Low-complexity Domain Research Articles

Optical characterization of molecular interaction strength in protein condensates.

Biomolecular condensates have been identified as a ubiquitous means of intracellular organization, exhibiting very diverse material properties. However, techniques to characterize these material properties and their underlying molecular interactions are scarce. Here, we introduce two optical techniques - Brillouin microscopy and quantitative phase imaging (QPI) - to address this scarcity. We establish Brillouin shift and linewidth as measures for average molecular interaction and dissipation strength, respectively, and we used QPI to obtain the protein concentration within the condensates. We monitored the response of condensates formed by FUS and by the low-complexity domain of hnRNPA1 (A1-LCD) to altering temperature and ion concentration. Conditions favoring phase separation increased Brillouin shift, linewidth, and protein concentration. In comparison to solidification by chemical crosslinking, the ion-dependent aging of FUS condensates had a small effect on the molecular interaction strength inside. Finally, we investigated how sequence variations of A1-LCD, that change the driving force for phase separation, alter the physical properties of the respective condensates. Our results provide a new experimental perspective on the material properties of protein condensates. Robust and quantitative experimental approaches such as the presented ones will be crucial for understanding how the physical properties of biological condensates determine their function and dysfunction.

Direct and indirect salt effects on homotypic phase separation

The low-complexity domain of hnRNPA1 (A1-LCD) phase separates in a salt-dependent manner. Unlike many intrinsically disordered proteins (IDPs) whose phase separation is suppressed by increasing salt concentrations, the phase separation of A1-LCD is promoted by >100 mM NaCl. To investigate the atypical salt effect on A1-LCD phase separation, we carried out all-atom molecular dynamics simulations of systems comprising multiple A1-LCD chains at NaCl concentrations from 50 to 1000 mM NaCl. The ions occupy first shell as well as more distant sites around the IDP chains, with Arg sidechains and backbone carbonyls the favored partners of Cl– and Na+, respectively. They play two direct roles in driving A1-LCD condensation. The first is to neutralize the high net charge of the protein (+9) by an excess of bound Cl– over Na+; the second is to bridge between A1-LCD chains, thereby fortifying the intermolecular interaction networks in the dense phase. At high concentrations, NaCl also indirectly strengthens π–π, cation–π, and amino–π interactions, by drawing water away from the interaction partners. Therefore, at low salt, A1-LCD is prevented from phase separation by net charge repulsion; at intermediate concentrations, NaCl neutralizes enough of the net charge while also bridging IDP chains to drive phase separation. This drive becomes even stronger at high salt due to strengthened π-type interactions. Based on this understanding, four classes of salt dependence of IDP phase separation can be predicted from amino-acid composition.

Direct and indirect salt effects on homotypic phase separation.

The low-complexity domain of hnRNPA1 (A1-LCD) phase separates in a salt-dependent manner. Unlike many intrinsically disordered proteins (IDPs) whose phase separation is suppressed by increasing salt concentrations, the phase separation of A1-LCD is promoted by >100 mM NaCl. To investigate the atypical salt effect on A1-LCD phase separation, we carried out all-atom molecular dynamics simulations of systems comprising multiple A1-LCD chains at NaCl concentrations from 50 to 1000 mM NaCl. The ions occupy first shell as well as more distant sites around the IDP chains, with Arg sidechains and backbone carbonyls the favored partners of Cl- and Na+, respectively. They play two direct roles in driving A1-LCD condensation. The first is to neutralize the high net charge of the protein (+9) by an excess of bound Cl- over Na+; the second is to bridge between A1-LCD chains, thereby fortifying the intermolecular interaction networks in the dense phase. At high concentrations, NaCl also indirectly strengthens π-π, cation-π, and amino-π interactions, by drawing water away from the interaction partners. Therefore, at low salt, A1-LCD is prevented from phase separation by net charge repulsion; at intermediate concentrations, NaCl neutralizes enough of the net charge while also bridging IDP chains to drive phase separation. This drive becomes even stronger at high salt due to strengthened π-type interactions. Based on this understanding, four classes of salt dependence of IDP phase separation can be predicted from amino-acid composition.

