Biology subjects

Dormann, D.

Publications and source records attributed to Dormann, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Kinase-phosphatase cycles drive self-sustained oscillations of protein condensates

Phase-separated protein condensates organize biochemical processes in cells. ATP-driven enzymatic reactions can change the physical properties and even trigger the dissolution of such condensates. Here we use molecular simulations to show how ATP-driven kinase and phosphatase reactions modulate the organization of TDP-43 low complexity domain (LCD) condensates. TDP-43 phosphorylation is enhanced by condensation, as CK1{delta} preferentially binds to and acts in TDP-43 LCD condensates, whereas dephosphorylation by the phosphatase calcineurin occurs predominantly in the dilute phase. This differential recruitment of kinases and phosphatases, together with the slow redistribution of kinases into newly formed condensates, triggers oscillatory appearance and disappearance of TDP-43 LCD condensates. The oscillations are self-sustained, persisting at constant chemical driving, showing that an antagonistic kinase-phosphatase pair can endow a condensate with autonomous dynamics rather than only setting its structure.

biophysics↗

bioRxiv2026

A Phosphorylation-Induced Micellization switch in the low complexity domain of TDP-43

Abstract textPhase separation (PS) of the low-complexity domain (LCD) of TDP-43 is linked to pathogenic aggregates in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP). Here, we show that extensive phosphorylation of the LCD C-terminus redirects its self-assembly. Coarse-grained Monte Carlo simulations predicted that 12 Ser phosphorylations partition the 148-residue LCD into a hydrophobic N-terminal and highly charged C-terminal block, favoring finite-sized micellization over macroscopic PS. In vitro, LCD phosphorylated by casein kinase 1 delta (CK1{delta}; mean of 12 phosphorylations by native mass spectrometry) and phosphomimetic 12D/12DD mutants formed spherical nanoparticles ({approx} 20-50 nm) above a low-micromolar critical micelle concentration, whereas the unphosphorylated LCD underwent reversible PS that matured into fibrils. Increasing ionic strength shifted the mutants toward anisotropic morphologies (worm-like 12D micelles and rigid 12DD nanocylinders). Turbidity assays and confocal imaging directly visualized the absence of PS in the phosphorylated form. Negative-stain and cryo-EM confirmed the spherical micellar architecture for the phosphorylated LCD and 12D/12DD mimics. Our data identify phosphorylation as a molecular switch tuning macrophase separation and fibril formation of TDP-43 LCD, providing a framework for an aggregation-protective role through microphase separation into size-limited micelles. Whether these assemblies are stable or kinetically trapped on pathological timescales remains unclear.

biophysics↗

bioRxiv2026

TDP-43 self-assembly is regulated by its disordered NLS-region

Cytosolic inclusions of the RNA-binding protein TDP-43 are a pathological hallmark of several neurodegenerative diseases, such as amyotrophic lateral sclerosis and frontotemporal dementia. Cellular or animal model systems often use TDP-43 mutated in its nuclear localization signal (NLS) to study its cytosolic mislocalization and aggregation. Here we show that the disordered NLS-region, in particular the basic amino acids, are crucial for self-assembly of full-length TDP-43 across size scales, ranging from small clusters to visible condensates and aggregates. Molecular dynamics simulations and NMR studies suggest that the NLS-region engages in inter-chain interactions with C-terminal aromatic residues as well as RRM1 and NTD interactions. We further demonstrate that a minimal NLS mutation (K82A) preserves TDP-43 condensation in vitro and in cells, while commonly used NLS mutations yield partially or strongly reduced self-assembly behaviors. Our data highlight TDP-43 K82A as ideal model system to study cytosolic TDP-43 aggregation in cell and animal models.

biochemistry↗

bioRxiv2026

Multi-Domain Interplay Controls Full-Length TDP-43 Phase Separation and Condensate Dynamics

