--------------------

Tracing the birth and evolution of a biomolecular condensate

Presenting author:
Marcus Jahnel

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:
Ramona Jühlen

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:
Linda Kartaschew

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:
Radhika Khatter

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:
Benjamin Lau

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:
Amelie Le Parc

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:
Miriam Linsenmeier

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

Presenting author:
Fatemeh Mamashli

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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Nucleosome spacing can fine-tune higher-order chromatin assembly

Presenting author:
Maria Julia Maristany

University of Cambridge, , 23A Gray Rd, CB13TA Cambridge [GB], mjm261@cam.ac.uk

Author(s):
Maria Julia Maristany, Lifeng Chen, Huabin Zhou, Stephen Farr, Jan Huertas, Brian Gibson, Jorge Espinosa, Sy Redding, Rosana Collepardo-Guevara, Michael Rosen

Chromatin architecture plays a crucial role in genome organization and gene regulation, with
higher-order assembly patterns influencing key biological functions. Cellular chromatin displays
heterogeneous structure and dynamics, essential to control diverse nuclear processes. Phase
separation has emerged as a key mechanism for organizing chromatin in vivo. In this work, we
employed molecular dynamics simulations of our minimal chromatin coarse-grained model,
accompanied by biochemistry assays, to examine, at single base-pair resolution, how
nucleosome spacing controls chromatin phase separation.
Our findings demonstrate that as internucleosomal DNA linkers lengthen from 25 bp to 30 bp,
the thermodynamic stability of chromatin condensates decreases, while nucleosome mobility
within these condensates increases. Our simulations reveal that these properties arise from a
balance between intra- and intermolecular nucleosome stacking, favored by chromatin
conformations produced by rigid linkers of 10-base pair periodicity. The ensemble of chromatin
conformations, predicted by our minimal chromatin model, are also consistent with
high-resolution Cryo-ET data of chromatin condensates under several linker length conditions.
Furthermore, we reveal the role of nucleosome remodelers in regulating chromatin
condensates. Thus, the intrinsic phase separation capacity of chromatin enables fine-tuning of
compaction and dynamics by regulatory factors, contributing to heterogeneous chromatin
organization in vivo.

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

Presenting author:
Patrick M. McCall

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

Presenting author:
Julia Meyer

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:
Samantha Milano

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:
Stefan Müller

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:
Michaela Müller-McNicoll

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 Müller-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:
Ellen Okuda, Mara Rudigier

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:
Carlos Emmanuel Panerio

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:
Marcell Papp

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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RNAs untangled: Shedding light onto complex structures of long RNAs

Presenting author:
Lukas Pekarek

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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Improving the accuracy of the self-organized polymer (SOP) model for Intrinsically disordered proteins by rescaling aromatic interactions

Presenting author:
Santosh Prajapati

Indian Institute of Science Bangalore, Molecular Biophysics Unit, C V Raman Road, 560012 Bangalore, Karnataka [IN], mbusantosh@gmail.com

Author(s):
Santosh Prajapati, Debayan Chakraborty, Anand Srivastavaa

Here, we present an improved version of the 2-site per amino-acid resolution Self-Organized polymer (SOP) model for studying Intrinsically Disordered Proteins (IDPs). The original SOP-IDP model works well for chains in good solvent that adopt expanded conformations [1,2]. However, we found that SOP-IDP force-field parameters need to be recalibrated for IDP chains whose Flory exponents deviate from the random-coil limit. For example, for hnRNPA1-LCD, the SOP-IDP force field produces conformational ensembles (with mean Rg of 3.26 nm) that are much more expanded as compared to the experiments (with mean Rg of 2.70 nm) with an error of ~20%. Other examples also suggest that there is scope for refining the SOP-IDP model [3,4]. Most condensate forming IDPs are rich in residues that can form π - π and cation-π interactions, which are weak but important interactions for biomolecular recognition and also for driving and stabilizing biomolecular condensates. In our improved model, we reparametrize the SOP-IDP force field to account for π - π and cation - π interaction at a very coarse-grained level such that the simulations provide experimentally-consistent behaviours for IDPs both in terms of single-chain and bulk properties – especially those rich in cation and aromatic residues. Our method addresses an immediate need in the IDP-biophysics and biomolecular condensate simulations community by providing a well-grounded prescription to simulate and generate faithful conformations of IDPs that are better suited to recapitulate experimental realities.

References:

1. Mugnai ML, Chakraborty D, Kumar A, Nguyen HT, Zeno W, Stachowiak JC, Straub JE, Thirumalai D, 2024, Sizes, conformational fluctuations, and SAXS profiles for Intrinsically Disordered Proteins, bioRxiv. (DOI:10.1101/2023.04.24.538147)
2. Baratam K, & Srivastava A (2024). SOP-MULTI: A Self-Organized Polymer-Based Coarse-Grained Model for Multidomain and Intrinsically Disordered Proteins with Conformation Ensemble Consistent with Experimental Scattering Data. Journal of Chemical Theory and Computation, 20(22), 10179-10198. (DOI:10.1021/acs.jctc.4c00579)
3. Erik W. Martin et al., Valence and patterning of aromatic residues determine the phase behaviour of prion-like domains. Science 367, 694-699(2020). (DOI:1126/science.aaw8653)
4. Bremer, Anne, et al. "Deciphering how naturally occurring sequence features impact the phase behaviours of disordered prion-like domains." Nature chemistry2 (2022): 196-207. (DOI: 10.1038/s41557-021-00840-w

