XB-ART-58621
Biochemistry
2021 Nov 30;6047:3566-3581. doi: 10.1021/acs.biochem.1c00465.
Show Gene links
Show Anatomy links
Clustering of Aromatic Residues in Prion-like Domains Can Tune the Formation, State, and Organization of Biomolecular Condensates.
Holehouse AS
,
Ginell GM
,
Griffith D
,
Böke E
.
???displayArticle.abstract???
In immature oocytes, Balbiani bodies are conserved membraneless condensates implicated in oocyte polarization, the organization of mitochondria, and long-term organelle and RNA storage. In Xenopus laevis, Balbiani body assembly is mediated by the protein Velo1. Velo1 contains an N-terminal prion-like domain (PLD) that is essential for Balbiani body formation. PLDs have emerged as a class of intrinsically disordered regions that can undergo various different types of intracellular phase transitions and are often associated with dynamic, liquid-like condensates. Intriguingly, the Velo1 PLD forms solid-like assemblies. Here we sought to understand why Velo1 phase behavior appears to be biophysically distinct from that of other PLD-containing proteins. Through bioinformatic analysis and coarse-grained simulations, we predict that the clustering of aromatic residues and the amino acid composition of residues between aromatics can influence condensate material properties, organization, and the driving forces for assembly. To test our predictions, we redesigned the Velo1 PLD to test the impact of targeted sequence changes in vivo. We found that the Velo1 design with evenly spaced aromatic residues shows rapid internal dynamics, as probed by fluorescent recovery after photobleaching, even when recruited into Balbiani bodies. Our results suggest that Velo1 might have been selected in evolution for distinctly clustered aromatic residues to maintain the structure of Balbiani bodies in long-lived oocytes. In general, our work identifies several tunable parameters that can be used to augment the condensate material state, offering a road map for the design of synthetic condensates.
???displayArticle.pubmedLink??? 34784177
???displayArticle.pmcLink??? PMC8638251
???displayArticle.link??? Biochemistry
Species referenced: Xenopus laevis
Genes referenced: dnai1 prnp velo1
???attribute.lit??? ???displayArticles.show???
|
|
|
|
|
|
|
|
|
|
Figure 1. Stickers-and-spacers framework that can be used to describe Velo1PLD. (A) The stickers-and-spacers framework subdivides biomolecules into sticker regions and spacer regions, whereby stickers contribute attractive interactions that drive phase transitions through multivalent interactions. (B) The Velo1 sequence architecture contains an N-terminal prion-like domain (Velo1PLD) and four fragments (F1–F4) as originally defined by Böke et al.39 The Velo1PLD sequence is shown explicitly with aromatic residues colored orange and all other residues colored black. (C) Questions of interest in this study are how sticker clustering (left) and spacer-related interactions (right) can alter the formation and equilibrium state of condensates formed by PLDs. |
|
Figure 2. Velo1PLD is enriched with aliphatic and aromatic residues and depleted of small polar residues and has highly clustered aromatic residues. (A) Log2 of fractions of different amino acids in Velo1PLD divided by equivalent fractions of the same amino acid across all X. laevis IDRs. (B) Log2 of fractions of different amino acids in Velo1PLD divided by equivalent fractions of the same amino acid across human PLDs that undergo liquidâliquid phase separation. (C) Graphical definition of the aromatic clustering parameter. (D) Assessment of aromatic clustering (y-axis) compared with the fraction of aromatic or aliphatic residues (x-axis). Among those of X. laevis PLDs, the clustering score Velo1PLD (black diamond) is higher than all but one, and among those of the PLDs from PhaseSepDB, the clustering score is higher than all but two. |
|
