Phase Behavior of TDP-43 and hnRNPH1: From Soluble and Aggregated States to Liquid-Like Droplets

Cytotoxic inclusions of TAR DNA-binding protein 43 (TDP-43) have been identified in various neurodegenerative diseases. Mediated by its low-complexity C-terminal domain (CTD), TDP-43 can undergo liquid-liquid phase separation (LLPS) and form aggregates and fibrils. These phase transitions are associated with pathological TDP-43 mislocalization and cytoplasmic aggregation. What determines whether and when TDP-43 adopts a liquid droplet state or forms solid aggregates, transiently or irreversibly? A full understanding of the underlying biophysical mechanisms will help explain the cellular toxicity of aberrant forms of TDP-43 and similar proteins, such as some of the large family of RNA-binding proteins. In this study, we generated recombinant full-length TDP-43 and maintained soluble-protein working solutions without the need of a solubility tag. By focusing on conditions that tune the electrostatic characteristics, we induced phase transitions among states of soluble TDP-43, solid aggregates, and liquid droplet phases in an expedient and controlled manner. We also obtained soluble working solutions of TDP-CTD and another crucial splicing factor, hnRNPH1, and investigated their phase behaviors as individual proteins and their interactions between each other during phase transitions. Using this system, we discovered a previously unreported aggregate-to-droplet transition and found evidence that phase separation of low-complexity domain (LCD)-containing proteins likely involves domain matching when they coaggregate out of solution.