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

Abstract

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. 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. Interests in phase separation of amyloidogenic proteins have intensified recently as key features in liquid–liquid phase separation (LLPS)—low sequence complexity and conformational disorder—are also prevalent in amyloid formation (1Boeynaems S. Alberti S. Fawzi N.L. Mittag T. Polymenidou M. Rousseau F. Schymkowitz J. Shorter J. Wolozin B. Bosch L.V.D. Tompa P. Fuxreiter M. Protein phase separation: A new phase in cell biology.Trends Cell Biol. 2018; 28: 420-435Google Scholar, 2Babinchak W.M. Surewicz W.K. Studying protein aggregation in the context of liquid-liquid phase separation using fluorescence and atomic force microscopy, fluorescence and turbidity assays, and FRAP.Bio Protoc. 2020; 10e3489Google Scholar, 3Zbinden A. Pérez-Berlanga M. De Rossi P. Polymenidou M. Phase separation and neurodegenerative diseases: A disturbance in the force.Dev. Cell. 2020; 55: 45-68Google Scholar). A number of pathological amyloids, including tau and α-synuclein, have been shown to phase separate and form liquid droplets in vitro (4Lin Y. Fichou Y. Zeng Z. Hu N.Y. Han S. Electrostatically driven complex coacervation and amyloid aggregation of tau are independent processes with overlapping conditions.ACS Chem. Neurosci. 2020; 11: 615-627Google Scholar, 5Ray S. Singh N. Kumar R. Patel K. Pandey S. Datta D. Mahato J. Panigrahi R. Navalkar A. Mehra S. Gadhe L. Chatterjee D. Sawner A.S. Maiti S. Bhatia S. et al.α-Synuclein aggregation nucleates through liquid-liquid phase separation.Nat. Chem. 2020; 12: 705-716Google Scholar). Furthermore, TAR DNA-binding protein 43 (TDP-43) and fused in sarcoma protein, proteins associated with phase-separated compartments (e.g., stress granules) in cells (6Dewey C.M. Cenik B. Sephton C.F. Johnson B.A. Herz J. Yu G. TDP-43 aggregation in neurodegeneration: Are stress granules the key?.Brain Res. 2012; 1462: 16-25Google Scholar, 7Murray D.T. Kato M. Lin Y. Thurber K.R. Hung I. McKnight S.L. Tycko R. Structure of FUS protein fibrils and its relevance to self-assembly and phase separation of low-complexity domains.Cell. 2017; 171: 615-627.e16Google Scholar), also have amyloid-forming domains (7Murray D.T. Kato M. Lin Y. Thurber K.R. Hung I. McKnight S.L. Tycko R. Structure of FUS protein fibrils and its relevance to self-assembly and phase separation of low-complexity domains.Cell. 2017; 171: 615-627.e16Google Scholar, 8Shuster S.O. Lee J.C. Tryptophan probes of TDP-43 C-terminal domain amyloid formation.J. Phys. Chem. B. 2021; 125: 3781-3789Google Scholar). The relationship between phase separation, protein aggregation, and disease remains to be elucidated. A prevailing hypothesis suggests that protein droplets could serve as loci of aggregation because of the hyperconcentrated pool of proteins (5Ray S. Singh N. Kumar R. Patel K. Pandey S. Datta D. Mahato J. Panigrahi R. Navalkar A. Mehra S. Gadhe L. Chatterjee D. Sawner A.S. Maiti S. Bhatia S. et al.α-Synuclein aggregation nucleates through liquid-liquid phase separation.Nat. Chem. 2020; 12: 705-716Google Scholar, 6Dewey C.M. Cenik B. Sephton C.F. Johnson B.A. Herz J. Yu G. TDP-43 aggregation in neurodegeneration: Are stress granules the key?.Brain Res. 2012; 1462: 16-25Google Scholar). This is evidenced by the observation that both phase-separated structures in vitro and in vivo can lose fluidity over time, preceding aggregation (5Ray S. Singh N. Kumar R. Patel K. Pandey S. Datta D. Mahato J. Panigrahi R. Navalkar A. Mehra S. Gadhe L. Chatterjee D. Sawner A.S. Maiti S. Bhatia S. et al.α-Synuclein aggregation nucleates through liquid-liquid phase separation.Nat. Chem. 2020; 12: 705-716Google Scholar, 9Lin Y. Protter D.S.W. Rosen M.K. Parker R. Formation and maturation of phase-separated liquid droplets by RNA-binding proteins.Mol. Cell. 2015; 60: 208-219Google Scholar, 10Ding Q. Chaplin J. Morris M.J. Hilliard M.A. Wolvetang E. Ng D.C.H. Noakes P.G. TDP-43 mutation affects stress granule dynamics in differentiated NSC-34 motoneuron-like cells.Front. Cell Dev. Biol. 2021; 9: 611601Google Scholar). Of note, recent in vitro work has suggested that TDP-43 amyloid aggregates can directly emerge from droplets as visualized by atomic force microscopy on dried samples, but how this transition occurs remains ill defined (11Babinchak W.M. Haider R. Dumm B.K. Sarkar P. Surewicz K. Choi J.