The role of liquid–liquid phase separation in aggregation of the TDP-43 low-complexity domain

Babinchak, W. Michael; Raza, Haider; Dumm, Benjamin K.; Sarkar, Prottusha; Surewicz, Krystyna; Choi, Joo Hee; Surewicz, Witold K.

doi:10.1074/jbc.ra118.007222

Cited by 282 publications

(344 citation statements)

References 67 publications

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“…The observed correlation between stability and zeta potential also has important pathophysiological implications, specifically for the transition of condensates from their liquid state to solid aggregates. It has been shown that FUS can transition into toxic aggregates associated with the onset and development of motor neuron disease more readily when it is contained in condensates (8), and this trend holds true for other proteins as well, including TDP-43 and other condensate forming systems (38,39). Recent theoretical work has also highlighted how condensates could behave as compartments for aggregate formation, and has even indicated how more aggregates could form within condensates of greater size (40).…”

Section: Discussionmentioning

confidence: 99%

Surface electrostatics govern the emulsion stability of biomolecular condensates

Welsh

Krainer

Espinosa

et al. 2020

Preprint

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Liquid–liquid phase separation underlies the formation of biomolecular condensates and membraneless compartments in living cells. Physically, condensed liquid biomolecular systems represent water-in-water emulsions with a very low surface tension. Such emulsions are commonly unstable towards coalescence, yet in order to be functional, they must persist inside the cell. This observation thus raises the fundamental question of the origin of the stability of such emulsions, and whether passive physical mechanisms exist that stabilize droplets against fusion or coalescence. Here, through measurement of condensate zeta potentials on a single droplet level, we show that surface electrostatic properties of condensates can be used to describe and assess the emulsion stability of condensed liquid biomolecular systems. We find that condensates formed from a representative set of peptide/nucleic acid and protein systems have zeta potentials in the stability range predicted by classical colloid theory. Specifically, we describe the electrostatic nature of PR25:PolyU and FUS condensates and show that their zeta potentials correlate well with their propensity to fuse, coalesce, and cluster. Further, we bring together experiments with multiscale molecular simulations and demonstrate that the differences in zeta potential and subsequent stability of biomolecular condensates are modulated by their internal molecular organization. Taken together, these findings shed light on the origin of the stability of biomolecular condensate systems, and connect the organization of molecular-level building blocks to the overall stability of phase-separated biomolecular systems.Significance StatementBiomolecular condensation is a phenomenon involved in the subcellular organization of living systems, yet little is known about the molecular forces that underlie their stability. Condensates consist of a biopolymer-rich phase dispersed as microdroplets in a polymer-poor continuous phase. Both phases are aqueous and, as such, the surface tension between them is low. However, how condensates are stabilized and maintain their integrity has remained unknown. Here we use a microfluidic platform to analyze the zeta potentials of condensates and show that they fit within the stability range of classical colloids. Furthermore, condensate zeta potentials correlate well with their propensity to fuse and coalesce, suggesting that emulsion theory is a new lens through which biomolecular condensation can be viewed and studied.

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Section: Discussionmentioning

confidence: 99%

Surface electrostatics govern the emulsion stability of biomolecular condensates

Welsh

Krainer

Espinosa

et al. 2020

Preprint

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“…However, TDP-43 aggregates are also observed -albeit at low frequency -in control samples 36 and, in vitro, TDP-43 can form both amyloid aggregates and liquid condensates [37][38][39][40][41] . Mutations in TDP-43 cause ~5% of familial ALS cases 17,42 , with these…”

Section: Main Textmentioning

confidence: 95%

“…TDP-43 aggregates are also present at autopsy in nearly all cases of frontotemporal dementia (FTD) that lack tau-containing inclusions (about half of all cases of FTD which is the second most common dementia) 33 . TDP-43 aggregates are also a hallmark of inclusion body myopathy and a secondary pathology in Alzheimer's, Parkinson's, and Huntington's disease [33][34][35] .However, TDP-43 aggregates are also observed -albeit at low frequency -in control samples 36 and, in vitro, TDP-43 can form both amyloid aggregates and liquid condensates [37][38][39][40][41] . Mutations in TDP-43 cause ~5% of familial ALS cases 17,42 , with these…”