DNA Sensor ABCF1 Phase Separates With cccDNA to Inhibit Hepatitis B Virus Replication.

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) contributes to viral persistence and recurrence, however, how the host innate immune system responds to cccDNA is still less known. Here, based on cccDNA-hepatic proteins interaction profiling, DNA sensor ATP-binding cassette subfamily F member 1 (ABCF1) is identified as a novel cccDNA-binding protein and host restriction factor for HBV replication. Mechanistically, ABCF1 recognizes cccDNA by KKx4 motif and forms phase-separated condensates by the poly-glutamine (PolyQ) region of the N-terminal intrinsically disordered low-complexity domain (LCD). Subsequently, ABCF1-cccDNA phase separation not only activates the type I/III interferon (IFN-I/III) pathway but also prevents Pol II accumulation on cccDNA to inhibit HBV transcription. In turn, to sustain viral replication, HBV reduces ABCF1 expression by HBx-mediated ubiquitination and degradation of SRY-box transcription factor 4（SOX4）, leading to defects in SOX4-mediated upregulation of ABCF1 transcription. Taken together, the study shows that ABCF1 interacts with cccDNA to form phase separation that dually drives innate immune signaling and HBV transcriptional inhibition. These findings shed new light on the understanding of host defense against cccDNA and provide a novel promising therapeutic strategy for HBV infection.

Heterogeneous Slowdown of Dynamics in the Condensate of an Intrinsically Disordered Protein.

The high concentration of proteins and other biological macromolecules inside biomolecular condensates leads to dense and confined environments, which can affect the dynamic ensembles and the time scales of the conformational transitions. Here, we use atomistic molecular dynamics (MD) simulations of the intrinsically disordered low complexity domain (LCD) of the human fused in sarcoma (FUS) RNA-binding protein to study how self-crowding inside a condensate affects the dynamic motions of the protein. We found a heterogeneous retardation of the protein dynamics in the condensate with respect to the dilute phase, with large-amplitude motions being strongly slowed by up to 2 orders of magnitude, whereas small-scale motions, such as local backbone fluctuations and side-chain rotations, are less affected. The results support the notion of a liquid-like character of the condensates and show that different protein motions respond differently to the environment.

Optical characterization of molecular interaction strength in protein condensates.

Biomolecular condensates have been identified as a ubiquitous means of intracellular organization, exhibiting very diverse material properties. However, techniques to characterize these material properties and their underlying molecular interactions are scarce. Here, we introduce two optical techniques - Brillouin microscopy and quantitative phase imaging (QPI) - to address this scarcity. We establish Brillouin shift and linewidth as measures for average molecular interaction and dissipation strength, respectively, and we used QPI to obtain the protein concentration within the condensates. We monitored the response of condensates formed by FUS and by the low-complexity domain of hnRNPA1 (A1-LCD) to altering temperature and ion concentration. Conditions favoring phase separation increased Brillouin shift, linewidth, and protein concentration. In comparison to solidification by chemical crosslinking, the ion-dependent aging of FUS condensates had a small effect on the molecular interaction strength inside. Finally, we investigated how sequence variations of A1-LCD, that change the driving force for phase separation, alter the physical properties of the respective condensates. Our results provide a new experimental perspective on the material properties of protein condensates. Robust and quantitative experimental approaches such as the presented ones will be crucial for understanding how the physical properties of biological condensates determine their function and dysfunction.

Molecular Interactions between Tau Protein and TIA1: Distinguishing Physiological Condensates from Pathological Fibrils.

