in the interaction of these condensates, regulatory elements, and developmental genes are seen while stem cells differentiate, the interplay of these components remains poorly understood. Here, we combine DNA-sequence-specific oligopaints and super-resolution microscopy to study gene-condensate interaction in the rapid induction of mesendodermal fate in zebrafish embryos. We apply the recombinant signaling protein activin A to induce the Nodal pathway, causing transcriptional response from mesendodermal genes within 30 minutes. Using STED super-resolution imaging and expansion microscopy, we aim to reveal Nodal-induced changes in nanoarchitecture and molecular composition of transcriptional condensates. Integrating DNA‑oligopaints, we further plan to monitor the 3D interaction of the induced genes and their associated regulatory regions (super-enhancers) with the transcriptional condensates. The planned combination of methods provides a controlled system to analyze condensate dynamics and condensate-gene interactions in the induction of an embryonic gene expression program. Ultimately, our goal is to reveal structural mechanisms underpinning precise transcriptional control in vivo, shedding light on how biomolecular condensates guide early developmental gene regulation.

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High-resolution in situ imaging reveals how a point mutation reshapes condensate material properties

Presenting author:

Max Planck Institute of Biophysics, Mechanisms of Cellular Quality Control, Max-von-Laue-Str. 3, 60438 Frankfurt am Main [DE], florian.wilfling@biophys.mpg.de

Author(s):
Florian Wilfling

Biomolecular condensates are critical for the spatial organization of many cellular processes. However, few biomolecular condensates have been visualized in their native state, limiting our understanding of condensate behaviour in vivo. Here we applied a correlative cryo-electron tomography pipeline to target S. cerevisiae Ape1 (aminopeptidase-1) complexes, a canonical target of selective autophagy. Using high-confidence template matching and subtomogram averaging, we resolved the first in situ structure of the 680kDa Ape1 dodecamer at 3.94Å resolution. Analysis of particle distributions revealed that Ape1 complexes are liquid-like assemblies. Furthermore, we were able to quantify striking changes in the material properties of Ape1 complexes upon introduction of a single point mutation. Tomography data was additionally used to inform molecular dynamics simulations, providing further insight into the organization and dynamics of the Ape1 complex. Our results demonstrate that cryo-electron tomography combined with high-confidence template matching is a powerful method for studying condensates within cells at molecular resolution, providing insights into the biophysical principles governing biological processes such as selective autophagy.

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The Relationship between LLPS of Transcription Factor and Transcriptional Activity

Presenting author:

University of Science and Technology of China, Department of Chemical Physics, No. 96 Jinzhai Road, Baohe District, 230026 Hefei [CN], sxakwjx@mail.ustc.edu.cn

Author(s):
Jiaxin Wu, Zhonghuai Hou

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Biomolecular condensates in virus-host interaction: interplay between stress granules and virus replication condensates

Presenting author:

, , YUNGU CAMPUS No. 600 Dunyu Road, Xihu District, 310030 HANGZHOU [CN], yangpeiguo@westlake.edu.cn

Author(s):
PEIGUO YANG

It has long been recognized that the intracellular replication of alphaviruses critically relies on several key host RBPs, including G3BP1/2 and FXR1/FXR2/FMR1, but how these RBPs modulate alphaviral replication and whether it would be possible to target these RBPs for antiviral treatment are less explored. Here, using SFV as a model, we report that SFV nsP3 exploits G3BP for its condensation and transforms antiviral stress granules into proviral nsP3-G3BP co-condensates. The gel-like co-condensates of nsP3 and G3BP enrich and protect viral genomic RNAs from host RNase degradation and serve as viral translational hubs to promote viral replication. The mode of nsP3-RBP co-condensation is prevalent across alphaviruses, and disruption of nsP3 condensates is an efficient antiviral approach. Thus, these findings uncover a general anti-alphavirus strategy based on the conserved reliance of nsP3-RBP co-condensation.

