scholarly journals Mechanistic inferences from analysis of measurements of protein phase transitions in live cells

2020 ◽  
Author(s):  
Ammon E. Posey ◽  
Kiersten M. Ruff ◽  
Jared M. Lalmansingh ◽  
Tejbir S. Kandola ◽  
Jeffrey J. Lange ◽  
...  
Keyword(s):  
Phase Transitions ◽  
Phase Separation ◽  
Phase Behavior ◽  
Driving Forces ◽  
Local Density ◽  
Expression Profiles ◽  
Resonance Energy ◽  
Nucleation Theory ◽  
Classical Nucleation Theory ◽  
Live Cells

AbstractThe combination of phase separation and disorder-to-order transitions can give rise to ordered, semi-crystalline fibrillar assemblies that underlie prion phenomena namely, the non-Mendelian transfer of information across cells. Recently, a method known as Distributed Amphifluoric Förster Resonance Energy Transfer (DAmFRET) was developed to study the convolution of phase separation and disorder-to-order transitions in live cells. In this assay, a protein of interest is expressed to a broad range of concentrations and the acquisition of local density and order, measured by changes in FRET, is used to map phase transitions for different proteins. The high-throughput nature of this assay affords the promise of uncovering sequence-to-phase behavior relationships in live cells. Here, we report the development of a supervised method to obtain automated and accurate classifications of phase transitions quantified using the DAmFRET assay. Systems that we classify as undergoing two-state discontinuous transitions are consistent with prion-like behaviors, although the converse is not always true. We uncover well-established and surprising new sequence features that contribute to two-state phase behavior of prion-like domains. Additionally, our method enables quantitative, comparative assessments of sequence-specific driving forces for phase transitions in live cells. Finally, we demonstrate that a modest augmentation of DAmFRET measurements, specifically time-dependent protein expression profiles, can allow one to apply classical nucleation theory to extract sequence-specific lower bounds on the probability of nucleating ordered assemblies. Taken together, our approaches lead to a useful analysis pipeline that enables the extraction of mechanistic inferences regarding phase transitions in live cells.

scholarly journals Ligand Effects on Phase Separation of Multivalent Macromolecules

2020 ◽  
Author(s):  
Kiersten M. Ruff ◽  
Furqan Dar ◽  
Rohit V. Pappu
Keyword(s):  
Phase Transitions ◽  
Phase Separation ◽  
Phase Behavior ◽  
Driving Forces ◽  
Regulatory Control ◽  
Cellular Processes ◽  
Ligand Effects ◽  
Direct Measurements ◽  
In Cells

AbstractBiomolecular condensates enable spatial and temporal control over cellular processes by concentrating biomolecules into non-stoichiometric assemblies. Many condensates form via reversible phase transitions of condensate-specific multivalent macromolecules known as scaffolds. Phase transitions of scaffolds can be regulated by changing the concentrations of ligands, which are defined as non-scaffold molecules that bind to specific sites on scaffolds. Here, we use theory and computation to uncover rules that underlie ligand-mediated control over scaffold phase behavior. We use the stickers-and-spacers model wherein reversible non-covalent crosslinks among stickers drive phase transitions of scaffolds, and spacers modulate the driving forces for phase transitions. We find that the modulatory effects of ligands are governed by: the valence of ligands; whether they bind directly to stickers versus spacers; and the relative affinities of ligand-scaffold versus scaffold-scaffold interactions. In general, all ligands have a diluting effect on the concentration of scaffolds within condensates. Whereas monovalent ligands destabilize condensates, multivalent ligands can stabilize condensates by binding directly to spacers or destabilize condensates by binding directly to stickers. Bipartite ligands that bind to stickers and spacers can alter the structural organization of scaffold molecules within condensates even when they have a null effect on condensate stability. Our work highlights the importance of measuring dilute phase concentrations of scaffolds as a function of ligand concentration in cells. This can reveal whether ligands modulate scaffold phase behavior by enabling or suppressing phase separation at endogeneous levels thereby regulating the formation and dissolution of condensates in vivo.SignificancePhase transitions of multivalent macromolecules known as scaffolds help drive the formation of functional biomolecular condensates in cells. The formation and dissolution of condensates is tightly regulated, as aberrant phase behavior is associated with disease. Here, we show that distinct types of ligands can exert control over the formation and dissolution of condensates by binding to distinct sites on scaffold molecules. We further show that the extent and direction of regulation can be inferred through direct measurements of how ligands impact scaffold phase boundaries. Our findings have broad implications for understanding and modeling ligand-mediated regulation of condensates in cells, and for designing novel molecules that exert regulatory control over condensates.