The interaction of tau protein with other key proteins essential for stress granule formation determines their functional and pathological impact. In a biological framework, the synergy between Alzheimer's associated tau protein and the stress granule core protein TIA1 is widely recognized. However, the molecular details of this association remain unclear. In this study, we throw light on the importance of the state in which the TIA1 exists in mediating its association with the tau protein. Investigations were carried out on the three repeat constructs of tau (K19) and different structures formed by TIA1. Specifically, the condensate formed by TIA1 full-length (TIA1-FL) protein as well as fibril formed by low complexity domain of TIA1 (TIA1-LCD). The dynamics of K19 inside TIA1-FL condensates and the aggregation kinetics of K19 in the presence of TIA1-LCD fibrils were examined using various biophysical techniques. Relaxation-based solution NMR spectroscopic investigations suggest a weak interaction with TIA1 condensates and indicated a reduction in the dynamics of K19 within these TIA1 condensates. In contrast, a significant interaction was observed between K19, and TIA1-LCD fibrils primarily mediated through 321KCGS324 and 306VQIVYKPVDLSKV317. Our findings emphasize that the interaction between Tau and TIA1 varies depending on whether TIA1 is in its physiological condensate form or its pathological fibril state.

Targeting the TDP-43 low complexity domain blocks spreading of pathology in a mouse model of ALS/FTD

Abnormal cytoplasmic localization and accumulation of pathological transactive response DNA binding protein of 43 kDa (TDP-43) underlies several devastating diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP). A key element is the correlation between disease progression and spatio-temporal propagation of TDP-43-mediated pathology in the central nervous system. Several lines of evidence support the concept of templated aggregation and cell to cell spreading of pathological TDP-43. To further investigate this mechanism in vivo, we explored the efficacy of capturing and masking the seeding-competent region of extracellular TDP-43 species. For this, we generated a novel monoclonal antibody (mAb), ACI-6677, that targets the pathogenic protease-resistant amyloid core of TDP-43. ACI-6677 has a picomolar binding affinity for TDP-43 and is capable of binding to all C-terminal TDP-43 fragments. In vitro, ACI-6677 inhibited TDP-43 aggregation and boosted removal of pathological TDP-43 aggregates by phagocytosis. When injecting FTLD-TDP brain extracts unilaterally in the CamKIIa-hTDP-43NLSm mouse model, ACI-6677 significantly limited the induction of phosphorylated TDP-43 (pTDP-43) inclusions. Strikingly, on the contralateral side, the mAb significantly prevented pTDP-43 inclusion appearance exemplifying blocking of the spreading process. Taken together, these data demonstrate for the first time that an immunotherapy targeting the protease-resistant amyloid core of TDP-43 has the potential to restrict spreading, substantially slowing or stopping progression of disease.

Pathological C-terminal phosphomimetic substitutions alter the mechanism of liquid-liquid phase separation of TDP-43 low complexity domain.

C-terminally phosphorylated TAR DNA-binding protein of 43 kDa (TDP-43) marks the proteinaceous inclusions that characterize a number of age-related neurodegenerative diseases, including amyotrophic lateral sclerosis, frontotemporal lobar degeneration and Alzheimer's disease. TDP-43 phosphorylation at S403/S404 and (especially) at S409/S410 is, in fact, accepted as a biomarker of proteinopathy. These residues are located within the low complexity domain (LCD), which also drives the protein's liquid-liquid phase separation (LLPS). The impact of phosphorylation at these LCD sites on phase separation of the protein is a topic of great interest, as these post-translational modifications and LLPS are both implicated in proteinopathies. Here, we employed a combination of experimental and simulation-based approaches to explore this question on a phosphomimetic model of the TDP-43 LCD. Our turbidity and fluorescence microscopy data show that phosphomimetic Ser-to-Asp substitutions at residues S403, S404, S409 and S410 alter the LLPS behavior of TDP-43 LCD. In particular, unlike the LLPS of unmodified protein, LLPS of the phosphomimetic variants displays a biphasic dependence on salt concentration. Through coarse-grained modeling, we find that this biphasic salt dependence is derived from an altered mechanism of phase separation, in which LLPS-driving short-range intermolecular hydrophobic interactions are modulated by long-range attractive electrostatic interactions. Overall, this in vitro and in silico study provides a physiochemical foundation for understanding the impact of pathologically relevant C-terminal phosphorylation on the LLPS of TDP-43 in a more complex cellular environment.