Figure 3. Sticker:spacer and spacer:spacer interaction strengths play a key role in determining the driving forces for phase separation. (A) Overview of the polymer models used in simulations. A 56-bead model is used (12 sticker beads, 44 spacer beads). Three parameters define the system: sticker:sticker, sticker:spacer, and spacer:spacer strength. (B) Summary of relative parameter ranges examined. The sticker:sticker strength is held fixed, and sticker:spacer and spacer:spacer interaction strengths are varied. (C) Simulations reveal that at a fixed starting volume fraction (Ï) of 0.0168 the emergence of a two-phase regime is symmetrically dependent on the sticker:spacer and spacer:spacer interaction strength. As the interaction strength increases, Ïsat decreases, and in parallel Ïden (the concentration inside the droplet) increases. (D) Varying interaction strengths can be recast as modulating the effective Flory Ï parameters or rescaling the critical temperature. As such, we can project the spacer:spacer interaction strength in the background of a fixed sticker:spacer interaction strength into a FloryâHuggins fit to analytically capture the interaction strength-dependent phase behavior. Points are simulation data, while lines are fits of data to FloryâHuggins theory. (E) Analogous analysis as in panel D but with a variable sticker:spacer strength in the background of a fixed spacer:spacer strength. Points are simulation data, while lines are fits of simulation data to FloryâHuggins theory. |
|
Figure 4. Apparent diffusion coefficient scales with droplet density. (A) Overview of the analysis approach as applied to the polymer architecture defined in Figure 3. Individual polymers are followed as they “diffuse†within a droplet and fit to extract the diffusive scaling exponent (α) and the apparent diffusion constant (Dapp). Dapp is determined only where simple Brownian diffusion is observed (i.e., α = 1), which occurs in almost all cases (see Figures S1–S3). (B) Dapp as a function of sticker:spacer and spacer:spacer strength. (C) Graphical representation of how density and Dapp relate to one another (left) and all data from panel B plotted as a single master curve of Dapp vs dense-phase volume fraction (ϕden). The linear fit to guide the eye leads to an apparent diffusion constant of 0 when the volume fraction is 1, reflecting the limit in which every lattice site is occupied such that no free sites are available for polymers to move into. |
|
Figure 5. Impact of sticker clustering can be altered by sticker:spacer strength. (A) Three polymers of equal length with equal composition but alternative sticker clustering. (B) Saturation concentration as a function of sticker clustering (x-axis) and sticker:spacer strength (top to bottom). These specific comparisons are shown also in panel D. (C) Fraction of chains in the largest cluster shown as a function of sticker:spacer, spacer:spacer, sticker clustering (top to bottom), and system temperature (left to right). (D) Saturation concentration as a function of sticker:spacer, spacer:spacer, sticker clustering (top to bottom), and system temperature (left to right). Numbers reflect the systems examined in panel B. (E) The spacer:spacer and spacer:sticker interaction strengths can determine the impact of sticker:spacer patterning by rescaling the definition of a sticker and spacer. Spacer strength here reflects the simultaneous titration of spacer:spacer and sticker:spacer interaction strength to match that of sticker:sticker strength. |
|
Figure 6. Dependence of intracondensate polymer apparent diffusion on spacer-mediated interactions that depends on sticker clustering. As the level of sticker clustering increases for polymers with a strong spacer:spacer interaction, there is little to no dependence of Dapp on sticker:spacer interactions. This suggests that in the limit of (relatively) strong spacer:spacer interactions, molecular rearrangement is dominated by spacer:spacer and sticker:sticker interactions. |
|