scholarly journals Ligand effects on phase separation of multivalent macromolecules

2021 ◽  
Vol 118 (10) ◽  
pp. e2017184118
Author(s):  
Kiersten M. Ruff ◽  
Furqan Dar ◽  
Rohit V. Pappu
Keyword(s):  
Phase Transitions ◽  
Phase Separation ◽  
Phase Behavior ◽  
Driving Forces ◽  
Cellular Processes ◽  
Ligand Effects ◽  
Null Effect ◽  
Cross Links ◽  
Reversible Phase

Biomolecular condensates enable spatial and temporal control over cellular processes by concentrating biomolecules into nonstoichiometric assemblies. Many condensates form via reversible phase transitions of condensate-specific multivalent macromolecules known as scaffolds. Phase transitions of scaffolds can be regulated by changing the concentrations of ligands, which are defined as nonscaffold molecules that bind to specific sites on scaffolds. Here, we use theory and computation to uncover rules that underlie ligand-mediated control over scaffold phase behavior. We use the stickers-and-spacers model wherein reversible noncovalent cross-links among stickers drive phase transitions of scaffolds, and spacers modulate the driving forces for phase transitions. We find that the modulatory effects of ligands are governed by the valence of ligands, whether they bind directly to stickers versus spacers, and the relative affinities of ligand–scaffold versus scaffold–scaffold interactions. In general, all ligands have a diluting effect on the concentration of scaffolds within condensates. Whereas monovalent ligands destabilize condensates, multivalent ligands can stabilize condensates by binding directly to spacers or destabilize condensates by binding directly to stickers. Bipartite ligands that bind to stickers and spacers can alter the structural organization of scaffold molecules within condensates even when they have a null effect on condensate stability. Our work highlights the importance of measuring dilute phase concentrations of scaffolds as a function of ligand concentration in cells. This can reveal whether ligands modulate scaffold phase behavior by enabling or suppressing phase separation at endogenous levels, thereby regulating the formation and dissolution of condensates in vivo.

scholarly journals On the Role of Poly-Glutamic Acid in the Early Stages of Iron(III) (Oxy)(hydr)oxide Formation

Minerals ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 715
Author(s):  
Miodrag J. Lukić ◽  
Felix Lücke ◽  
Teodora Ilić ◽  
Katharina Petrović ◽  
Denis Gebauer
Keyword(s):  
Fourier Transform ◽  
Infrared Spectroscopy ◽  
Phase Separation ◽  
Fourier Transform Infrared Spectroscopy ◽  
Fourier Transform Infrared ◽  
Nucleation Theory ◽  
Classical Nucleation Theory ◽  
Oxide Formation ◽  
Early Stages ◽  
Titration Assay

Nucleation of minerals in the presence of additives is critical for achieving control over the formation of solids in biomineralization processes or during syntheses of advanced hybrid materials. Herein, we investigated the early stages of Fe(III) (oxy)(hydr)oxide formation with/without polyglutamic acid (pGlu) at low driving force for phase separation (pH 2.0 to 3.0). We employed an advanced pH-constant titration assay, X-ray diffraction, thermal analysis with mass spectrometry, Fourier Transform infrared spectroscopy, and scanning electron microscopy. Three stages were observed: initial binding, stabilization of Fe(III) pre-nucleation clusters (PNCs), and phase separation, yielding Fe(III) (oxy)(hydr)oxide. The data suggest that organic–inorganic interactions occurred via binding of olation Fe(III) PNC species. Fourier Transform Infrared Spectroscopy (FTIR) analyses revealed a plausible interaction motif and a conformational adaptation of the polypeptide. The stabilization of the aqueous Fe(III) system against nucleation by pGlu contrasts with the previously reported influence of poly-aspartic acid (pAsp). While this is difficult to explain based on classical nucleation theory, alternative notions such as the so-called PNC pathway provide a possible rationale. Developing a nucleation theory that successfully explains and predicts distinct influences for chemically similar additives like pAsp and pGlu is the Holy Grail toward advancing the knowledge of nucleation, early growth, and structure formation.