-K. Surewicz W.K. The role of liquid–liquid phase separation in aggregation of the TDP-43 low-complexity domain.J. Biol. Chem. 2019; 294: 6306-6317Google Scholar). Thus, it is essential to evaluate protein conformation state(s) inside droplets and to monitor how they change with time to determine a possible mechanistic connection to amyloid formation. Here, we investigated conformational changes of the C-terminal domain (CTD) of TDP-43 (TDP-43CTD) during the aging process of droplets with spatial resolution by pairing a Raman spectrometer with an inverted microscope. TDP-43CTD was chosen because its phase separation and aggregation into amyloid fibrils have been established (8Shuster S.O. Lee J.C. Tryptophan probes of TDP-43 C-terminal domain amyloid formation.J. Phys. Chem. B. 2021; 125: 3781-3789Google Scholar, 11Babinchak W.M. Haider R. Dumm B.K. Sarkar P. Surewicz K. Choi J.-K. Surewicz W.K. The role of liquid–liquid phase separation in aggregation of the TDP-43 low-complexity domain.J. Biol. Chem. 2019; 294: 6306-6317Google Scholar, 12Conicella A.E. Zerze G.H. Mittal J. Fawzi N.L. ALS mutations disrupt phase separation mediated by α-helical structure in the TDP-43 low-complexity C-terminal domain.Structure. 2016; 24: 1537-1549Google Scholar). Specifically, a TDP-43CTD mutant (W334F/W385F/W412F, referred to as Wfree) was used because of its improved solubility and purification yield (Fig. 1A). Raman spectroscopy was utilized because of experimental simplicity; it is an intrinsic (i.e., probe free) measurement and provides direct information on protein secondary structure, in which α-helix, β-sheet, and disordered regions exhibit characteristic amide backbone frequencies (13Movasaghi Z. Rehman S. Rehman D.I.U. Raman spectroscopy of biological tissues.Appl. Spectrosc. Rev. 2007; 42: 493-541Google Scholar, 14Devitt G. Howard K. Mudher A. Mahajan S. Raman spectroscopy: An emerging tool in neurodegenerative disease research and diagnosis.ACS Chem. Neurosci. 2018; 9: 404-420Google Scholar). LLPS of Wfree was initiated by buffer exchange; droplets are evident immediately using bright-field microscopy (Fig. 1B, top). Upon incubation, the protein exhibits a canonical sigmoidal aggregation curve as evaluated by thioflavin-T (ThT), an amyloid-specific fluorophore (Fig. 1C) (15Biancalana M. Koide S. Molecular mechanism of thioflavin-T binding to amyloid fibrils.Biochim. Biophys. Acta. 2010; 1804: 1405-1412Google Scholar). Postaggregation at 48 h, bright-field (Fig. 1B, bottom) images show the persistence of droplets along with large fibrous aggregates, and transmission electron microscopy (TEM; Fig. 1D) reveals the existence of amyloid fibrils. Clearly, Wfree phase separates and aggregates into amyloid fibrils under the same solution conditions. However, it is unclear what role LLPS plays in this amyloid formation process. To address this question, protein secondary structural changes of droplets were measured by Raman spectroscopy. Phase-separated droplets are first visualized by bright-field microscopy. Then, Raman spectra are measured at selected locations and monitored up to 48 h. Bright-field images taken immediately (t0) and after 4 (t4 h) and 24 h (t24 h) indicate that droplet distortions appear at 4 h and become pervasive at 24 h (Fig. 2A), highlighting a time-dependent transformation. Correspondingly, Raman spectra collected from droplets show unique features (Fig. 2B). At t0, the amide-I band is broad, which is characteristic of complex mixture of secondary structural components (16Flynn J.D. Lee J.C. Raman fingerprints of amyloid structures.Chem. Commun. 2018; 54: 6983-6986Google Scholar). By 4 h, however, a sharper component emerges at 1669 cm−1, consistent with β-sheet structure development (13Movasaghi Z. Rehman S. Rehman D.I.U. Raman spectroscopy of biological tissues.Appl. Spectrosc. Rev. 2007; 42: 493-541Google Scholar, 16Flynn J.D. Lee J.C. Raman fingerprints of amyloid structures.Chem. Commun. 