Uncovering Intermolecular Interactions Driving the Liquid-Liquid Phase Separation of the TDP-43 Low-Complexity Domain via Atomistic Dimerization Simulations.

Liquid-liquid phase separation (LLPS) of transactive response DNA-binding protein of 43 kDa (TDP-43), which exerts multiple functions in the splicing, trafficking, and stabilization of RNA, mediates the formation of membraneless condensates with crucial physiological roles, while its aberrant LLPS is linked to multiple neurodegenerative diseases. However, due to the heterogeneous and dynamic nature of LLPS, major gaps remain in understanding the precise intermolecular interactions driving LLPS and how specific mutations alter LLPS dynamics. Here, we investigated the molecular mechanisms underlying the LLPS of the TDP-43 low-complexity domain (LCD) by simulating the dimerization process using all-atom discrete molecular dynamics with microsecond-long simulations. Our results showed that the TDP-43 LCD was intrinsically disordered, with helical structures consistent with prior nuclear magnetic resonance studies. Phase separation propensity was assessed by simulating the dimerization of the TDP-43 LCD and four mutants, showing that A321G, W334G, and M337V inhibited self-association, while G335D promoted it, fully consistent with experimental reports. During the dimerization process, two peptides experienced both elastic and nonelastic collisions, and the self-associated dimer featured both high- and low-contact states. These results suggested that the dimerization process of the TDP-43 LCD was accordingly dynamic and heterogeneous. Additionally, we identified crucial regions containing hydrophobic clusters and aromatic residues in the N-terminus, central region, and C-terminus that were essential for the self-association of the TDP-43 LCD. These residues with high binding affinities can act as stickers to form peptide networks in LLPS. Together, our simulation provides a comprehensive picture of the intermolecular interactions driving the phase separation of the TDP-43 LCD, offering insights into both physiological functions and pathological mechanisms.

Prion-like Proteins in Plants: Key Regulators of Development and Environmental Adaptation via Phase Separation.

Prion-like domains (PrLDs), a unique type of low-complexity domain (LCD) or intrinsically disordered region (IDR), have been shown to mediate protein liquid-liquid phase separation (LLPS). Recent research has increasingly focused on how prion-like proteins (PrLPs) regulate plant growth, development, and stress responses. This review provides a comprehensive overview of plant PrLPs. We analyze the structural features of PrLPs and the mechanisms by which PrLPs undergo LLPS. Through gene ontology (GO) analysis, we highlight the diverse molecular functions of PrLPs and explore how PrLPs influence plant development and stress responses via phase separation. Finally, we address unresolved questions about PrLP regulatory mechanisms, offering prospects for future research.

Heteromeric amyloid filaments of ANXA11 and TDP-43 in FTLD-TDP type C

Neurodegenerative diseases are characterized by the abnormal filamentous assembly of specific proteins in the central nervous system1. Human genetic studies have established a causal role for protein assembly in neurodegeneration2. However, the underlying molecular mechanisms remain largely unknown, which is limiting progress in developing clinical tools for these diseases. Recent advances in cryo-electron microscopy have enabled the structures of the protein filaments to be determined from the brains of patients1. All neurodegenerative diseases studied to date have been characterized by the self-assembly of proteins in homomeric amyloid filaments, including that of TAR DNA-binding protein 43 (TDP-43) in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) types A and B3,4. Here we used cryo-electron microscopy to determine filament structures from the brains of individuals with FTLD-TDP type C, one of the most common forms of sporadic FTLD-TDP. Unexpectedly, the structures revealed that a second protein, annexin A11 (ANXA11), co-assembles with TDP-43 in heteromeric amyloid filaments. The ordered filament fold is formed by TDP-43 residues G282/G284–N345 and ANXA11 residues L39–Y74 from their respective low-complexity domains. Regions of TDP-43 and ANXA11 that were previously implicated in protein–protein interactions form an extensive hydrophobic interface at the centre of the filament fold. Immunoblots of the filaments revealed that the majority of ANXA11 exists as an approximately 22 kDa N-terminal fragment lacking the annexin core domain. Immunohistochemistry of brain sections showed the colocalization of ANXA11 and TDP-43 in inclusions, redefining the histopathology of FTLD-TDP type C. This work establishes a central role for ANXA11 in FTLD-TDP type C. The unprecedented formation of heteromeric amyloid filaments in the human brain revises our understanding of amyloid assembly and may be of significance for the pathogenesis of neurodegenerative diseases.