Figure 7. Sticker clustering tunes intradroplet organization. (A) Assortativity, a measure of the spatial mixing between stickers and spacers, is measured for the entire system as a function of sticker strength, spacer strength, and sticker clustering. For each system, the phase boundary is shown as a dashed line for reference. For the well-clustered sequences, significant deviations from a value of 0 are observed. (B) To understand the origins of these large assortativity values, we generated snapshots from distinct regions in panel A (orange beads are stickers, and black beads spacers). These revealed the intradroplet organization of stickers into local clusters and subdomains. For sequences with the most well-clustered stickers, we observe an assortativity of >0 at subsaturating concentrations due to the presence of small labile clusters. |
|
Figure 8. Architecture of Velo1 and amino acid sequences of F1 fragment variants. (A) Relative position of the F1 fragment. (B) Amino acid sequence of the wild type sequence and rationally designed F1 variants tested. |
|
Figure 9. Rationally designed Velo1 designs tune cellular localization and material state. (A) mRNAs encoding Velo1 designs and wild type Velo1 fragment F1 (Figure 8) fused to GFP were microinjected into stage I X. laevis oocytes. Oocytes were left to recover and express injected mRNAs overnight and imaged the next day. The cell membrane and nucleus are outlined in a white dashed line. (B) Internal rearrangement of fluorescent wild type or redesigned Velo1 (F1-GFP) particles after photobleaching in the Balbiani body. Note that Velo1Ali2S/Repat did not localize to the Balbiani body. (C) Overview, DIC image, and schematic of the oocyte with its Balbiani body in the Ali2S design. (D) Internal rearrangement of fluorescent redesigned Velo1 (F1-GFP) particles after photobleaching in the nucleus. Note that Velo1WT does not localize to the nucleus. (E) The fluorescent recoveries of the photobleached Velo1WT or redesigned Velo1 (F1-GFP) particles in Balbiani bodies in panel B and two other biological replicates were quantified over time. (F) The fluorescent recoveries of photobleached Velo1 designs in nuclei in panel C and two other biological replicates were quantified over time. For panels D and E, the fluorescence in the bleached region was quantified over time and normalized by an unbleached neighboring region. At least three oocytes per biological replicate were plotted. Scale bars are as indicated in the figure. |
References [+] :
Alberti,
A systematic survey identifies prions and illuminates sequence features of prionogenic proteins.
2009, Pubmed
Alberti, A systematic survey identifies prions and illuminates sequence features of prionogenic proteins. 2009, Pubmed
Banani, Biomolecular condensates: organizers of cellular biochemistry. 2017, Pubmed
Berry, RNA transcription modulates phase transition-driven nuclear body assembly. 2015, Pubmed
Boeynaems, Spontaneous driving forces give rise to protein-RNA condensates with coexisting phases and complex material properties. 2019, Pubmed
Boeynaems, Phase Separation of C9orf72 Dipeptide Repeats Perturbs Stress Granule Dynamics. 2017, Pubmed
Boeynaems, Protein Phase Separation: A New Phase in Cell Biology. 2018, Pubmed
Boke, Amyloid-like Self-Assembly of a Cellular Compartment. 2016, Pubmed , Xenbase
Boke, The balbiani body and the concept of physiological amyloids. 2017, Pubmed
Bolognesi, The mutational landscape of a prion-like domain. 2019, Pubmed
Boothby, Tardigrades Use Intrinsically Disordered Proteins to Survive Desiccation. 2017, Pubmed
Boulay, Cancer-Specific Retargeting of BAF Complexes by a Prion-like Domain. 2017, Pubmed
Brady, Structural and hydrodynamic properties of an intrinsically disordered region of a germ cell-specific protein on phase separation. 2017, Pubmed
Brangwynne, Germline P granules are liquid droplets that localize by controlled dissolution/condensation. 2009, Pubmed