2018; 54: 6983-6986Google Scholar), which increases modestly in the following 20 h. This increase in β-sheet character as the droplets age is recapitulated by a red-shifted amide-III peak. To quantify the differences, the amide-I regions were decomposed into individual peaks (Table S1 and Fig. 2C): aromatic side chains (∼1604 cm−1), water (Fig. S1), and secondary structure components—α-helix (∼1657 cm−1), β-sheet (∼1669 cm−1), and disordered (∼1683 cm−1). At t0, 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 (17Murthy A.C. Dignon G.L. Kan Y. Zerze G.H. Parekh S.H. Mittal J. Fawzi N.L. Molecular interactions underlying liquid-liquid phase separation of the FUS low complexity domain.Nat. Struct. Mol. Biol. 2019; 26: 637-648Google Scholar, 18Murakami K. Kajimoto S. Shibata D. Kuroi K. Fujii F. Nakabayashi T. Observation of liquid–liquid phase separation of ataxin-3 and quantitative evaluation of its concentration in a single droplet using Raman microscopy.Chem. Sci. 2021; 12: 7411-7418Google Scholar). Helical structure for TDP-43CTD droplets has been previously reported (12Conicella A.E. Zerze G.H. Mittal J. Fawzi N.L. ALS mutations disrupt phase separation mediated by α-helical structure in the TDP-43 low-complexity C-terminal domain.Structure. 2016; 24: 1537-1549Google Scholar, 19Conicella A.E. Dignon G.L. Zerze G.H. Schmidt H.B. D'Ordine A.M. Kim Y.C. Rohatgi R. Ayala Y.M. Mittal J. Fawzi N.L. TDP-43 α-helical structure tunes liquid-liquid phase separation and function.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 5883-5894Google Scholar); however, the Raman data indicate a greater helical content than would be expected for the short transient helix (<10%) characterized by NMR. Upon aging for 4 h, conformations with increased β-sheet structure are observed with some decrease in the disordered component but no significant change in helical content (Table S2). Interestingly, a loss of water content is also evident. Formation of β-sheet structure suggests that protein aggregation is occurring, which is supported by the observation of dehydration. In the following 20 h, there were insignificant spectroscopic changes. These trends were consistent across independent experiments (Fig. S2 and Table S3). The presence of α-helical conformation is unexpected as TDP-43CTD aggregates are characterized to be β-sheet rich (8Shuster S.O. Lee J.C. Tryptophan probes of TDP-43 C-terminal domain amyloid formation.J. Phys. Chem. B. 2021; 125: 3781-3789Google Scholar, 20Li Q. Babinchak W.M. Surewicz W.K. Cryo-EM structure of amyloid fibrils formed by the entire low complexity domain of TDP-43.Nat. Commun. 2021; 12: 1620Google Scholar). The stabilization of the polypeptide structure inside the droplets by 4 h is also intriguing as this corresponds to the lag phase of aggregation. To interrogate this further, we turned to confocal fluorescence microscopy and fluorescence recovery after photobleaching (FRAP) experiments using ThT to provide information on whether amyloid aggregation and solidification has occurred, respectively. There is obvious structural maturation in which a higher ThT intensity is measured at 24 h, consistent with amyloid formation (Fig. 3, A and B). Upon photobleaching, the 4 h droplets quickly recover (Figs. 3C and S3), indicating freely diffusing fluorophores in exchange with the bulk solution. In contrast, there is sequestered ThT at 24 h, suggestive of solidification. Notably, emissive filamentous aggregates are also now apparent at 24 h. These results show that the droplets are evolving from 4 to 24 h, even though the protein secondary structures within them remain similar. TEM characterization at the ultrastructural level also indicates differences between the two times. At 4 h, only a few filamentous aggregates are observed (Fig. 3D), which become numerous, larger, bundled fibrils by 24 h (Fig. 3E). We note that these filaments at 4 h are not associated with droplets and appear similar to the intermediate fibrils previously observed in non–phase-separating conditions (i.e., no salt) for TDP-43CTD (8Shuster S.O. Lee J.C. Tryptophan probes of TDP-43 C-terminal domain amyloid formation.J. Phys. Chem. B. 2021; 125: 3781-3789Google Scholar). This led us to question whether the aggregation process that leads to large fibril bundles visible in the bright-field and confocal fluorescence images is in fact distinct from droplet solidification. To test this hypothesis, we once again turned to Raman spectroscopy to delineate any spectral differences between the fibrous aggregates and the droplets at 48 h. Because both solidified droplets and filamentous aggregates (referred to as fibrils) are present in large numbers, measurements can be made within droplets and fibrils in the same field of view (Fig. 4A). Both the amide-III (Fig. 4B) and amide-I (Fig. 4C) regions display