TDP43 and huntingtin Exon-1 undergo a conformationally specific interaction that strongly alters the fibril formation of both proteins

Protein aggregation is a common feature of many neurodegenerative diseases. In Huntington's disease, mutant huntingtin is the primary aggregating protein, but the aggregation of other proteins, such as TDP43, is likely to further contribute to toxicity. Moreover, mutant huntingtin is also a risk factor for TDP pathology in ALS. Despite this co-pathology of huntingtin and TDP43, it remains unknown whether these amyloidogenic proteins directly interact with each other. Using a combination of biophysical methods, we show that the aggregation-prone regions of both proteins, huntingtin exon-1 (Httex1) and the TDP43 low complexity domain (TDP43-LCD), interact in a conformationally specific manner. This interaction significantly slows Httex1 aggregation, while it accelerates TDP43-LCD aggregation. A key intermediate responsible for both effects is a complex formed by liquid TDP43-LCD condensates and Httex1 fibrils. This complex shields seeding competent surfaces of Httex1 fibrils from Httex1 monomers, which are excluded from the condensates. In contrast, TDP43-LCD condensates undergo an accelerated liquid-to-solid transition upon exposure to Httex1 fibrils. Cellular studies show co-aggregation of untagged Httex1 with TDP43. This interaction causes mislocalization of TDP43, which has been linked to TDP43 toxicity. The protection from Httex1 aggregation in lieu of TDP43-LCD aggregation is interesting, as it mirrors what has been found in disease models, namely that TDP43 can protect from huntingtin toxicity, while mutant huntingtin can promote TDP43 pathology. These results suggest that direct protein interaction could, at least in part, be responsible for the linked pathologies of both proteins.

DNAJA2 and Hero11 mediate similar conformational extension and aggregation suppression of TDP-43.

Many RNA-binding proteins (RBPs) contain low-complexity domains (LCDs) with prion-like compositions. These long intrinsically disordered regions regulate their solubility, contributing to their physiological roles in RNA processing and organization. However, this also makes these RBPs prone to pathological misfolding and aggregation that are characteristic of neurodegenerative diseases. For example, TAR DNA-binding protein 43 (TDP-43) forms pathological aggregates associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). While molecular chaperones are well-known suppressors of these aberrant events, we recently reported that highly disordered, hydrophilic, and charged heat-resistant obscure (Hero) proteins may have similar effects. Specifically, Hero proteins can maintain the activity of other proteins from denaturing conditions in vitro, while their overexpression can suppress cellular aggregation and toxicity associated with aggregation-prone proteins. However, it is unclear how these protective effects are achieved. Here, we used single-molecule FRET to monitor the conformations of the aggregation-prone prion-like LCD of TDP-43. While we observed high conformational heterogeneity in wild-type LCD, the ALS-associated mutation A315T promoted collapsed conformations. In contrast, an Hsp40 chaperone, DNAJA2, and a Hero protein, Hero11, stabilized extended states of the LCD, consistent with their ability to suppress the aggregation of TDP-43. Our results link single-molecule effects on conformation to macro effects on bulk aggregation, where a Hero protein, like a chaperone, can maintain the conformational integrity of a client protein to prevent its aggregation.

Observation of the Liquid-Liquid Phase Separation of FUS-LC Using Vacuum-Ultraviolet Circular Dichroism Spectroscopy.