Bremer, Deciphering how naturally occurring sequence features impact the phase behaviours of disordered prion-like domains. 2022, Pubmed
Burke, Residue-by-Residue View of In Vitro FUS Granules that Bind the C-Terminal Domain of RNA Polymerase II. 2015, Pubmed
Celetti, The liquid state of FG-nucleoporins mimics permeability barrier properties of nuclear pore complexes. 2020, Pubmed
Cho, Mediator and RNA polymerase II clusters associate in transcription-dependent condensates. 2018, Pubmed
Choi, LASSI: A lattice model for simulating phase transitions of multivalent proteins. 2019, Pubmed
Choi, Generalized models for bond percolation transitions of associative polymers. 2020, Pubmed
Choi, Physical Principles Underlying the Complex Biology of Intracellular Phase Transitions. 2020, Pubmed
Conicella, ALS Mutations Disrupt Phase Separation Mediated by α-Helical Structure in the TDP-43 Low-Complexity C-Terminal Domain. 2016, Pubmed
Conicella, TDP-43 α-helical structure tunes liquid-liquid phase separation and function. 2020, Pubmed
Das, Comparative roles of charge, π, and hydrophobic interactions in sequence-dependent phase separation of intrinsically disordered proteins. 2020, Pubmed
Das, A Lattice Model of Charge-Pattern-Dependent Polyampholyte Phase Separation. 2018, Pubmed
Dasmeh, The length scale of multivalent interactions is evolutionarily conserved in fungal and vertebrate phase-separating proteins. 2022, Pubmed
Dorone, A prion-like protein regulator of seed germination undergoes hydration-dependent phase separation. 2021, Pubmed
Dzuricky, De novo engineering of intracellular condensates using artificial disordered proteins. 2020, Pubmed
Elbaum-Garfinkle, The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics. 2015, Pubmed
Emenecker, Sequence determinants of in cell condensate morphology, dynamics, and oligomerization as measured by number and brightness analysis. 2021, Pubmed
Emenecker, Metapredict: a fast, accurate, and easy-to-use predictor of consensus disorder and structure. 2021, Pubmed
Feric, Coexisting Liquid Phases Underlie Nucleolar Subcompartments. 2016, Pubmed , Xenbase
Fisher, Tunable multiphase dynamics of arginine and lysine liquid condensates. 2020, Pubmed
Franzmann, Phase separation of a yeast prion protein promotes cellular fitness. 2018, Pubmed
Frey, FG-rich repeats of nuclear pore proteins form a three-dimensional meshwork with hydrogel-like properties. 2006, Pubmed
Frey, A saturated FG-repeat hydrogel can reproduce the permeability properties of nuclear pore complexes. 2007, Pubmed
Guillén-Boixet, RNA-Induced Conformational Switching and Clustering of G3BP Drive Stress Granule Assembly by Condensation. 2020, Pubmed
Han, Cell-free formation of RNA granules: bound RNAs identify features and components of cellular assemblies. 2012, Pubmed
Harmon, Intrinsically disordered linkers determine the interplay between phase separation and gelation in multivalent proteins. 2017, Pubmed
Hastings, Designer Condensates: A Toolkit for the Biomolecular Architect. 2021, Pubmed
Hertig, The primary human oocyte: some observations on the fine structure of Balbiani's vitelline body and the origin of the annulate lamellae. 1968, Pubmed
Hofweber, Phase Separation of FUS Is Suppressed by Its Nuclear Import Receptor and Arginine Methylation. 2018, Pubmed
Holehouse, Functional Implications of Intracellular Phase Transitions. 2018, Pubmed
Holehouse, CIDER: Resources to Analyze Sequence-Ensemble Relationships of Intrinsically Disordered Proteins. 2017, Pubmed
Hubstenberger, Translation repressors, an RNA helicase, and developmental cues control RNP phase transitions during early development. 2013, Pubmed
Hughes, Atomic structures of low-complexity protein segments reveal kinked β sheets that assemble networks. 2018, Pubmed
Hyman, Liquid-liquid phase separation in biology. 2014, Pubmed
Jamieson-Lucy, The vertebrate Balbiani body, germ plasm, and oocyte polarity. 2019, Pubmed
Kato, Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels. 2012, Pubmed