distinctive spectral features for droplets and fibrils. In the amide-III region, there is a single peak in the droplets, which resolves into two peaks at 1236 and 1249 cm−1 in the fibrils. A new peak also appears at 1298 cm−1 in the fibrils, supportive of different conformations. The difference spectrum of the amide-I region (Fig. 4C, inset) highlights an enhancement of the β-sheet peak (1665 cm−1) in the fibrils as compared with the droplets. Based on the fits, there is more than a twofold increase (24–60%) of the β-sheet component along with a comparable decrease (39–13%) of the α-helical component in the fibrils (Fig. S4 and Table S4). This change in secondary structural composition along with a small red-shift of the amide-I band (∼2 cm−1) is highly reproducible (Fig. S5 and Table S5), substantiating that the fibrils formed in bulk solution are distinct from the aggregates inside droplets. While it is plausible that these differences could represent incomplete aggregation in droplets compared with fibrils, the respective FRAP data would indicate otherwise, as neither the droplets or fibrils have observable exchange with the bulk solution (Fig. 4D). We cannot, however, rule out interplay between the two species (e.g., nucleation off the side of a hardened droplet) as they occur in the same area with fibrils appearing near, around, and even on top of droplets. Moreover, droplets may also have divergent paths, possibly with some droplets remaining liquid and transforming into fibrils. However, all droplets examined here solidified. Interestingly, seeded aggregation of TDP-43CTD was shown to be delayed under LLPS conditions (21Pakravan D. Michiels E. Bratek-Skicki A. De Decker M. Van Lindt J. Alsteens D. Derclaye S. Van Damme P. Schymkowitz J. Rousseau F. Tompa P. Van Den Bosch L. Liquid–liquid phase separation enhances TDP-43 LCD aggregation but delays seeded aggregation.Biomolecules. 2021; 11: 548Google Scholar), suggestive of underlying structural incompatibility of the aggregates. This then could explain the observed differences in ThT activities of the aggregates in droplets and fibrils, reflecting fibril polymorphism as reported in other amyloids (22Watson M.D. Lee J.C. N-terminal acetylation affects α-synuclein fibril polymorphism.Biochemistry. 2019; 58: 3630-3633Google Scholar). In summary, this work demonstrates that Raman spectroscopy is a simple and powerful approach to study protein conformational changes in LLPS. We have directly observed droplet maturation of TDP-43CTD, offering detailed structural information with spatial context, which would be by bulk TDP-43CTD in droplets a mixture of α-helical and disordered but as time β-sheet structure consistent with amyloid formation within droplets. In a of filamentous aggregates is outside the droplets, after which time the solidified droplets are not in exchange with the solution. These results that TDP-43CTD aggregation in conditions is with aggregation first within droplets, by the formation of amyloids in solution from the remaining pool of both of aggregates are amyloid in TDP-43CTD structures in droplets and fibrils are and separate on unique Raman spectroscopic features and ThT intensity polymorphism is in some disease as α-synuclein in with and L. L. G. J. B. K. A. R. and characterization of two α-synuclein Commun. Scholar, P. J.D. L. N. of α-synuclein aggregated species and possible in J. Mol. Sci. 2020; Scholar), this intriguing whether different structures could be in the of amyotrophic lateral and A. G. A. L. F. Van S. R. The structural differences between α-synuclein of and with 2020; Scholar). There is for of similar Raman not only with other proteins but also by from as and to information on LLPS. Raman spectroscopy is with M.D. J.D. Lee J.C. Raman spectral of α-synuclein amyloid fibrils in Chem. 2021; Scholar, K. L. and of aggregates by Raman of Sci. 2020; this could be to evaluate the composition of biomolecules in phase-separated compartments and in all used were from TDP-43CTD was a from Fawzi (12Conicella A.E. Zerze G.H. Mittal J. Fawzi N.L. ALS mutations disrupt phase separation mediated by α-helical structure in the TDP-43 low-complexity C-terminal domain.Structure. 2016; 24: 1537-1549Google Scholar). The of an N-terminal by to mutations and were a using the following and or TDP-43CTD was into and as previously (8Shuster S.O. Lee J.C. 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