Observation of liquid–liquid phase separation of ataxin-3 and quantitative evaluation of its concentration in a single droplet using Raman microscopy

Murakami, Kohji; Kajimoto, Shinji; Shibata, Daiki; Kuroi, Kunisato; Fujii, Fumihiko; Nakabayashi, Takakazu

doi:10.1039/d0sc06095j

Cited by 42 publications

(62 citation statements)

References 58 publications

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“…So far, LLPS is usually characterized in vitro by differential interference contrast (DIC) and/or fluorescence microscopy, fluorescence correlation spectroscopy 51 , turbidity, and absorption at 280 nm 52 . Microfluidic approaches 53 – 55 to characterize the phase diagram have also been proposed as well as NMR 56 , quantitative phase microscopy 57 and Raman microscopy/spectroscopy 58 , 59 techniques. However, easily approachable HTP methods for characterizing LLPS are still lacking.…”

Section: Introductionmentioning

confidence: 99%

Capillary flow experiments for thermodynamic and kinetic characterization of protein liquid-liquid phase separation

et al. 2021

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Liquid-liquid phase separation or LLPS of proteins is a field of mounting importance and the value of quantitative kinetic and thermodynamic characterization of LLPS is increasingly recognized. We present a method, Capflex, which allows rapid and accurate quantification of key parameters for LLPS: Dilute phase concentration, relative droplet size distributions, and the kinetics of droplet formation and maturation into amyloid fibrils. The binding affinity between the polypeptide undergoing LLPS and LLPS-modulating compounds can also be determined. We apply Capflex to characterize the LLPS of Human DEAD-box helicase-4 and the coacervate system ssDNA/RP3. Furthermore, we study LLPS and the aberrant liquid-to-solid phase transition of α-synuclein. We quantitatively measure the decrease in dilute phase concentration as the LLPS of α-synuclein is followed by the formation of Thioflavin-T positive amyloid aggregates. The high information content, throughput and the versatility of Capflex makes it a valuable tool for characterizing biomolecular LLPS.

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Section: Introductionmentioning

confidence: 99%

Capillary flow experiments for thermodynamic and kinetic characterization of protein liquid-liquid phase separation

et al. 2021

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“…A direct measurement of the concentration in the condensates using the 1 H- 15 N heteronuclear quantum coherence spectrum showed a much higher value of 27.7 mM for the RNA-binding protein FUS [ 10 ]. A similar concentration (around 14 mM) was obtained using Raman spectroscopy of condensates formed by ataxin-3, a protein that forms aggregates in the neurodegenerative Machado–Joseph disease [ 11 ]. The total protein concentration in the cells has been estimated to be around 2.5 mM [ 12 ].…”

Section: Introductionmentioning

confidence: 69%

Liquid–Liquid Phase Separation in Cancer Signaling, Metabolism and Anticancer Therapy

Igelmann

Lessard²,

Ferbeyre

2022

Cancers

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The cancer state is thought to be maintained by genetic and epigenetic changes that drive a cancer-promoting gene expression program. However, recent results show that cellular states can be also stably maintained by the reorganization of cell structure leading to the formation of biological condensates via the process of liquid–liquid phase separation. Here, we review the data showing cancer-specific biological condensates initiated by mutant oncoproteins, RNA-binding proteins, or lincRNAs that regulate oncogenic gene expression programs and cancer metabolism. Effective anticancer drugs may specifically partition into oncogenic biological condensates (OBC).

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“…At t 0 , the protein secondary structure inside the droplets is composed of 45% α-helical, 5% β-sheet, and 50% disordered conformation ( Table S2 ). Previous Raman studies on phase-separated proteins also have indicated the presence of disordered conformation for the low complexity domain of fused in sarcoma and ataxin-3 ( 17 , 18 ). Helical structure for TDP-43 CTD droplets has been previously reported ( 12 , 19 ); however, the Raman data indicate a greater helical content than would be expected for the short transient helix (<10%) characterized by NMR.…”

Section: Resultsmentioning

confidence: 97%

Watching liquid droplets of TDP-43CTD age by Raman spectroscopy

Shuster¹,

Lee²

2022

Journal of Biological Chemistry

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Liquid–liquid phase separation (LLPS) is a biological phenomenon wherein a metastable and concentrated droplet phase of biomolecules spontaneously forms. A link may exist between LLPS of proteins and the disease-related process of amyloid fibril formation; however, this connection is not fully understood. Here, we investigated the relationship between LLPS and aggregation of the C-terminal domain of TAR DNA-binding protein 43, an amyotrophic lateral sclerosis–related protein known to both phase separate and form amyloids, by monitoring conformational changes during droplet aging using Raman spectroscopy. We found that the earliest aggregation events occurred within droplets as indicated by the development of β-sheet structure and increased thioflavin-T emission. Interestingly, filamentous aggregates appeared outside the solidified droplets at a later time, suggestive that amyloid formation is a heterogeneous process under LLPS solution conditions. Furthermore, the secondary structure content of aggregated structures inside droplets is distinct from that in de novo fibrils, implying that fibril polymorphism develops as a result of different environments (LLPS versus bulk solution), which may have pathological significance.

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Observation of liquid–liquid phase separation of ataxin-3 and quantitative evaluation of its concentration in a single droplet using Raman microscopy

Abstract: Liquid–liquid phase separation (LLPS) plays an important role in a variety of biological processes and is also associated with protein aggregation in neurodegenerative diseases. Quantification of LLPS is necessary to...

Cited by 42 publications

References 58 publications

Capillary flow experiments for thermodynamic and kinetic characterization of protein liquid-liquid phase separation

Capillary flow experiments for thermodynamic and kinetic characterization of protein liquid-liquid phase separation

Liquid–Liquid Phase Separation in Cancer Signaling, Metabolism and Anticancer Therapy

Watching liquid droplets of TDP-43CTD age by Raman spectroscopy

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