An intrinsically disordered pathological prion variant Y145Stop converts into self-seeding amyloids via liquid–liquid phase separation

2021 ◽  
Vol 118 (45) ◽  
pp. e2100968118
Author(s):  
Aishwarya Agarwal ◽  
Sandeep K. Rai ◽  
Anamika Avni ◽  
Samrat Mukhopadhyay
Keyword(s):  
Phase Transitions ◽  
Phase Separation ◽  
Liquid Phase ◽  
Phase Behavior ◽  
Intrinsically Disordered Proteins ◽  
Liquid Phase Separation ◽  
Disordered Proteins ◽  
Cell Physiology ◽  
Intrinsically Disordered ◽  
Liquid Liquid Phase Separation

Biomolecular condensation via liquid–liquid phase separation of intrinsically disordered proteins/regions (IDPs/IDRs) along with other biomolecules is proposed to control critical cellular functions, whereas aberrant phase transitions are associated with a range of neurodegenerative diseases. Here, we show that a disease-associated stop codon mutation of the prion protein (PrP) at tyrosine 145 (Y145Stop), resulting in a truncated, highly disordered, N-terminal IDR, spontaneously phase-separates into dynamic liquid-like droplets. Phase separation of this highly positively charged N-terminal segment is promoted by the electrostatic screening and a multitude of weak, transient, multivalent, intermolecular interactions. Single-droplet Raman measurements, in conjunction with an array of bioinformatic, spectroscopic, microscopic, and mutagenesis studies, revealed a highly mobile internal organization within the liquid-like condensates. The phase behavior of Y145Stop is modulated by RNA. Lower RNA:protein ratios promote condensation at a low micromolar protein concentration under physiological conditions. At higher concentrations of RNA, phase separation is abolished. Upon aging, these highly dynamic liquid-like droplets gradually transform into ordered, β-rich, amyloid-like aggregates. These aggregates formed via phase transitions display an autocatalytic self-templating characteristic involving the recruitment and binding-induced conformational conversion of monomeric Y145Stop into amyloid fibrils. In contrast to this intrinsically disordered truncated variant, the wild-type full-length PrP exhibits a much lower propensity for both condensation and maturation into amyloids, hinting at a possible protective role of the C-terminal domain. Such an interplay of molecular factors in modulating the protein phase behavior might have much broader implications in cell physiology and disease.

A Phase Transformation Model for the Austenitisation of Martensite in Dual-Phase Steel

2007 ◽  
Vol 539-543 ◽  
pp. 4608-4613 ◽  
Author(s):  
Richard G. Thiessen ◽  
Jilt Sietsma ◽  
I.M. Richardson
Keyword(s):  
Dual Phase Steel ◽  
Driving Forces ◽  
Field Method ◽  
Nucleation And Growth ◽  
Transformation Model ◽  
Nucleation Theory ◽  
Phase Field Method ◽  
Classical Nucleation Theory ◽  
Dual Phase ◽  
Unique Approach

This work presents a unique approach for the modelling of the austenitisation of martensite in dual-phase steels within the phase-field method. Driving forces for nucleation and growth are derived from thermodynamic databases. Routines for nucleation are based on a discretisation of the classical nucleation theory. Validation is given via dilatometric experiments.

scholarly journals Negative regulation of a ribonucleoprotein condensate driven by dilute phase oligomerization

2021 ◽  
Author(s):  
Ian Seim ◽  
Ammon E. Posey ◽  
Wilton T. Snead ◽  
Benjamin M. Stormo ◽  
Daphne Klotsa ◽  
...  
Keyword(s):  
Phase Separation ◽  
Phase Behavior ◽  
Material Properties ◽  
Driving Forces ◽  
Negative Regulation ◽  
Regulatory Control ◽  
Sequence Motif ◽  
Rich Region ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions

