(Summary by Feng Yu)
Noriyuki Kodera, Toshio Ando
TL;DR: High-speed atomic force microscopy (HS-AFM) can reveal disordered protein conformational change in different physiological conditions including liquid droplet condensate with sub-nanometer accuracy.
Current experimental methods have limitations on revealing intrinsically disordered protein (local) structures. The NMR is the most common method for decoding IDP local structure but can not measure the structure of fully disordered regions. Other methods may not be able to reveal the real-time structural change or are limited by the distance between residues.
The author developed a new method utilising HS-AFM to uncover IDP structure change in different physiological conditions. To test their method, they measured 9 different fully disordered sequences and the result follows the power law as previously reported. Then, they apply this method to the Atg13 protein which consists of a folded HORMA domain and a long disordered N-terminal tail. They fused maltose-binding protein to the C-terminal of Atg13 (Atg13-MBP) to compare with wild-type Atg13. They observed the HORMA region in Atg13-MBP is mostly disordered and undergoes disorder-to-order transition from time to time. It indicates the N-terminal long disordered tail on the wild type Atg13 stabilized the folding of the HORMA region.
Binding between Atg13 and Atg17 can form the pre-autophagosomal structure during the canonical macroautophagy process. They added Atg13 and Atg17 into a solution containing KCl and observed the aggregation formed on the mica. When the solution was changed to NaCl solution, they observed the formation of large condensation with a diameter of 100-400nm. The HS-AFM images demonstrate that the cross-link between Atg13 and Atg17 dimers drives the condensate formation.
They further investigated the cross-link between other low-complexity IDP domains. Sup35, a translation terminator in the yeast cell. The N-terminal domain and middle domain of Sup35 (Sup35NM) are involved in amyloid formation. Monomeric Sup35NM has two highly dynamic disordered tails. After incubation for 30 min under a high concentration, the Sup35NM formed spherical oligomers. The dynamic disordered tail caused a 0.72nm gap between the spherical oligomer and surrounding monomers which indicates the tail may perform an important role in the oligomer formation.
In a word, the author describes how HS-AFM can be useful to quantify the IDP structural change, especially in different physiological conditions.
Toshio Ando is a professor at the Nano Life Science Institute, Kanazawa University.
(Summary by Sara Mingu)
Amanda M. Gleixner, Brandie Morris Verdone, Charlton G. Otte, Eric N. Anderson, Nandini Ramesh, Olivia R. Shapiro, Jenna R. Gale, Jocelyn C. Mauna, Jacob R. Mann, Katie E. Copley, Elizabeth L. Daley, Juan A. Ortega, Maria Elena Cicardi, Evangelos Kiskinis, Julia Kofler, Udai B. Pandey, Davide Trotti & Christopher J. Donnelly
TL;DR: Poly-GR accumulation in in C9-ALS/FTD disease model causes cytoplasmic mislocalization and sequestration of Nup62 and TDP-43. Nup62 and TDP-43 interaction promotes insolubility and liquid-to-solid transition.
ALS and FTD are well-known neurodegenerative diseases with many common characteristics. They share a common neuropathology, characterized by FUS and TDP43 cytoplasmic inclusions, as well as a common genetic cause, a G4C2 hexanucleotide expansion in the C9orf72 gene (C9-ALS/FTD). In particular, C9-ALS/FTD toxicity involves RNA-protein accumulations (RNA foci) and the translation of dipeptide repeats (GR, GA, PR, PA and GP); resulting in defects in various cellular processes - among which nucleocytoplasmic transport is one of the most highlighted.
Nucleocytoplasmic transport is regulated by intrinsically disordered, FG-rich protein called FG-Nups. In this study, Gleixner et al. focus on the relationship between C9-ALS/FTD and Nup62, a critical FG-Nup with important roles in nucleocytoplasmic transport and in the nuclear pore complex permeability barrier. Using a variety of methods and combining in vitro and in vivo models, the authors could show that in C9-ALS/FTD models, Nup62 and TDP-43 mislocalize to the cytoplasm. GR (polyglycine arginine) repeats form cytoplasmic, RNA-rich, stress-granule-like condensates which promote Nup62 mislocalization and additionally sequester TDP-43 and other FG-Nups such as Nup54. Furthermore, condensates where Nup62 and TDP-43 was found to colocalize, displayed solid-like characteristics with increased stability and non-spherical shape. In conclusion, this study relates Nup62 with C9-ALS/FTD-related TDP-43 inclusions and suggests a role for Nup62 cytoplasmic mislocalization to disease pathology.
Christopher Donnelly is an Assistant Professor in the Department of Neurobiology at the University of Pittsburgh (USA).
Surface electrostatics govern the emulsion stability of biomolecular condensates
(Summary by Noah Wake)
Timothy J. Welsh, Georg Krainer, Jorge R. Espinosa, Jerelle A. Joseph, Akshay Sridhar, Marcus Jahnel, William E. Arter, Kadi L. Saar, Simon Alberti, Rosana Collepardo-Guevara, and Tuomas P. J. Knowles
TL;DR: The physicochemical properties that drive and stabilize biological condensates are poorly understood, especially those that concern droplet fusion. Welsh et al. demonstrate a correlation between single-condensate zeta potential and droplet stability against fusion.