Kim, Mutations in prion-like domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS. 2013, Pubmed
Kloc, The Balbiani body and germ cell determinants: 150 years later. 2004, Pubmed
Koster, Glass formation and desiccation tolerance in seeds. 1991, Pubmed
Krainer, Reentrant liquid condensate phase of proteins is stabilized by hydrophobic and non-ionic interactions. 2021, Pubmed
Kwon, Phosphorylation-regulated binding of RNA polymerase II to fibrous polymers of low-complexity domains. 2013, Pubmed
Labokha, Systematic analysis of barrier-forming FG hydrogels from Xenopus nuclear pore complexes. 2013, Pubmed , Xenbase
Lancaster, PLAAC: a web and command-line application to identify proteins with prion-like amino acid composition. 2014, Pubmed
Li, Phase transitions in the assembly of multivalent signalling proteins. 2012, Pubmed
Lin, Formation and Maturation of Phase-Separated Liquid Droplets by RNA-Binding Proteins. 2015, Pubmed
Lin, Intrinsically disordered sequences enable modulation of protein phase separation through distributed tyrosine motifs. 2017, Pubmed
Lin, Sequence-Specific Polyampholyte Phase Separation in Membraneless Organelles. 2016, Pubmed
Lin, Phase Separation and Single-Chain Compactness of Charged Disordered Proteins Are Strongly Correlated. 2017, Pubmed
Lyon, A framework for understanding the functions of biomolecular condensates across scales. 2021, Pubmed
Martin, Intrinsically disordered protein regions and phase separation: sequence determinants of assembly or lack thereof. 2020, Pubmed
Martin, Valence and patterning of aromatic residues determine the phase behavior of prion-like domains. 2020, Pubmed
Mateju, An aberrant phase transition of stress granules triggered by misfolded protein and prevented by chaperone function. 2017, Pubmed
McCarty, Complete Phase Diagram for Liquid-Liquid Phase Separation of Intrinsically Disordered Proteins. 2019, Pubmed
Milles, Plasticity of an ultrafast interaction between nucleoporins and nuclear transport receptors. 2015, Pubmed
Mir, How the mother can help: studying maternal Wnt signaling by anti-sense-mediated depletion of maternal mRNAs and the host transfer technique. 2008, Pubmed , Xenbase
Molliex, Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization. 2015, Pubmed
Moses, Revealing the Hidden Sensitivity of Intrinsically Disordered Proteins to their Chemical Environment. 2020, Pubmed
Munder, A pH-driven transition of the cytoplasm from a fluid- to a solid-like state promotes entry into dormancy. 2016, Pubmed
Murakami, ALS/FTD Mutation-Induced Phase Transition of FUS Liquid Droplets and Reversible Hydrogels into Irreversible Hydrogels Impairs RNP Granule Function. 2015, Pubmed
Murray, Structure of FUS Protein Fibrils and Its Relevance to Self-Assembly and Phase Separation of Low-Complexity Domains. 2017, Pubmed
Murthy, Molecular interactions underlying liquid-liquid phase separation of the FUS low-complexity domain. 2019, Pubmed
Newman, Mixing patterns in networks. 2003, Pubmed
Niaki, Loss of Dynamic RNA Interaction and Aberrant Phase Separation Induced by Two Distinct Types of ALS/FTD-Linked FUS Mutations. 2020, Pubmed
Nott, Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles. 2015, Pubmed
Pak, Sequence Determinants of Intracellular Phase Separation by Complex Coacervation of a Disordered Protein. 2016, Pubmed
Patel, A Liquid-to-Solid Phase Transition of the ALS Protein FUS Accelerated by Disease Mutation. 2015, Pubmed
Powers, Nucleo-cytoplasmic Partitioning of ARF Proteins Controls Auxin Responses in Arabidopsis thaliana. 2019, Pubmed
Putnam, A gel phase promotes condensation of liquid P granules in Caenorhabditis elegans embryos. 2019, Pubmed
Qamar, FUS Phase Separation Is Modulated by a Molecular Chaperone and Methylation of Arginine Cation-π Interactions. 2018, Pubmed , Xenbase
Quiroz, Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers. 2015, Pubmed
Rana, Phase separation vs aggregation behavior for model disordered proteins. 2021, Pubmed