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Deciphering Munc13 assembly and dynamics through mass spectrometry

Presenting author:
Zahra Riazimand

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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Proteomic profiling of cytoplasmic stress-induced liquid condensates of SMN

Presenting author:
Yannick Riedel

Universität Bonn, Institute of Gentics, Westumer Straße 8B, 53489 Sinzig [DE], y.riedel@uni-bonn.de

Author(s):
Yannick Riedel, Jessica Dressler, Maximilian Schilling, Oliver Gruss

The survival of motor neuron (SMN) protein, best known for its role in spinal muscular atrophy (SMA), is essential for assembling uridine-rich small nuclear ribonucleoproteins (UsnRNPs) for RNA splicing. SMN functions as part of a macromolecular complex with Gemin2–8 and UNRIP, facilitating snRNP assembly in both the cytoplasm and nucleus, where it condenses into nuclear Cajal bodies through liquid-liquid phase separation (LLPS) in an mTOR-dependent manner. SMN also participates in the formation of cytoplasmic stress granules (SGs) via RNA-binding regions. However, the role of SMN’s LLPS capacity in cytoplasmic processes remains poorly understood.

Here, we show that hyperosmotic or mechanical stress induces the formation of cytoplasmic SMN condensates, termed S-bodies, in human cells. Live-cell imaging reveals that S-bodies are dynamic, rapidly moving along microtubules during stress, and are distinct from SGs, incorporating additional proteins and RNAs beyond the canonical SMN complex. Notably, the SMA-linked SMN variant lacking exon 7 (SMNΔEx7), which lacks LLPS capability, fails to form S-bodies and instead preferentially accumulates in SGs during stress recovery. In mouse embryonic fibroblasts (MEFs) modeling SMA, treatment with an approved SMA drug restores LLPS ability, enabling efficient S-body formation.

During recovery, SMN forms Janus-like droplets with CLNS1A, suggesting a regulated molecular handoff that facilitates the repair or rebuilding of snRNPs after stress. Proteomic analysis of isolated S-bodies using proximity labeling and sedimentation approaches reveals that these condensates are enriched in RNA helicases, indicating a role in RNA metabolism.

Collectively, our findings identify a novel cytoplasmic function of SMN in the formation of stress-induced S-bodies, which appear to transiently arrest and reactivate key steps in snRNP biogenesis during stress. These results suggest that defective phase separation of SMN underlies SMA pathology, implicating SMA as a disorder of impaired LLPS.

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

Presenting author:
Hao Ruan

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:
Archita Sarkar

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:
Frederik Saulich

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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Enhanced and disease specific drug delivery using Polyoma-virus deduced engineered protein nanoparticles

Presenting author:
Chantal Karin Schmitt, Michael Schäfer

University of Cologne - Institute of Biochemistry, , Zülpicherstr. 47, 50674 Cologne [DE], cschm123@smail.uni-koeln.de

Author(s):
Chantal Karin Schmitt, Michael Schäfer, Isabella Maier, Volker Stoldt, Sebastian Franken, Alexander Glassmann

Nanomaterials for drug delivery have attracted considerable interest in biomedical research due to
their capacity to improve tissue specificity and reduce off-target effects. Conventional artificial
nanomaterials, such as liposomes, polymers, or drug conjugates, often present significant limitations, including complex fabrication requirements, instability, degradation-associated toxicity, and challenging conjugation chemistries. The need for efficient targeted delivery systems, especially for nucleic acid-based therapies in oncology and rare genetic disorders, remains largely unmet as current successful applications are predominantly restricted to vaccines and selected liver-targeted therapies. Viral capsid protein-based nanoparticles offer advantages in terms of biocompatibility and biodegradability. Recent advances in genetic and chemical engineering have enabled the generation of engineered protein nanoparticles with tailored cell tropisms and adaptable cargo loading. The production and modification of protein nanoparticles (PNs) based on human polyomavirus VP1 capsid proteins utilize US-patented methodologies (US15/533,377). Unlike other viral-derived nanoparticles, these PNs self-assemble after recombinant expression in E. coli and facilitate payload incorporation via in vitro assembly. This research aims to identify which polyomavirus VP1 variants offer optimal encapsulation and targeted delivery capabilities. Additionally, functionalization with paramagnetic beads may enable the selective isolation of cells, such as circulating tumour cells, in complex biological samples.

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

Presenting author:
Timo N. Schneider

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:
Nils Schumann

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:
Halyna Shcherbata

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:
Tsvetomir Ivanov

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:
Matej Siketanc

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:
Iris Smokers

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:
Tabea Stark

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:
Lukas Stelzl

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:
Leonhard Thews

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:
Fatmanur Tiryaki

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:
Florian Valero

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:
Claire Vargas

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:
Thibaut Vignane

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:
Sarah Wasilewski

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:
Robin Weinmann

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:
Mona Wellhäusser

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:
Florian Wilfling

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:
Jiaxin Wu

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