Biomolecular condensates have become extensively studied owing to their diverse functional repertoire in the cell. In disease states, the solid-to-liquid transition of biomolecular condensates have been increasingly implicated in the emergence of various neuropathies. However, the material properties of these liquid droplets have been demonstrated to significantly impact the functional properties of condensates in the cell, for example, stress adaptation and signalling depend on the ability of the droplets to resist fusion for timescales of up to hours. It is, therefore, important to gain a better understanding of the mechanisms influencing the material properties of droplets formed by various protein systems.
Using a microfluidics approach, Welsh et al. measure the zeta potentials of single condensates and correlate the results with the observation of droplet fusion via light microscopy. They find that, while the zeta potential and size distributions were polydisperse for the systems they studied, a clear correlation could be formed across their samples (a highly charged synthetic peptide (PR)25 mimicking a dipeptide repeat derived from the hexanucleotide expansion of C9orf72, FUS, and FUS G156E disease mutant) which suggests that electrostatic interactions of individual condensates may play a role in modulating their stability against fusion over various timescales. Using further multiscale molecular simulations, they find that (PR)25 and FUS both have homogenous charge distributions in the condensate core, however, (PR)25 tended to concentrate more charged PR tails at the surface of the condensates. These results demonstrate a possible mechanism in which condensates predominantly formed through electrostatic interactions may be able to respond to particular solvent environments by concentrating charged residues to the surface of condensates to stabilize them against fusion.
Simon Alberti is a Professor of Cellular Biochemistry at the Dresden University of Technology (Germany).
Rosana Collepardo-Guevara is an Associate Professor in the Department of Chemistry at the University of Cambridge (UK).
Rosana Collepardo-Guevara is an Associate Professor in the Department of Chemistry at the University of Cambridge (UK).
(Summary by Dr. Aritra Chowdhury)
Mrityunjoy Kar, Furqan Dar, Timothy J. Welsh, Laura T. Vogel, Ralf Kühnemuth, Anupa Majumdar, Georg Krainer, Titus M. Franzmann, Simon Alberti, Claus A. M. Seidel, Tuomas P. J. Knowles, Anthony A. Hyman, and Rohit V. Pappu
TL;DR: RNA binding IDPs are shown to form clusters of differnet sizes in concentrations below the critical concentration for liquid liquid phase separation.
Liquid-liquid phase separation driven by IDPs has come to the fore in the last decade, as a key mechanism in cellular organisation and function. Flory-Huggins formalism for homo-polymers provides the simplest underpinning for such phase transitions, which predicts a critical concentration (csat) above which the system splits into a dilute phase with a concentration equal to csat and a concentrated phase. Under this framework, below csat the dilute phase is almost exclusively dominated by monomeric molecules. The observations of Kar et. al., in this paper challenges this notion by elegantly demonstrating cluster formation in generic RNA binding IDPs-FUS, EWSR1 and TAF-15, in concentrations below csat.
Using (dynamic light scattering) DLS the authors demonstrated cluster formation in solutions of FUS below csat, which showed a concentration dependant increase in size. These clusters are not manifestations of micro-phase separation or micellization, neither are they nulcetion clusters seen in phase separating systems at concentration greater than csat. The clusters span a range of sizes, and the size distribution is heavily tailed. Using different techniques such as DLS, TEM (Transmission Electron Microscopy), NPT (Nano Particle Tracking) and MFD (Multi-parameter Fluorescence Detection) which are sensitive to different cluster sizes, the size distribution of the cluster can be assessed; while NPT also allows quantification of the fraction of molecules forming clusters which increases with concentration. The generality of cluster formation is emondtrated for other RNA binding IDPs such as EWSR1 and TAF-15. The authors also show the extent of coupling between cluster formation and phase separation using different perturbations such as modulation of solvent conditions and mutations; phase separation and cluster formation seems to be intimately associated in many cases. To understand the physical basis of cluster formation the authors perform lattice simulations with homopolymers and hetero-polymers; the former having uniform attractive monomer-monomer interaction energies, while the latter has specific monomers (stickers) interspersed with other monomers (spacers) with only attractive sticker-sticker interaction.The simulations reveal that for hetero-polymeric sequences, anisotropic sticker-sticker interaction gives rise to cluster formation with heavily-tailed size distribution even in absence of phase separation which is not seen for homopolymers.
These results provide new insights into dilute phase behaviour of phase separating IDPs and potential regulatory mechanisms of liquid-liquid phase separation. Furthemore; the results highlight the need for going beyond an apparent two state view of phase transitions of IDPs and development of theory and experimental modalities that can characterise such cluster formation both in-vitro and in-cella.
Rohit V. Pappu is the Gene K. Beare Distinguished Professor of Engineering at the McKelvey School of Engineering, a Professor of Biomedical Engineering and the Director of Center for Science & Engineering of Living Systems at the Washington University in St. Louis (USA).
Anthony Hyman is a Director and Research Group Leader the Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG).