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ALS/FTD Mutation-Induced Phase Transition of FUS Liquid Droplets and Reversible Hydrogels into Irreversible Hydrogels Impairs RNP Granule Function.

Murakami T, Qamar S, Lin JQ, Schierle GS, Rees E, Miyashita A, Costa AR, Dodd RB, Chan FT, Michel CH, Kronenberg-Versteeg D, Li Y, Yang SP, Wakutani Y, Meadows W, Ferry RR, Dong L, Tartaglia GG, Favrin G, Lin WL, Dickson DW, Zhen M, Ron D, Schmitt-Ulms G, Fraser PE, Shneider NA, Holt C, Vendruscolo M, Kaminski CF, St George-Hyslop P - Neuron (2015)

Bottom Line: One consequence is impairment of new protein synthesis by cytoplasmic RNP granules in axon terminals, where RNP granules regulate local RNA metabolism and translation.Nuclear FUS granules may be similarly affected.Inhibiting formation of these fibrillar hydrogel assemblies mitigates neurotoxicity and suggests a potential therapeutic strategy that may also be applicable to ALS/FTD associated with mutations in other RNA binding proteins.

View Article: PubMed Central - PubMed

Affiliation: Tanz Centre for Research in Neurodegenerative Diseases, and Departments of Medicine, Medical Biophysics and Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5S 3H2, Canada.

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Recombinant FUS Generates Gel-Like Assemblies, the Reversibility of which Is Reduced by ALS/FTD Mutants(A) Purified recombinant FUS(LC) forms clear solution at 23°C (Top left panel) but forms reversible gel-like structures at 4°C (Top center panel). Rewarming to 23°C reverts FUS(LC) to a liquid state (top left panel). Repeated cycling of wild-type FUS(LC) eventually results in the formation of irreversible gels (top right panel).(B) ∼4 to 5 assembly/disassembly cycles can be achieved by wild-type FUS(LC) (black bar) and by FUS(LC) containing benign polymorphic variants (P21H and N63S, white bars). ALS/FTD mutations (gray bars) dramatically reduce the number of cycles to ≤1 before irreversibility (∗∗p < 0.01).(C) Top panel: Coomassie gel (left) and western blot (right) of RIPA- and 8 M urea solubilisation of FUS(LC) reveal that liquid state FUS(LC) is fully soluble in RIPA buffer. Irreversible FUS(LC) is partly soluble in RIPA, but fully soluble in 8 M urea. Bottom panel: Transmission electron microscopy of human FUS inclusions ([Ci]–[Civ]) and irreversible recombinant protein gels ([Cv]–[Cviii]). Both are composed of loose networks of ∼14 nm fibrils. Scale bars are 500 nm ([Cv] and [Cvi]) and 100 nm ([Cvii] and [Cviii]).(D) Analysis of liquid, reversible gel, and irreversible gel using fluorescent particle tracking revealed that liquid (left cluster) and reversible gels (black bars, middle cluster) have viscosities similar to those of P-granules. Mutant reversible gels (gray bars, middle cluster) were more viscous than wild-type reversible gels. Irreversible gels, regardless of whether made from wild-type or ALS/FTD mutant FUS exhibit very high viscosities. N.S., not significant; ∗∗p < 0.01.Error bars are SEM.
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fig5: Recombinant FUS Generates Gel-Like Assemblies, the Reversibility of which Is Reduced by ALS/FTD Mutants(A) Purified recombinant FUS(LC) forms clear solution at 23°C (Top left panel) but forms reversible gel-like structures at 4°C (Top center panel). Rewarming to 23°C reverts FUS(LC) to a liquid state (top left panel). Repeated cycling of wild-type FUS(LC) eventually results in the formation of irreversible gels (top right panel).(B) ∼4 to 5 assembly/disassembly cycles can be achieved by wild-type FUS(LC) (black bar) and by FUS(LC) containing benign polymorphic variants (P21H and N63S, white bars). ALS/FTD mutations (gray bars) dramatically reduce the number of cycles to ≤1 before irreversibility (∗∗p < 0.01).(C) Top panel: Coomassie gel (left) and western blot (right) of RIPA- and 8 M urea solubilisation of FUS(LC) reveal that liquid state FUS(LC) is fully soluble in RIPA buffer. Irreversible FUS(LC) is partly soluble in RIPA, but fully soluble in 8 M urea. Bottom panel: Transmission electron microscopy of human FUS inclusions ([Ci]–[Civ]) and irreversible recombinant protein gels ([Cv]–[Cviii]). Both are composed of loose networks of ∼14 nm fibrils. Scale bars are 500 nm ([Cv] and [Cvi]) and 100 nm ([Cvii] and [Cviii]).(D) Analysis of liquid, reversible gel, and irreversible gel using fluorescent particle tracking revealed that liquid (left cluster) and reversible gels (black bars, middle cluster) have viscosities similar to those of P-granules. Mutant reversible gels (gray bars, middle cluster) were more viscous than wild-type reversible gels. Irreversible gels, regardless of whether made from wild-type or ALS/FTD mutant FUS exhibit very high viscosities. N.S., not significant; ∗∗p < 0.01.Error bars are SEM.

Mentions: To quantitatively assess the biophysical features that govern phase transition of FUS from soluble monomer to reversible liquid droplet and then to more stable assemblies, and to assess the consequences of these phase transitions, we adapted a previously published method (Han et al., 2012, Kato et al., 2012) that provides a simple and tractable platform to produce a working model of reversible (physiological) and irreversible (pathological) assembly of FUS into higher-order structures. Briefly, when 0.25–1 mM solutions of wild-type FUS(LC) in 200–500 mM NaCl are cooled to 4°C, they condensed into gels. Upon rewarming to 23°C, the gels re-dissolve into a clear solution (Figure 5A). This liquid→gel→liquid cycle can be assayed by placing the protein solution into a siliconized microtube and then varying the temperature between 4°C and 23°C. Liquid assemblies readily fall under gravity when the tube is inverted. Gelled assemblies remain at the bottom of the tube (Figure 5A).


ALS/FTD Mutation-Induced Phase Transition of FUS Liquid Droplets and Reversible Hydrogels into Irreversible Hydrogels Impairs RNP Granule Function.

Murakami T, Qamar S, Lin JQ, Schierle GS, Rees E, Miyashita A, Costa AR, Dodd RB, Chan FT, Michel CH, Kronenberg-Versteeg D, Li Y, Yang SP, Wakutani Y, Meadows W, Ferry RR, Dong L, Tartaglia GG, Favrin G, Lin WL, Dickson DW, Zhen M, Ron D, Schmitt-Ulms G, Fraser PE, Shneider NA, Holt C, Vendruscolo M, Kaminski CF, St George-Hyslop P - Neuron (2015)