To reveal the structural mechanism by which the low-complexity domain of the fused in sarcoma protein (FUS-LC) mediates liquid-liquid phase separation (LLPS), we conducted a vacuum-ultraviolet circular dichroism (VUV-CD) spectroscopic study, a technique to analyze the secondary structures of proteins. The VUV-CD measurements were performed at the BL12 VUV-CD station at the Hiroshima Synchrotron Radiation Center (HiSOR) in Japan. CD spectra were measured between 180 and 260 nm while controlling the temperature of samples from 37°C to 5°C to obtain the LLPS of FUS-LC. The CD spectrum obtained at 37°C exhibited a large negative peak at 195 nm and a small negative shoulder near 220 nm. The peak intensity around 195 nm decreased as the sample temperature decreased. The spectral changes originated from the LLPS formation.

Conformations of a Low-Complexity Protein in Homogeneous and Phase-Separated Frozen Solutions.

Liquid-liquid phase separation (LLPS) in solutions of proteins with intrinsically disordered domains has attracted recent attention because of its relevance to multiple biological processes and its inherent interest from the standpoint of protein biophysics. The high protein concentrations and abundant intermolecular interactions within protein-rich, phase-separated "droplets" suggests that conformational distributions of intrinsically disordered proteins may differ in homogeneous and phase-separated solutions. To investigate whether detectable differences exist, we performed experiments on the low-complexity domain of the FUS protein (FUS-LC) in which FUS-LC solutions were first equilibrated at temperatures well above or well below their LLPS transition temperatures, then rapidly frozen and examined at very low temperatures by solid state nuclear magnetic resonance (ssNMR) spectroscopy. The ssNMR data for homogeneous and phase-separated frozen solutions of FUS-LC were found to be nearly identical, showing that LLPS is not accompanied by substantial changes in the local conformational distributions of this intrinsically disordered protein.

Oxidative stress elicits the remodeling of vimentin filaments into biomolecular condensates

The intermediate filament protein vimentin performs an essential role in cytoskeletal interplay and dynamics, mechanosensing and cellular stress responses. In pathology, vimentin is a key player in tumorigenesis, fibrosis and infection. Vimentin filaments undergo distinct and versatile reorganizations, and behave as redox sensors. The vimentin monomer possesses a central α-helical rod domain flanked by N- and C-terminal low complexity domains. Interactions between this type of domains play an important function in the formation of phase-separated biomolecular condensates, which in turn are critical for the organization of cellular components. Here we show that several oxidants, including hydrogen peroxide and diamide, elicit the remodeling of vimentin filaments into small particles. Oxidative stress elicited by diamide induces a fast dissociation of filaments into circular, motile dots, which requires the presence of the single vimentin cysteine residue, C328. This effect is reversible, and filament reassembly can occur within minutes of oxidant removal. Diamide-elicited vimentin droplets recover fluorescence after photobleaching. Moreover, fusion of cells expressing differentially tagged vimentin allows the detection of dots positive for both tags, indicating that vimentin dots merge upon cell fusion. The aliphatic alcohol 1,6-hexanediol, known to alter interactions between low complexity domains, readily dissolves diamide-elicited vimentin dots at low concentrations, in a C328 dependent manner, and hampers reassembly. Taken together, these results indicate that vimentin oxidation promotes a fast and reversible filament remodeling into biomolecular condensate-like structures, and provide primary evidence of its regulated phase separation. Moreover, we hypothesize that filament to droplet transition could play a protective role against irreversible damage of the vimentin network by oxidative stress.

Deciphering the molecular toolkit: regulatory elements governing shell biomineralization in marine molluscs.

