Ambadipudi S, Biernat J, Riedel D, Mandelkow E, Zweckstetter M.
Liquid-liquid phase separation of the microtubule-binding repeats of the Alzheimer-related protein Tau.
Nat Commun. 2017 Aug 17;8(1):275.
PubMed.
No doubt tau protein is the culprit in its namesake diseases—the tauopathies, which include Alzheimer’s disease. Among the longstanding head-scratching problems in tau pathobiology is how tau, one of the most soluble proteins known, transforms into a neurofibrillary tangle, one of the most insoluble protein complexes known. Like a master magician who deceives us into questioning the laws of physics as objects appear and disappear over impossible distances, so tau undergoes this elusive transition in what, until now, has been obscured from view. This new work from Ambadipudi et al. and our previous study (see Zhang et al., 2017) build strong arguments that the tau transition state in vitro involves liquid-liquid phase separation (LLPS). The discovery that tau can phase separate into droplets opens the door to the rich world of coacervation. Complex coacervation requires an associative interaction between oppositely charged macromolecules or macromolecular domains to create a distinct phase that is immiscible in, while in equilibrium with, the surrounding liquid (De Jong and Bonner, Protoplasma, 24:198, 1935). Complex coacervates reside in a finely tuned state stabilized by sufficiently strong (intra- or inter-) polymer-polymer interactions, often of electrostatic nature, but not so strong as to cause polymer dehydration and precipitation. Although the concentration of macromolecules in complex coacervates can reach levels not sustainable in aqueous solutions, protein structure, dynamics, and function appear to be minimally altered as illustrated in one example by the retention of enzymatic activity (Xia et al., Biopolymers, 41:359, 1997). Many biopolymers are also capable of LLPS by self-coacervation, whose process, however, is facilitated when the polymer contains oppositely charged blocks of domains, and can be considered a mechanism closely related to complex coacervation (Das and Pappu 2013).
The key tuning parameters conducive to coacervation of the K18 tau construct (the repeat region of 4R-tau) according to Ambadipudi et al. and of the N-terminus truncated 4R-tau according to Zhang et al. include temperature, ionic strength, pH, excluded volume and protein concentration, as well as an important kinetic parameter, the time course over which coacervation conditions occur. Salt tunes the effective polyelectrolyte charges and pH affects the degree of ionization of the functional groups. When taken together, complex coacervation of tau variants is found to be an entropy-driven process by displaying a lower critical separation temperature (LCST). In the case of tau, when the conditions are finely tuned, these critical parameters approach physiological conditions in terms of temperature, ionic strength, pH, and crowding pressure. Ambadipudi et al. excluded the influence of intramolecular and intermolecular cross-linking through two native cysteine residues by mimicking the reducing environment inside cells. Monitoring the formation of droplet-like structures and their characteristic fusion due to their low interfacial tension is quite straightforward by differential interference contrast microscopy. The presence of K18 in the droplets is determined by labeling this tau variant with the fluorescent dye Alexa-488.
The discovery that tau can liquid-liquid phase separate immediately raises the question whether this phase transition is on pathway to tau fibrils. In both Ambadipudi et al. and in our study, a small increase in thioflavin T (ThT) fluorescence suggests the formation of β-sheet structures, but only after long incubation times. Using two different methods—NMR in the case of Ambadipudi et al. and EPR (electron paramagnetic resonance) in ours—the studies show that tau appears to retain its solution structure inside the droplet consistent with coacervation. The NMR spectra leaves some ambiguity as to whether tau is dynamic within the droplets or fast exchanging in and out of the droplets. With its faster timescale on the order of nanoseconds for dynamics versus micro- to milliseconds for exchange, EPR suggests that tau is actually in a dynamic state within droplets. Importantly with regard to the potential for fibril formation, the observed molecular contacts of K18 are direct evidence for crowding of the repeat domain of tau and its aggregation-prone hexapeptides. Ambadipudi et al. build on this observation by adding heparin, which is known to promote tau aggregation, to the droplets. In an elegant set of experiments they show that optimal temperature, pH, and ionic conditions for heparin-induced fibril formation resembled those of droplet formation, and that even though heparin is a very strong aggregator, its potency falls off sharply when temperature and ionic conditions are no longer conducive to droplet formation. Finally, they show that phosphorylation within the repeat domain by MARK2 enhances LLPS. This finding is important because it is known that phosphorylation of tau precedes the formation of insoluble tau deposits.