TDP-43 (TAR DNA-binding protein 43) is a 414-amino acid protein with a structured N-terminal domain (NTD), two RNA recognition motifs (RRM1 and RRM2), and a long disordered C-terminal low complexity domain (LCD). TDP-43 forms phase-separated condensates as part of its physiological function in RNA processing. However, aberrant TDP-43 condensation, changes in condensate material properties, and subsequent aggregation are linked to the development of neurodegenerative diseases. Using explicit-solvent, near-atomic resolution coarse-grained simulations we demonstrate how the interplay among different domains drives phase separation of the full-length protein.We directly capture how the secondary structure of a conserved helix in the LCD modulates phase separation, and follow the effect of phosphomimicking mutations on the condensation of full-length TDP-43. C-terminal phosphomimicking mutations rewire the interactions of the LCD by increasing solvation locally and enhancing Na+ binding to the LCD. Our simulations and in vitro experiments emphasize the importance of the aromatic residues in the LCD but also of N-terminal residues 1-101 including the NTD for full-length TDP-43 condensation. With a G[o]-type approach we capture not just conformational flexibility but also specific dimer formation through NTD-NTD interactions and how they modulate the phase behavior as well as the dynamic and interfacial properties of full-length TDP-43 condensates.

biophysics↗

bioRxiv2026

Phase separation behavior of TDP-43 governs its protein interactome and regulation of altern

TDP-43 is a nuclear RNA-binding protein that regulates RNA metabolism, including alternative splicing. Its aggregation is a major pathological hallmark of several neurodegenerative diseases. TDP-43 undergoes phase separation (PS) and this condensation behavior may be linked to aggregate formation. Whether and how PS governs TDP-43 RNA regulatory functions remains poorly understood. Here we utilized rationally designed mutations in the TDP-43 low complexity domain to tune TDP-43 PS, yielding a panel of TDP-43 variants with reduced propensity to form condensates (PS-deficient), and a panel forming irreversible, undynamic condensates (solid-like) in vitro and in cells. Two complementary interactomics approaches identified PS-dependent interactions between TDP-43 and key RNA regulatory factors, including splicing regulators and the RNA helicase UPF1, which show increased interactions with solid-like variants. Our results highlight that TDP-43 PS regulates RNA and protein homeostasis by modulating a subset of TDP-43-dependent alternative splicing events and by reshaping interactions with RNA regulatory factors.

biochemistry↗

bioRxiv2026

DNA Protonuclei as Programmable Nuclear Mimics Reveal Environmental Context on Protein Phase Separation

Understanding protein phase separation in cellular environments remains a major challenge, as ex vivo assays often fail to capture the influence of environmental context - such as crowding, multimodal interactions, and the dynamic properties of the cytosol or nucleus. Here, we introduce programmable DNA-based protonuclei (PN) as nucleus-inspired compartments to probe phase separation of the neurodegeneration-linked protein FUS. We show that FUS partitioning and condensate formation are highly sensitive to nucleic acid sequence, spatial confinement, and viscoelastic properties of the PN core. Notably, classical test-tube affinity assays fail to predict protein behavior within the crowded and multivalent PN environment. By tuning DNA crosslinking, we modulate condensate dynamics and suppress liquid-to-solid transitions of FUS - a hallmark of disease. These findings demonstrate that multivalent, confined environments fundamentally reshape protein-nucleic acid interactions and phase behavior. The PN platform complements test-tube assays and complex cellular settings and enables to dissect nuclear condensates under controllable conditions.

synthetic biology↗

bioRxiv2026

The sequence context of RG/RGG motifs determines condensate formation, transportin-1 binding and chaperoning