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Phase behaviour of RNA-binding proteins and the key mutations that drives it

Presenting author:

Johannes Gutenberg University of Mainz, Institute of Physics, Staudingerweg 9, 55099 Mainz [DE], mahesh.yadav@uni-mainz.de

Author(s):
Mahesh Yadav

Biomolecular condensates are phenomena that emerges through liquid-liquid phase separation of disordered proteins. In this work, we study one such protein known as Fused in Sarcoma (FUS), a multi-domain protein with regions rich in arginine and glycine residues, referred to as RG-rich domains, which are involved in a wide range of essential cellular processes. Unique sequence patterns such as RGGRGRGG...RGRGGGRGG.. allow FUS to interact with itself and with nucleic acids. The role of these repeats in phase separation can be significantly diminished by point mutations e.g., RtoK, RtoA, which disrupt the naturally occurring pattern. Although lysine carries a positive charge similar to arginine, it lacks the guanidine group, limiting its interaction network. The alanine substitution completely breaks the interaction network and impair the FUS’s ability to form droplets. Beyond phase separation, we also investigate the time-dependent viscoelastic properties of FUS condensates as altered by mutations. In many cases, condensates that exhibit liquid-like (Newtonian fluid) behavior transition into a non-Newtonian regime known as a Maxwell fluid, which exhibits elastic characteristics. From application point of view, Non-Newtonian condensates may act as a pro- tein reservoirs that accelerate biochemical reactions. On the other hand, elastic or gel-like protein-RNA condensate could provide structural support to shape chromatin organization in the nucleus.

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Probing scaffold–client interactions within biomolecular condensates using coarse-grained molecular dynamics simulation

Presenting author:

, , 3-14-1, Hiyoshi, Kohoku-ku, , 223-8522 Yokohama, Kanagawa [JP], ikki8638@keio.jp

Author(s):
Ikki Yasuda, Eiji Yamamoto, Kenji Yasuoka, Kresten Lindorff-Larsen

Biomolecular condensates function as a compartmentalization mechanism of biomolecules without membranes, playing important roles in the regulation of cellular activities. While various types of biomolecular condensates exist in cells, they can selectively partition specific molecules. This is facilitated by the physicochemical properties of both scaffold and client molecules, but the underlying molecular mechanisms remain not fully understood. In this work, we aim to clarify the molecular partitioning mechanisms of condensates mediated by intrinsically disordered regions. Using coarse-grained molecular dynamics simulations that partially capture chemical differences of amino acids and nucleotides, we study the differential partitioning in two types of condensates, hydrophobic-residue-rich and charged-residue-rich condensates. We elucidate the relationship between partitioning and molecular interaction energies, and subsequently develop a sequence-based model to predict the partitioning.

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Tuning TDP-43 condensation behavior to understand the physiological relevance of TDP-43 phase transitions

Presenting author:

Johannes Gutenberg University Mainz, , Hanns-Dieter-Hüsch-Weg 15, 55128 Mainz [DE], yezadoro@uni-mainz.de

Author(s):
Yelyzaveta Zadorozhna, Federico Uliana, Dorothee Dormann

TDP-43 is an RNA-binding protein with important roles in RNA metabolism. It is commonly found in inclusions in patients affected by several neurodegenerative disorders, including ALS, FTD and AD. TDP-43 can undergo phase separation (PS), and partition into biomolecular condensates in cells. Dysregulation of this process is believed to favor the formation of pathological aggregates, and potentially contribute to disease progression. PS of TDP-43 most likely has physiological importance; however, it remains unclear which RNA regulatory functions of TDP-43 might require its ability to form condensates.

We designed a panel of TDP-43 PS mutants exhibiting different propensities to undergo PS, and to form condensates of different material properties. We characterize these mutant proteins using in vitro phase separation assays as well as in HeLa cells by expressing them in an inducible manner in the absence of endogenous TDP-43. We show that TDP-43 PS behavior can be tuned in vitro and in cells by specific mutations. By applying immunoprecipitation coupled to mass spectrometry to our cellular models, we identify that PS-prone mutants exhibit a global increase in protein interactors. We further validate potentially affected TDP-43-regulated functions using genomic approaches. Our preliminary results shed light on the link between TDP-43 condensation status and its roles in RNA processing, which are often dysregulated in disease.

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Heterochromatome-wide analyses reveal MBD2 as a phase separation scaffold for heterochromatin compartmentalization and composition

Presenting author:

TU Darmstadt, Department of Biology, Schnittspahnstr. 10, Darmstadt, 64287 Darmstadt [DE], hui.zhang@tu-darmstadt.de

Author(s):
Hui Zhang, Enes Ugur, Christian Hake, Frederik Lermyte, Heinrich Leonhardt, M. Cristina Cardoso