The intricate process of shell biomineralization in marine molluscs is governed by a complex interplay of regulatory elements, encompassing secretomes, transporters, and noncoding RNA. This review delves into recent advancements in understanding these regulatory mechanisms, emphasizing their significance in elucidating the functions and evolutionary dynamics of the molluscan shell biomineralization process. Central to this intricate orchestration are secretomes with diverse functional domains, selectively exported to the extrapallial space, which directly regulate crystal growth and morphology. Transporters are crucial for substrate transportation in the calcification and maintenance of cellular homeostasis. Beyond proteins and transporters, noncoding RNA molecules are integral components influencing shell biomineralization. This review underscores the nonnegligible roles played by these genetic elements at the molecular level. To comprehend the complexity of biomineralization in mollusc, we explore the origin and evolutionary history of regulatory elements, primarily secretomes. While some elements have recently evolved, others are ancient genes that have been co-opted into the biomineralization toolkit. These elements undergo structural and functional evolution through rapidly evolving repetitive low-complexity domains and domain gain/loss/rearrangements, ultimately shaping a distinctive set of secretomes characterized by both conserved features and evolutionary innovations. This comprehensive review enhances our understanding of molluscan biomineralization at the molecular and genetic levels.

Sequence-specific interactions determine viscoelasticity and aging dynamics of protein condensates.

Biomolecular condensates are viscoelastic materials. Here, we investigate the determinants of sequence-encoded and age-dependent viscoelasticity of condensates formed by the prion-like low-complexity domain of the protein hnRNP A1 and its designed variants. We find that the dominantly viscous forms of the condensates are metastable Maxwell fluids. A Rouse-Zimm model that accounts for the network-like organization of proteins within condensates reproduces the measured viscoelastic moduli. We show that the strengths of aromatic inter-sticker interactions determine sequence-specific amplitudes of elastic and viscous moduli, and the timescales over which elastic properties dominate. These condensates undergo physical ageing on sequence-specific timescales. This is driven by mutations to spacer residues that weaken the metastability of dominantly viscous phases. The ageing of condensates is accompanied by disorder-to-order transitions, leading to the formation of non-fibrillar, beta-sheet-containing, semi-crystalline, elastic, Kelvin-Voigt solids. Our results suggest that sequence grammars, which refer to amino acid identities of stickers versus spacers in prion-like low-complexity domains, have evolved to afford control over metastabilities of dominantly viscous fluid phases of condensates. This selection is likely to render barriers for conversion from metastable fluids to globally stable solids insurmountable on functionally relevant timescales.

Heteromeric amyloid filaments of ANXA11 and TDP-43 in FTLD-TDP Type C.

Neurodegenerative diseases are characterised by the abnormal filamentous assembly of specific proteins in the central nervous system 1 . Human genetic studies established a causal role for protein assembly in neurodegeneration 2 . However, the underlying molecular mechanisms remain largely unknown, which is limiting progress in developing clinical tools for these diseases. Recent advances in electron cryo-microscopy (cryo-EM) have enabled the structures of the protein filaments to be determined from patient brains 1 . All diseases studied to date have been characterised by the self-assembly of a single intracellular protein in homomeric amyloid filaments, including that of TAR DNA-binding protein 43 (TDP-43) in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) Types A and B 3,4 . Here, we used cryo-EM to determine filament structures from the brains of individuals with FTLD-TDP Type C, one of the most common forms of sporadic FTLD-TDP. Unexpectedly, the structures revealed that a second protein, annexin A11 (ANXA11), co-assembles with TDP-43 in heteromeric amyloid filaments. The ordered filament fold is formed by TDP-43 residues G282/284-N345 and ANXA11 residues L39-L74 from their respective low-complexity domains (LCDs). Regions of TDP-43 and ANXA11 previously implicated in protein-protein interactions form an extensive hydrophobic interface at the centre of the filament fold. Immunoblots of the filaments revealed that the majority of ANXA11 exists as a ∼22 kDa N-terminal fragment (NTF) lacking the annexin core domain. Immunohistochemistry of brain sections confirmed the co-localisation of ANXA11 and TDP-43 in inclusions, redefining the histopathology of FTLD-TDP Type C. This work establishes a central role for ANXA11 in FTLD-TDP Type C. The unprecedented formation of heteromeric amyloid filaments in human brain revises our understanding of amyloid assembly and may be of significance for the pathogenesis of neurodegenerative diseases.