Intrinsically disordered arginine-glycine-rich (RG/RGG) regions are highly abundant in the eukaryotic proteome. Proteins containing these motifs participate in fundamental cellular processes, including nuclear import, transcriptional regulation, biomolecular condensate formation, and apoptosis. Mutations or dysfunction of RG/RGG proteins have been implicated in neurodegenerative diseases and cancer. Although some RG/RGG proteins have been shown to drive condensate formation, localize to membrane-less organelles, interact with nuclear import receptors, or undergo arginine methylation, these properties are not shared uniformly across the proteome. The considerable diversity in RG/RGG motif length and amino acid composition raises the question of which sequence features determine their functional behaviour. To address this, we conducted a systematic bioinformatics and experimental analysis, combining synthetic and natural peptides with studies on the RNA-binding protein FUS as a model system. Our results reveal that the sequence composition of RG/RGG motifs is a key determinant of their capacity for RNA-mediated condensate formation, stress granule recruitment, and transportin-1-mediated chaperoning and nuclear import. These findings provide new insight into the sequence grammar of disordered RG/RGG regions and how it encodes the multifunctionality of these proteins in cellular regulation.

molecular biology↗

bioRxiv2026

The Structured RNA-binding Domains and Condensation Capacity of FUS Shape its RNA-binding Landscape and Function.

RNA-binding proteins (RBPs) are key regulators of gene expression and often contain intrinsically disordered regions that drive biomolecular condensation. Yet, how condensation affects RBP specificity and function remains poorly defined. Here, we use strategically designed point mutations to selectively impair canonical RNA-binding and condensation of the amyotrophic lateral sclerosis (ALS)-linked RBP Fused in Sarcoma (FUS). Using automated high-content imaging, we show that both properties shape nuclear ribonucleoprotein condensates and govern distinct aspects of FUS function in the DNA damage response. Transcriptome-wide mapping of FUS-RNA interactions reveals that the canonical RNA-binding domains recognise G-rich and C-rich motifs, whereas condensation selectively enhances binding to G-rich and structured sequences, often acting in concert with canonical RNA-binding to control transcriptional programs and splicing decisions. These findings provide a mechanistic framework for how FUS integrates condensation with sequence-specific RNA recognition to orchestrate nuclear organisation, genome stability and RNA metabolism. Furthermore, our rigorously validated mutants offer a platform for future mechanistic dissection of ALS pathogenesis and the development of targeted therapeutic strategies. HighlightsO_LIFUS condensation and RNA-binding can be functionally uncoupled with targeted point mutations C_LIO_LICondensation drives FUS recruitment and assembly of nuclear RNP condensates C_LIO_LIFUS condensation and RNA-binding play distinct roles in the DNA damage response C_LIO_LIThe structured RNA-binding domains confer specificity to G-rich and C-rich motifs C_LIO_LICondensation enhances FUS binding to G-rich, structured RNA elements C_LIO_LIFUS condensation and canonical RNA-binding synergistically regulate gene expression C_LI

molecular biology↗

bioRxiv2025

Clustering within a single-component biomolecular condensate

Biomolecular condensates (BMCs) are assemblies of hundreds to many thousands of macromolecules within cells that are organized without physical barriers. Condensate function is dictated not only by its molecular composition, but also by substructural organization and molecular mobility. One hypothesis for the onset of multiple protein aggregation diseases is that the increased densities of specific proteins within BMCs promotes the formation of solid inclusions. However, deciphering the internal structural and functional properties of BMCs at the nanoscale and identifying the initiating events of inclusion formation requires tools with high spatiotemporal precision. Here we show using single molecule and other microscopy approaches that single component Fused in Sarcoma (FUS) condensates exhibit confinement and contain clusters with higher FUS density even at early timepoints. Upon aging, condensates displayed altered physical properties and reduced monomer mobility, and yet most FUS monomers diffused throughout the condensate within seconds. While an increase in connectivity over time explains reduced mobility, the large fraction of molecules retaining high mobility suggests a sponge-like structure rather than a system-spanning network. These findings indicate that a pseudo-equilibrium between distinct structural connectivities can exist within simple condensates, suggesting the potential for substantial structural and functional complexity of BMCs at the nanoscale.