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confidence: 99%

The mutational landscape of a prion-like domain

Bolognesi

Faure

Seuma

et al. 2019

Preprint

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Specific insoluble protein aggregates are the hallmarks of many neurodegenerative diseases 1-5 . For example, cytoplasmic aggregates of the RNA-binding protein are observed in 97% of cases of Amyotrophic Lateral Sclerosis (ALS) 6,7 . However, it is still unclear for ALS and other diseases whether it is the insoluble aggregates or other forms of the mutated proteins that cause these diseases that are actually toxic to cells [8][9][10][11][12][13] . Here we address this question for TDP-43 by systematically mutating 14 the protein and quantifying the effects on cellular toxicity. We generated >50,000 mutations in the intrinsically disordered prion-like domain (PRD) and observed that changes in hydrophobicity and aggregation potential are highly predictive of changes in toxicity. Surprisingly, however, increased hydrophobicity and cytoplasmic aggregation actually reduce cellular toxicity. Mutations have their strongest effects in a central region of the PRD, with variants that increase toxicity promoting the formation of more dynamic liquidlike condensates. The genetic interactions in double mutants reveal that specific structures exist in this 'unstructured' region in vivo. Our results demonstrate that deep mutagenesis is a powerful approach for probing the sequence-function relationships of intrinsically disordered proteins as well as their in vivo structural conformations.Moreover, we show that aggregation of TDP-43 is not harmful but actually protects cells, most likely by titrating the protein away from a toxic liquid-like phase.3 Main textThe conversion of specific proteins into insoluble aggregates is a hallmark of many neurodegenerative disorders, including Alzheimer's, Parkinson's, Huntington's, and Amyotrophic Lateral Sclerosis (ALS) with dominantly inherited mutations in aggregateforming proteins causing rare familial forms of these diseases 3,6,15 . However, both in humans and in animal models, there is often only a weak association between the presence of aggregates and disease progression 16,17 . Indeed, many therapeutic approaches that reduce the formation of aggregates have failed at different stages of development 12,18,19 . On the other hand, there is increasing evidence that alternative protein assemblies generated during or in parallel to the aggregation process, may be toxic [8][9][10][11]20 . Despite evidence that cellular damage may be induced either before, after or independent of the formation of insoluble aggregates, the latter are still widely assumed to be pathogenic in many neurodegenerative diseases 21,22 .For many proteins, aggregation depends critically on intrinsically disordered regions with a low sequence complexity resembling that of infectious yeast prions. These prion-like domains (PRDs) are also enriched in proteins that can form liquid-like cellular condensates 23-25 with the PRDs necessary and sufficient for liquid-demixing 26,27 . At least in vitro, insoluble aggregates can nucleate from more liquid phases 27-29 , leading to the suggestion that liquid de-mixed states can ma...

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“…Furthermore, limited observations indicate that tau LLPS may also occur in neurons (14). Because the environment inside phase separated droplets is distinct from that of the surrounding aqueous phase, LLPS may have a major effect on the formation of pathological protein aggregates in AD and other neurodegenerative disorders (17)(18)(19)(20).…”

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confidence: 99%

Zinc promotes liquid–liquid phase separation of tau protein

Singh

Boyko

et al. 2020

Journal of Biological Chemistry

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Tau is a microtubule-associated protein that plays a major role in Alzheimer's disease (AD) and other tauopathies. Recent reports indicate that, in the presence of crowding agents, tau can undergo liquid-liquid phase separation (LLPS), forming highly dynamic liquid droplets. Here, using recombinantly expressed proteins, turbidimetry, fluorescence microscopy imaging, and fluorescence recovery after photobleaching (FRAP) assays, we show that the divalent transition metal zinc strongly promotes this process, shifting the equilibrium phase boundary to lower protein or crowding agent concentrations. We observed no tau LLPS-promoting effect for any other divalent transition metal ions tested, including Mn 2+ , Fe 2+ , Co 2+ , Ni 2+ , and Cu 2+ . We also demonstrate that multiple zinc-binding sites on tau are involved in the LLPS-promoting effect and provide insights into the mechanism of this process. Zinc concentration is highly elevated in AD brains, and this metal ion is believed to be an important player in the pathogenesis of this disease. Thus, the present findings bring a new dimension to understanding the relationship between zinc homeostasis and the pathogenic process in AD and related neurodegenerative disorders. ____________________________________ https://www.jbc.org/cgi/

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The role of liquid–liquid phase separation in aggregation of the TDP-43 low-complexity domain

Cited by 282 publications

References 67 publications

Surface electrostatics govern the emulsion stability of biomolecular condensates

Surface electrostatics govern the emulsion stability of biomolecular condensates

The mutational landscape of a prion-like domain

Zinc promotes liquid–liquid phase separation of tau protein

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