Recombinant FUS Generates Gel-Like Assemblies, the Reversibility of which Is Reduced by ALS/FTD Mutants(A) Purified recombinant FUS(LC) forms clear solution at 23°C (Top left panel) but forms reversible gel-like structures at 4°C (Top center panel). Rewarming to 23°C reverts FUS(LC) to a liquid state (top left panel). Repeated cycling of wild-type FUS(LC) eventually results in the formation of irreversible gels (top right panel).(B) ∼4 to 5 assembly/disassembly cycles can be achieved by wild-type FUS(LC) (black bar) and by FUS(LC) containing benign polymorphic variants (P21H and N63S, white bars). ALS/FTD mutations (gray bars) dramatically reduce the number of cycles to ≤1 before irreversibility (∗∗p < 0.01).(C) Top panel: Coomassie gel (left) and western blot (right) of RIPA- and 8 M urea solubilisation of FUS(LC) reveal that liquid state FUS(LC) is fully soluble in RIPA buffer. Irreversible FUS(LC) is partly soluble in RIPA, but fully soluble in 8 M urea. Bottom panel: Transmission electron microscopy of human FUS inclusions ([Ci]–[Civ]) and irreversible recombinant protein gels ([Cv]–[Cviii]). Both are composed of loose networks of ∼14 nm fibrils. Scale bars are 500 nm ([Cv] and [Cvi]) and 100 nm ([Cvii] and [Cviii]).(D) Analysis of liquid, reversible gel, and irreversible gel using fluorescent particle tracking revealed that liquid (left cluster) and reversible gels (black bars, middle cluster) have viscosities similar to those of P-granules. Mutant reversible gels (gray bars, middle cluster) were more viscous than wild-type reversible gels. Irreversible gels, regardless of whether made from wild-type or ALS/FTD mutant FUS exhibit very high viscosities. N.S., not significant; ∗∗p < 0.01.Error bars are SEM.
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fig5: Recombinant FUS Generates Gel-Like Assemblies, the Reversibility of which Is Reduced by ALS/FTD Mutants(A) Purified recombinant FUS(LC) forms clear solution at 23°C (Top left panel) but forms reversible gel-like structures at 4°C (Top center panel). Rewarming to 23°C reverts FUS(LC) to a liquid state (top left panel). Repeated cycling of wild-type FUS(LC) eventually results in the formation of irreversible gels (top right panel).(B) ∼4 to 5 assembly/disassembly cycles can be achieved by wild-type FUS(LC) (black bar) and by FUS(LC) containing benign polymorphic variants (P21H and N63S, white bars). ALS/FTD mutations (gray bars) dramatically reduce the number of cycles to ≤1 before irreversibility (∗∗p < 0.01).(C) Top panel: Coomassie gel (left) and western blot (right) of RIPA- and 8 M urea solubilisation of FUS(LC) reveal that liquid state FUS(LC) is fully soluble in RIPA buffer. Irreversible FUS(LC) is partly soluble in RIPA, but fully soluble in 8 M urea. Bottom panel: Transmission electron microscopy of human FUS inclusions ([Ci]–[Civ]) and irreversible recombinant protein gels ([Cv]–[Cviii]). Both are composed of loose networks of ∼14 nm fibrils. Scale bars are 500 nm ([Cv] and [Cvi]) and 100 nm ([Cvii] and [Cviii]).(D) Analysis of liquid, reversible gel, and irreversible gel using fluorescent particle tracking revealed that liquid (left cluster) and reversible gels (black bars, middle cluster) have viscosities similar to those of P-granules. Mutant reversible gels (gray bars, middle cluster) were more viscous than wild-type reversible gels. Irreversible gels, regardless of whether made from wild-type or ALS/FTD mutant FUS exhibit very high viscosities. N.S., not significant; ∗∗p < 0.01.Error bars are SEM.
Mentions: To quantitatively assess the biophysical features that govern phase transition of FUS from soluble monomer to reversible liquid droplet and then to more stable assemblies, and to assess the consequences of these phase transitions, we adapted a previously published method (Han et al., 2012, Kato et al., 2012) that provides a simple and tractable platform to produce a working model of reversible (physiological) and irreversible (pathological) assembly of FUS into higher-order structures. Briefly, when 0.25–1 mM solutions of wild-type FUS(LC) in 200–500 mM NaCl are cooled to 4°C, they condensed into gels. Upon rewarming to 23°C, the gels re-dissolve into a clear solution (Figure 5A). This liquid→gel→liquid cycle can be assayed by placing the protein solution into a siliconized microtube and then varying the temperature between 4°C and 23°C. Liquid assemblies readily fall under gravity when the tube is inverted. Gelled assemblies remain at the bottom of the tube (Figure 5A).

Bottom Line: One consequence is impairment of new protein synthesis by cytoplasmic RNP granules in axon terminals, where RNP granules regulate local RNA metabolism and translation.Nuclear FUS granules may be similarly affected.Inhibiting formation of these fibrillar hydrogel assemblies mitigates neurotoxicity and suggests a potential therapeutic strategy that may also be applicable to ALS/FTD associated with mutations in other RNA binding proteins.

View Article: PubMed Central - PubMed

Affiliation: Tanz Centre for Research in Neurodegenerative Diseases, and Departments of Medicine, Medical Biophysics and Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5S 3H2, Canada.

Show MeSH
Related in: MedlinePlus