biophysics↗

bioRxiv2025

A step towards animal models with self-sustained fungal bioluminescence

Engineering animal models with self-sustained luminescence could enable non-invasive longitudinal monitoring of molecular events in living animals. To create animal models that report physiology with autoluminescence, both luciferin biosynthesis enzymes and the luciferase need to be optimised. Previous work on engineering the autoluminescence pathway from fungi resulted in the development of nnLuz_v3, a version of Neonothopanus nambi luciferase with enhanced thermal stability. Here, we generated an nnLuz_v3 reporter of endogenous Cyp1a1 expression as a measure of aryl hydrocarbon receptor (AHR) activation, assessing the performance of nnLuz_v3 in vivo at physiologically relevant expression levels. As AHR dynamically responds to metabolic, environmental and dietary changes it provides a validated platform to assess novel luminescence approaches. In Cyp1a1-nnLuz mice bioluminescence signal was stable, allowing the generation of well-resolved luminescence images both on standard in vivo imaging equipment and consumer-grade cameras. Using mice and nematode models, we demonstrated limited oral availability of the fungal luciferin, potentially compatible with delivering the substrate via food or the microbiome. Our results are an encouraging first step in the generation of an autoluminescent mammalian model of a molecular event and encourage optimisation of other enzymes of the fungal luciferase pathway.

genetics↗

bioRxiv2025

Direct binding of TDP-43 and Tau drives their co-condensation, but suppresses Tau fibril formation and seeding

Neuronal Tau aggregates are a hallmark of Alzheimers disease (AD), but more than half of the patients exhibit additional TDP-43 inclusions and some have co-aggregates of both proteins. The presence of Tau/TDP-43 co-pathology is associated with increased disease severity, although the causal relationship remains unclear. Here we demonstrate that Tau and TDP-43 mutually promote each others condensation through direct interaction in vitro, forming irregularly shaped or multiphasic co-condensates with lower TDP-43 mobility, but higher Tau dynamics. While Tau promotes TDP-43 aggregation in vitro, TDP-43 suppresses formation of Tau fibrils and instead causes formation of oligomeric Tau and Tau/TDP-43 species. These co-assemblies hinder Tau seeding in a biosensor assay specific for proteopathic Tau seeds. Consistent with this data, SarkoSpin extracts from AD brains with Tau/TDP-43 co-pathology exhibit reduced Tau seeding compared to Tau-only AD brains. In contrast, patient-derived extracts from AD brains with Tau/TDP-43 co-pathology are highly potent in seeding TDP-43 neoaggregates in a TDP-43 reporter cell line. Our results suggest that direct interaction of TDP-43 and Tau may suppress Tau pathology, while promoting TDP-43 pathology. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/662960v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1ed5dfaorg.highwire.dtl.DTLVardef@b4ef07org.highwire.dtl.DTLVardef@b8dbe8org.highwire.dtl.DTLVardef@6d9b49_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

CRISPRa-mediated disentanglement of the Dux-MERVL axis in the 2C-like state, totipotency and cell death

Transposable elements (TEs) provide sequences that are powerful cis-regulatory drivers of gene expression programmes. This is particularly apparent during early development when many TEs become de-repressed. MERVL elements are highly yet transiently upregulated in mouse totipotent 2-cell (2C) embryos during major zygotic genome activation (ZGA), and in 2C-like cells in vitro. One of the most powerful activators of MERVL is the pioneer transcription factor, Dux. However, apparent differences lie in the requirement for Dux versus MERVL activation in embryos, for unclear reasons. Moreover, sustained Dux activation causes cell toxicity in multiple cell types, which may or may not be linked to MERVL activation. Using a CRISPR-activation, 2C-GFP reporter system, we have unpicked the relative role of Dux and MERVL in ZGA, totipotent-like characteristics and cell toxicity. We find that direct MERVL activation comprises only a portion of the Dux-dependent transcriptome, and which is sufficient for expanded fate potential, but not other totipotency features. Conversely, Dux-induced pathology is independent of MERVL activation and involves induction of the pro-apoptotic factor, Noxa. Our study highlights the complexity of the Dux-MERVL transcriptional network and uncovers a new player in Dux-driven pathology.

