{"doi":"10.1016/j.jbc.2021.101528","title":"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: 705-7","journal":"Journal of Biological Chemistry","year":2021,"id":166472,"datarank":1.1964388963765993,"base_score":3.5553480614894135,"endowment":3.5553480614894135,"self_citation_contribution":0.5333022092234121,"citation_network_contribution":0.6631366871531873,"self_endowment_contribution":0.5333022092234121,"citer_contribution":0.6631366871531873,"corpus_percentile":null,"corpus_rank":null,"citation_count":34,"citer_count":30,"citers_with_citation_signal":23,"citers_with_endowment":23,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9633,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2021-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":297793,"name":"Jennifer C. Lee","orcid":"0000-0003-0506-8349","position":1,"is_corresponding":false},{"id":692135,"name":"Sydney O. Shuster","orcid":"0000-0003-0996-6779","position":0,"is_corresponding":true}],"reference_count":30,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-18T23:45:49.644768Z","pmid":"34953857","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}