Ranganathan, Dynamic metastable long-living droplets formed by sticker-spacer proteins. 2020, Pubmed
Riback, Stress-Triggered Phase Separation Is an Adaptive, Evolutionarily Tuned Response. 2017, Pubmed
Ribbeck, The permeability barrier of nuclear pore complexes appears to operate via hydrophobic exclusion. 2002, Pubmed
Sanders, Competing Protein-RNA Interaction Networks Control Multiphase Intracellular Organization. 2020, Pubmed
Sankaranarayanan, Adaptable P body physical states differentially regulate bicoid mRNA storage during early Drosophila development. 2021, Pubmed
Schmidt, Nup98 FG domains from diverse species spontaneously phase-separate into particles with nuclear pore-like permselectivity. 2015, Pubmed , Xenbase
Schmidt, Phase separation-deficient TDP43 remains functional in splicing. 2019, Pubmed
Schmidt, In Vivo Formation of Vacuolated Multi-phase Compartments Lacking Membranes. 2016, Pubmed
Schmidt, Transport Selectivity of Nuclear Pores, Phase Separation, and Membraneless Organelles. 2016, Pubmed
Schuster, Controllable protein phase separation and modular recruitment to form responsive membraneless organelles. 2018, Pubmed , Xenbase
Schuster, Identifying sequence perturbations to an intrinsically disordered protein that determine its phase-separation behavior. 2020, Pubmed
Shimizu, Desiccation-induced structuralization and glass formation of group 3 late embryogenesis abundant protein model peptides. 2010, Pubmed
Shin, Liquid phase condensation in cell physiology and disease. 2017, Pubmed
Snead, The Control Centers of Biomolecular Phase Separation: How Membrane Surfaces, PTMs, and Active Processes Regulate Condensation. 2019, Pubmed
Sørensen, Effective concentrations enforced by intrinsically disordered linkers are governed by polymer physics. 2019, Pubmed
Statt, Model for disordered proteins with strongly sequence-dependent liquid phase behavior. 2020, Pubmed
Vernon, Pi-Pi contacts are an overlooked protein feature relevant to phase separation. 2018, Pubmed
Wang, A Prion-like Domain in Transcription Factor EBF1 Promotes Phase Separation and Enables B Cell Programming of Progenitor Chromatin. 2020, Pubmed
Wang, Regulation of RNA granule dynamics by phosphorylation of serine-rich, intrinsically disordered proteins in C. elegans. 2014, Pubmed
Wang, A Molecular Grammar Governing the Driving Forces for Phase Separation of Prion-like RNA Binding Proteins. 2018, Pubmed
Weber, Sequence-encoded material properties dictate the structure and function of nuclear bodies. 2017, Pubmed
Weber, Getting RNA and protein in phase. 2012, Pubmed
Weiner, Motif-pattern dependence of biomolecular phase separation driven by specific interactions. 2021, Pubmed
Woodruff, Centrosomes. Regulated assembly of a supramolecular centrosome scaffold in vitro. 2015, Pubmed
Woodruff, Organization and Function of Non-dynamic Biomolecular Condensates. 2018, Pubmed
Xiang, The LC Domain of hnRNPA2 Adopts Similar Conformations in Hydrogel Polymers, Liquid-like Droplets, and Nuclei. 2015, Pubmed
Yang, Single Amino Acid Substitutions in Stickers, but Not Spacers, Substantially Alter UBQLN2 Phase Transitions and Dense Phase Material Properties. 2019, Pubmed
Yang, Analyses on clustering of the conserved residues at protein-RNA interfaces and its application in binding site identification. 2020, Pubmed
Yang, G3BP1 Is a Tunable Switch that Triggers Phase Separation to Assemble Stress Granules. 2020, Pubmed
Yoo, Cellular sensing by phase separation: Using the process, not just the products. 2019, Pubmed
Yoshizawa, Nuclear Import Receptor Inhibits Phase Separation of FUS through Binding to Multiple Sites. 2018, Pubmed
You, PhaSepDB: a database of liquid-liquid phase separation related proteins. 2020, Pubmed
Zeng, Connecting Coil-to-Globule Transitions to Full Phase Diagrams for Intrinsically Disordered Proteins. 2020, Pubmed
Zervoudis, The effects of protein charge patterning on complex coacervation. 2021, Pubmed