developmental biology↗

bioRxiv2024

FUS controls muscle differentiation and structure through LLPS mediated recruitment of MEF2 and ETV5

FUS is an RNA binding protein mutated in amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by progressive muscle weakness. We show that ALS-associated FUS mutations lead to ultrastructural defects in muscle of FUS-ALS patients, with disruption of sarcomeres and mitochondria. Studies in mouse and Drosophila models demonstrate an evolutionary-conserved cell autonomous function of FUS in muscle development. Mechanistically, FUS is required for transcription of MEF2 dependent genes, binds to the promoter of genes bound by ETS transcription factors in particular ETV5 and co-activates transcription of MEF2 dependent genes with ETV5. FUS phase separates with ETV5 and MEF2A, and MEF2A binding to FUS is potentiated by ETV5. Last, Etv5 haploinsufficiency exacerbates muscle weakness in a mouse model of FUS-ALS. These findings establish FUS as an essential protein for skeletal muscle structure through its phase separation-dependent recruitment of ETV5 and MEF2, defining a novel pathway compromised in FUS-ALS.

neuroscience↗

bioRxiv2024

Molecular simulations of enzymatic phosphorylation of disordered proteins and their condensates

Understanding the condensation and aggregation of intrinsically disordered proteins in a non-equilibrium environment is crucial for unraveling many biological processes. Active enzymes catalyse many processes by consuming chemical fuels such as ATP. Enzymes called kinases phosphorylate disordered regions of proteins and thus profoundly affect their properties and interactions. Protein phosphorylation is implicated in neurodegenerative diseases and may modulate pathogenesis. However, how protein sequence and molecular recognition of a disordered protein by kinases determine phosphorylation patterns is not understood. In principle, molecular dynamics simulations hold the promise to resolve how phosphorylation affects disordered proteins and their assemblies. In practice, chemically-detailed simulations of enzymatic reactions and the dynamics of enzymes are highly challenging, in particular it is difficult to verify whether implementations of driven simulations are thermodynamically consistent. We can now address this problem with residue-level coarse-grained molecular dynamics simulations, integrating Metropolis Monte Carlo steps to model chemical reactions. Importantly, we show how to verify by Markov-state modeling that the realisation of a non-equilibrium steady state satisfies local-detailed balance. We investigate TDP-43 phosphorylation by the kinase CK1{delta} in simulations, examining patterns of phosphorylation and assessing its preventive role in chain aggregation, which may be a cytoprotective mechanism in neurodegenerative diseases. We find that the degree of residue phosphorylation is determined by sequence preference and charges, rather than by the position in the chain. The phosphorylation frequency is also affected by the phosphorylation patterns, since the interactions between CK1{delta} and TDP-43 actively change after each reaction. For TDP-43, our simulations show condensates dissolution through phosphorylation with kinases binding to the condensates and phosphorylating TDP-43 in the condensates.

biophysics↗

bioRxiv2024

Rapid depletion and super-resolution microscopy reveal an unexpected role of the nuclear-speckle protein SRSF5 in paraspeckle assembly and dynamics during cellular stress