The in vitro studies lay the groundwork for the next big step: how might these phenomena operate in vivo, where life is not only many-fold more complicated but rife with emergent properties. Our previous work on tau droplets suggested that RNA could serve as a counter ion and thereby loosely tied tau LLPS to intracellular membraneless organelles such as RNA granules (Knowles et al., 1996), as well as to a set of RNA-binding proteins involved in neurodegeneration. However, Ambadipudi et al. show that tau is capable of self-coacervation with its phosphorylation domain serving as the counter ion. In moving this work to an in vivo setting, those sites where tau can become tightly packed and demix from the surrounding cytoplasm to a membraneless oragnelles is the next hurdle.
Songi Han was the co-author of this comment.
References:
Zhang X, Lin Y, Eschmann NA, Zhou H, Rauch JN, Hernandez I, Guzman E, Kosik KS, Han S.
RNA stores tau reversibly in complex coacervates.
PLoS Biol. 2017 Jul;15(7):e2002183. Epub 2017 Jul 6
PubMed.
Das RK, Pappu RV.
Conformations of intrinsically disordered proteins are influenced by linear sequence distributions of oppositely charged residues.
Proc Natl Acad Sci U S A. 2013 Aug 13;110(33):13392-7. Epub 2013 Jul 30
PubMed.
Knowles RB, Sabry JH, Martone ME, Deerinck TJ, Ellisman MH, Bassell GJ, Kosik KS.
Translocation of RNA granules in living neurons.
J Neurosci. 1996 Dec 15;16(24):7812-20.
PubMed.
Comments
University of California, Santa Barbara
Tau Packs Heat
No doubt tau protein is the culprit in its namesake diseases—the tauopathies, which include Alzheimer’s disease. Among the longstanding head-scratching problems in tau pathobiology is how tau, one of the most soluble proteins known, transforms into a neurofibrillary tangle, one of the most insoluble protein complexes known. Like a master magician who deceives us into questioning the laws of physics as objects appear and disappear over impossible distances, so tau undergoes this elusive transition in what, until now, has been obscured from view. This new work from Ambadipudi et al. and our previous study (see Zhang et al., 2017) build strong arguments that the tau transition state in vitro involves liquid-liquid phase separation (LLPS). The discovery that tau can phase separate into droplets opens the door to the rich world of coacervation. Complex coacervation requires an associative interaction between oppositely charged macromolecules or macromolecular domains to create a distinct phase that is immiscible in, while in equilibrium with, the surrounding liquid (De Jong and Bonner, Protoplasma, 24:198, 1935). Complex coacervates reside in a finely tuned state stabilized by sufficiently strong (intra- or inter-) polymer-polymer interactions, often of electrostatic nature, but not so strong as to cause polymer dehydration and precipitation. Although the concentration of macromolecules in complex coacervates can reach levels not sustainable in aqueous solutions, protein structure, dynamics, and function appear to be minimally altered as illustrated in one example by the retention of enzymatic activity (Xia et al., Biopolymers, 41:359, 1997). Many biopolymers are also capable of LLPS by self-coacervation, whose process, however, is facilitated when the polymer contains oppositely charged blocks of domains, and can be considered a mechanism closely related to complex coacervation (Das and Pappu 2013).
