TDP-43 and HSP70 phase separate into anisotropic, intranuclear liquid spherical annuli

Abstract

One Sentence Summary:Acetylation of TDP-43 drives its phase separation into spherical annuli that form a liquid-insidea-liquid-inside-a-liquid. AbstractThe RNA binding protein TDP-43 naturally phase separates within cell nuclei and forms cytoplasmic aggregates in age-related neurodegenerative diseases. Here we show that acetylation-mediated inhibition of TDP-43 binding to RNA produces co-de-mixing of acetylated and unmodified TDP-43 into symmetrical, intranuclear spherical annuli whose shells and cores have liquid properties. Shells are anisotropic, like liquid crystals. Consistent with our modelling predictions that annulus formation is driven by components with strong self-interactions but weak interaction with TDP-43, the major components of annuli cores are identified to be HSP70 family proteins, whose chaperone activity is required to maintain liquidity of the core. Proteasome inhibition, mimicking reduction in proteasome activity during aging, induces TDP-43-containing annuli in neurons in rodents. Thus, we identify that TDP-43 phase separation is regulated by acetylation, proteolysis, and ATPase-dependent chaperone activity of HSP70.A seminal discovery in the last decade has been recognition that large biological molecules, especially RNA binding proteins, can undergo liquid-liquid phase separation (LLPS), resembling oil droplets in vinegar. Under certain physical conditions, proteins, nucleic acids, or a mixture of both in a complex solution can form two phases, a condensed de-mixed phase and more dilute aqueous phase (1). Inside the cell, proteins and/or nucleic acids de-mix into a condensed phase to form membraneless organelles which have been proposed to promote biological functions (2).One membraneless organelle is the nucleolus, first described in the early 1800's and now recognized to be composed of proteins that undergo LLPS (3-5). Discovery that P-granules are de-mixed compartments with liquid behaviour brought widespread recognition to LLPS and its ability to mediate subcellular compartmentalization in a biological context (6). Phase separation has been also proposed for heterochromatin and RNA bodies (1, 6, 7). LLPS potentially underlies the operational principle governing formation of important organelles and structures, such as centrosomes, nuclear pore complexes, and super enhancers (8-10).Few mechanisms to modulate intracellular LLPS have been identified. Disease-causing mutations of proteins such as FUS (11) and HNRNPA2B1 (12) alter their LLPS behaviors in vitro, but the physiological or pathological relevance of their intracellular LLPS has not been fully elucidated. Random, multivalent interactions among intrinsically disordered, low complexity domains (LCDs) of proteins are a major driving force for LLPS in vitro. Oligomerization of other domains is thought to be required for LLPS in vivo (13). The inherent randomness of multivalent interactions (14) favors a model of disordered alignment -a conventional liquid phase in which molecules randomly move. The evidence is compelling that multi...

Enhanced detection of nucleotide repeat mRNA with hybridization chain reaction

“…Alternatively, repeat RNAs may interact with nuclear stress bodies. These complexes result from nuclear re-localization of heat shock factors, including HSF1 and HSP70, as well as RNA factors, including TDP-43, with satellite III repeat RNAs (Udan-Johns et al 2014; Metz et al 2004;Frottin et al 2019;Yu et al 2020). We observed a significant increase in co-localization between nuclear TDP-43 and G4C2 RNA.…”

Shape Recovery of Deformed Biomolecular Droplets: Dependence on Condensate Viscoelasticity

“…(72) for a Newtonian fluid. In the limit ) → 0, the two recovery rates become infinite and 6 , *. 6 and *1 6 get closer and closer to each other as ) / A approaches 1, i.e., when the Newtonian component of the complex shear modulus becomes dominant.…”

“…7 , i.e., the time constant of the initial decay, which we predict to be significantly affected by the shear relaxation rate [see *. 6 given by Eq.…”

“…The kinematic boundary condition [Eq. (6)] yields *̇( ) * (cos ) = 9 at = . (26) Subscripts and denote the components of the velocity.…”

“…Phase-separated biomolecular condensates exhibit different extents of liquidity, which may be crucial for cellular functions and correlated with diseases such as neurodegeneration and cancer [1][2][3][4][5][6][7]. Driven by interfacial tension (or capillarity), condensates that are more liquid-like retain a spherical shape and hence appear as micro-sized droplets.…”