Nuclear speckles (NS) and paraspeckles (PS) are adjacent condensates with distinct protein composition, with serine-arginine-rich splicing factors (SRSFs) concentrated in NS. Surprisingly, we find that SRSF5 is present in both. Combining super-resolution imaging, proximity proteomics and iCLIP, we show that SRSF5 binds with PS core proteins to the PS-scaffold RNA NEAT1 and locates between PS spheres. Acute SRSF5 depletion results in reduced PS with differently packaged NEAT1. Under stress, SRSF5s association with PS increases, and without SRSF5, PS cluster assembly is impaired. Interfering with binding to purine-rich RNAs even causes PS-NS fusion. In an intriguing over-compensation, longer SRSF5 depletion reduces TDP-43 levels via premature polyadenylation, leading to NEAT1 isoform switching and more PS. We propose that SRSF5 forms a stress-specific PS shell and acts as a glue for PS clusters. Additionally, we uncover SRSF5 as a novel regulator of TDP-43 and demonstrate how acute depletion distinguishes direct from compensatory effects. HighlightsO_LINS protein SRSF5 associates with PS shells and enriches between PS spheres C_LIO_LISRSF5 binds the PS-scaffold RNA NEAT1 and ensures proper NEAT1 packaging C_LIO_LISRSF5 association with PS increases under stress and promotes cluster formation C_LIO_LIInterfering with binding to purine-rich RNAs causes the fusion of PS and NS C_LIO_LIAcute SRSF5 depletion reveals compensatory effects on PS assembly and dynamics C_LIO_LISRSF5 regulates TDP-43 levels via premature polyadenylation C_LI

cell biology↗

bioRxiv2024

Intrinsically disordered RNA-binding motifs cooperate to catalyze RNA folding and drive phase separation

RNA-binding proteins are essential for gene regulation and the spatial organization of cells. Here, we report that the yeast ribosome biogenesis factor Loc1p is an intrinsically disordered RNA-binding protein with eight repeating positively charged, unstructured nucleic acid binding (PUN) motifs. While a single of these previously undefined motifs stabilizes folded RNAs, multiple copies strongly cooperate to catalyze RNA folding. In the presence of RNA, these multivalent PUN motifs drive phase separation. Proteome-wide searches in pro-and eukaryotes for proteins with similar arrays of PUN motifs reveal a strong enrichment in RNA-mediated processes and DNA remodeling. Thus, PUN motifs are potentially involved in a large variety of RNA-and DNA-related processes by concentrating them in membrane-less organelles. The general function and wide distribution of PUN motifs across species suggests that in an ancient "RNA world" PUN-like motifs may have supported the correct folding of early ribozymes.

biochemistry↗

bioRxiv2023

PURA Syndrome-causing mutations impair PUR-domain integrity and affect P-body association

Mutations in the human PURA gene cause the neuro-developmental PURA syndrome. In contrast to several other mono-genetic disorders, almost all reported mutations in this nucleic acid binding protein result in the full disease penetrance. In this study, we observed that patient mutations across PURA impair its previously reported co-localization with processing bodies. These mutations either destroyed the folding integrity, RNA binding or dimerization of PURA. We also solved the crystal structures of the N- and C-terminal PUR domains of human PURA and combined them with molecular dynamics simulations and NMR measurements. The observed unusually high dynamics and structural promiscuity of PURA indicated that this protein is particularly susceptible to mutations impairing its structural integrity. It offers an explanation why even conservative mutations across PURA result in the full penetrance of symptoms in patients with PURA syndrome.

molecular biology↗

bioRxiv2021

Nucleolar-based Dux repression is essential for 2-cell stage exit

Upon fertilisation, the mammalian embryo must switch from dependence on maternal transcripts to transcribing its own genome, and in mice involves the transient upregulation of MERVL transposons and MERVL-driven genes at the 2-cell stage. The mechanisms and requirement for MERVL and 2-cell (2C) gene upregulation are poorly understood. Moreover, this MERVL-driven transcriptional program must be rapidly shut off to allow 2C exit and developmental progression. Here, we report that robust ribosomal RNA (rRNA) synthesis and nucleolar maturation are essential for exit from the 2C state. 2C-like cells and 2C embryos show similar immature nucleoli with altered structure and reduced rRNA output. We reveal that nucleolar disruption via blocking Pol I activity or preventing nucleolar phase separation enhances conversion to a 2C-like state in embryonic stem cells (ESCs) by detachment of the MERVL activator Dux from the nucleolar surface. In embryos, nucleolar disruption prevents proper Dux silencing and leads to 2-4 cell arrest. Our findings reveal an intriguing link between rRNA synthesis, nucleolar maturation and gene repression during early development.

developmental biology↗