The key tuning parameters conducive to coacervation of the K18 tau construct (the repeat region of 4R-tau) according to Ambadipudi et al. and of the N-terminus truncated 4R-tau according to Zhang et al. include temperature, ionic strength, pH, excluded volume and protein concentration, as well as an important kinetic parameter, the time course over which coacervation conditions occur. Salt tunes the effective polyelectrolyte charges and pH affects the degree of ionization of the functional groups. When taken together, complex coacervation of tau variants is found to be an entropy-driven process by displaying a lower critical separation temperature (LCST). In the case of tau, when the conditions are finely tuned, these critical parameters approach physiological conditions in terms of temperature, ionic strength, pH, and crowding pressure. Ambadipudi et al. excluded the influence of intramolecular and intermolecular cross-linking through two native cysteine residues by mimicking the reducing environment inside cells. Monitoring the formation of droplet-like structures and their characteristic fusion due to their low interfacial tension is quite straightforward by differential interference contrast microscopy. The presence of K18 in the droplets is determined by labeling this tau variant with the fluorescent dye Alexa-488.
The discovery that tau can liquid-liquid phase separate immediately raises the question whether this phase transition is on pathway to tau fibrils. In both Ambadipudi et al. and in our study, a small increase in thioflavin T (ThT) fluorescence suggests the formation of β-sheet structures, but only after long incubation times. Using two different methods—NMR in the case of Ambadipudi et al. and EPR (electron paramagnetic resonance) in ours—the studies show that tau appears to retain its solution structure inside the droplet consistent with coacervation. The NMR spectra leaves some ambiguity as to whether tau is dynamic within the droplets or fast exchanging in and out of the droplets. With its faster timescale on the order of nanoseconds for dynamics versus micro- to milliseconds for exchange, EPR suggests that tau is actually in a dynamic state within droplets. Importantly with regard to the potential for fibril formation, the observed molecular contacts of K18 are direct evidence for crowding of the repeat domain of tau and its aggregation-prone hexapeptides. Ambadipudi et al. build on this observation by adding heparin, which is known to promote tau aggregation, to the droplets. In an elegant set of experiments they show that optimal temperature, pH, and ionic conditions for heparin-induced fibril formation resembled those of droplet formation, and that even though heparin is a very strong aggregator, its potency falls off sharply when temperature and ionic conditions are no longer conducive to droplet formation. Finally, they show that phosphorylation within the repeat domain by MARK2 enhances LLPS. This finding is important because it is known that phosphorylation of tau precedes the formation of insoluble tau deposits.
The in vitro studies lay the groundwork for the next big step: how might these phenomena operate in vivo, where life is not only many-fold more complicated but rife with emergent properties. Our previous work on tau droplets suggested that RNA could serve as a counter ion and thereby loosely tied tau LLPS to intracellular membraneless organelles such as RNA granules (Knowles et al., 1996), as well as to a set of RNA-binding proteins involved in neurodegeneration. However, Ambadipudi et al. show that tau is capable of self-coacervation with its phosphorylation domain serving as the counter ion. In moving this work to an in vivo setting, those sites where tau can become tightly packed and demix from the surrounding cytoplasm to a membraneless oragnelles is the next hurdle.
Songi Han was the co-author of this comment.
References:
Zhang X, Lin Y, Eschmann NA, Zhou H, Rauch JN, Hernandez I, Guzman E, Kosik KS, Han S. RNA stores tau reversibly in complex coacervates. PLoS Biol. 2017 Jul;15(7):e2002183. Epub 2017 Jul 6 PubMed.
Das RK, Pappu RV. Conformations of intrinsically disordered proteins are influenced by linear sequence distributions of oppositely charged residues. Proc Natl Acad Sci U S A. 2013 Aug 13;110(33):13392-7. Epub 2013 Jul 30 PubMed.
Knowles RB, Sabry JH, Martone ME, Deerinck TJ, Ellisman MH, Bassell GJ, Kosik KS. Translocation of RNA granules in living neurons. J Neurosci. 1996 Dec 15;16(24):7812-20. PubMed.
View all comments by Kenneth KosikMake a Comment
To make a comment you must login or register.