Heterochromatin is essential for nuclear integrity, genome stability, and gene regulation. However, the mechanisms governing heterochromatin compartmentalization remain poorly understood. Here, we integrated quantitative spatial proteomics, phase separation assays, and phase separation prediction tools to identify and characterize candidate phase separation scaffold proteins involved in heterochromatin compartmentalization. We in vitro reconstituted phase-separated heterochromatin condensates using heterochromatin fractions isolated from mouse brain. Mass spectrometric analysis yielded around 1000 proteins within them from which 250 were predicted to have scaffold phase separation properties using machine learning-based phase separation protein prediction tools. From these, 20 proteins, including methyl-CpG binding domain protein 2 (MBD2), were localized to pericentric heterochromatin compartments using gene ontology annotation. We demonstrated that MBD2 undergoes liquid-liquid phase separation via coiled coil-mediated homo-oligomerization, forming liquid-like condensates that regulate heterochromatin compartmentalization. Moreover, we found that MBD2-driven phase separation excludes histone acetyltransferase and recruits histone deacetylases. This study advances our understanding of heterochromatin compartmentalization and highlights the role of MBD2 in heterochromatin dynamics and composition functionally regulating chromatin states.

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Molecular simulations of enzymatic phosphorylation of disordered proteins and their condensates

Presenting author:

Johannes Gutenberg University Mainz, , Am Linsenberg, 29, 55131 Mainz [DE], zippoema@uni-mainz.de

Author(s):
Emanuele Zippo, Lukas Stelzl

Condensation and aggregation of disordered proteins in cellular non-equilibrium environments are strongly shaped by enzymes. Kinases like Casein kinase 1δ (CK1δ) phosphorylate proteins by consuming ATP, modulating interactions and affecting condensate stability. For the neurodegeneration-linked protein TDP-43, hyperphosphorylation by CK1δ may be cytoprotective, but how the kinase interacts with condensates remains unclear.

Using coarse-grained simulations with Monte Carlo moves to model phosphorylation, we study how CK1δ modifies TDP-43 and drives condensate dissolution. We find phosphorylation is non-uniform: serine residues in the C-terminal region of the TDP-43 low-complexity domain are more frequently modified than those in the N-terminal region, consistent with experiments. This arises from sequence composition, local context, and serine spacing. Phosphorylation is also cooperative—each event alters CK1δ–TDP-43 interactions, promoting further modifications.¹

Additionally, the intrinsically disordered region of CK1δ interacts favorably with TDP-43, enhancing its recruitment into condensates. Once localized, CK1δ phosphorylates TDP-43 and weakens intermolecular interactions, leading to condensate dissolution.

While this work focuses on condensate dissolution, the framework sets the stage for exploring more complex non-equilibrium regulation, including condensate size control and droplet division, in future studies.

¹ Zippo, E., et al. Nat Commun 16, 4649 (2025).

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The role of MED1 condensates in EBV-related B cell lymphomagenesis

Presenting author:

Max Delbrück Center (MDC), , Robert-Rössle-Straße 10, 13125 Berlin [DE], yue.zong@mdc-berlin.de

Author(s):
Yue Zong, Raku Son, Yasuhiro Murakawa, Oliver Popp, Phillipp Mertin, Klaus Rajewsky

EBV is strongly associated with B cell malignancies, particularly in immunosuppressed individuals. Previous research of our group has shown that the expression of a single EBV oncogene, LMP1, in B cells of T cell deficient mice is sufficient to induce rapid, fatal lymphoproliferation and lymphomagenesis. To understand the molecular mechanisms of LMP1 induced B cell lymphomagenesis, we investigated the formation of MED1 condensates in LMP1 B cells and observed that LMP1 can induce MED1 condensates over time. To further study the MED1 condensates in LMP1 B cells, we plan to identify the transcription factors (TFs) and enhancers recruited to MED1 condensates. For the identification of TFs in LMP1-induced MED1 condensates, we have established an endogenous BioID2 knock-in strategy for in situ MED1 proximate protein labeling in a B cell lymphoma cell line. This system will be applied to LMP1 primary B cells. To identify the LMP1-induced enhancers in MED1 condensates, we have analyzed the activated enhancers in LMP1 B cells via cap analysis of gene expression (CAGE) based eRNA sequencing. By intersecting LMP1 activated enhancers with published MED1 binding DNA loci, we can now identify LMP1 induced enhancers recruited to MED1 condensates. We plan to verify the functional importance of TFs and enhancers in MED1 condensates by CRISPR-Cas9 mediated gene knock out in LMP1 B cells. This study aims at elucidating the role of MED1 condensates in LMP1 induced B cell lymphomagenesis.

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