AbstractRibonucleoprotein bodies are exemplars of membraneless biomolecular condensates that can form via spontaneous or driven phase transitions. The fungal protein Whi3 forms compositionally distinct ribonucleoprotein condensates that are implicated in key processes such as cell-cycle control and cell polarity. Whi3 has a modular architecture that includes a Q-rich intrinsically disordered region and a tandem RNA recognition module. Here, we uncover localized order-to-disorder transitions within a 21-residue stretch of the Q-rich region. This region, which can form alpha-helical conformations, is shown to modulate protein density within Whi3-RNA condensates by driving dilute phase oligomerization. Specifically, enhancing helicity within this region enhances oligomerization in the dilute phase. This weakens the associations among disordered Q-rich regions thereby diluting the concentration of Whi3 in condensates. The opposite behavior is observed when helicity within the 21-residue stretch of the Q-rich region is abrogated. Thus, dilute phase oligomers, driven by a specific sequence motif, lead to negative regulation of the stoichiometry of protein versus RNA in the dense phase. Our findings stand in contrast to other systems where oligomerization is known to enhance the drive for phase separation. Our results highlight distinctive regulatory effects over phase behavior due to local order-to-disorder transitions within intrinsically disordered regions. This provides a way to leverage molecular scale conformational preferences and coupled intermolecular associations to regulate mesoscale phase behavior and material properties of condensates.SignificanceA large sub-class of biomolecular condensates are linked to RNA regulation and known as ribonucleoprotein (RNP) bodies. While extensive work has identified driving forces of biomolecular condensates, relatively little is known about negative regulation of assembly. Here, using a fungal RNP component, Whi3, we show that its intrinsically-disordered, Q-rich region exerts regulatory control over condensate formation through a cryptic helical region that enables the formation of dilute phase oligomers. These oligomers detour Whi3 proteins from condensates, thereby impacting the driving forces for phase separation, the protein-to-RNA ratio in condensates, and the material properties of condensates. Our findings show how nanoscale conformational equilibria can enable control over micron-scale phase equilibria.

scholarly journals Imaging the Homogeneous Nucleation During the Melting of Superheated Colloidal Crystals

Science ◽  
2012 ◽  
Vol 338 (6103) ◽  
pp. 87-90 ◽  
Author(s):  
Ziren Wang ◽  
Feng Wang ◽  
Yi Peng ◽  
Zhongyu Zheng ◽  
Yilong Han
Keyword(s):  
Phase Transitions ◽  
Homogeneous Nucleation ◽  
Critical Size ◽  
Colloidal Crystals ◽  
Nucleation Theory ◽  
Classical Nucleation Theory ◽  
Nucleation Process ◽  
Particle Exchange ◽  
Superheat Limit ◽  
Shape And Size

The nucleation process is crucial to many phase transitions, but its kinetics are difficult to predict and measure. We superheated and melted the interior of thermal-sensitive colloidal crystals and investigated by means of video microscopy the homogeneous melting at single-particle resolution. The observed nucleation precursor was local particle-exchange loops surrounded by particles with large displacement amplitudes rather than any defects. The critical size, incubation time, and shape and size evolutions of the nucleus were measured. They deviate from the classical nucleation theory under strong superheating, mainly because of the coalescence of nuclei. The superheat limit agrees with the measured Born and Lindemann instabilities.

Kinetic Aspects of Island Nucleation Derived from Near Equilibrium Growth Experiments

1999 ◽  
Vol 583 ◽  
Author(s):  
S. H. Christiansen ◽  
M. Becker ◽  
H. Wawra ◽  
M. Albrecht ◽  
H. P. Strunk
Keyword(s):  
Growth Temperature ◽  
Growth Rates ◽  
Driving Forces ◽  
Substrate Surface ◽  
High Growth ◽  
Nucleation Theory ◽  
Classical Nucleation Theory ◽  
Island Formation ◽  
Island Nucleation ◽  
Equilibrium Growth

AbstractUsing liquid phase epitaxy from Bi solution, a by its nature a near equilibrium growth process, we study the kinetics of island formation in the heteroepitaxial system SiGe/Si(001) as dependent on growth temperature, growth rate and composition (which also determines the lattice misfit between layer and substrate). As a main result island formation can be described by classical nucleation theory, moreover, it can be described as any other crystallization process such as solid state crystallization of amorphous silicon or crystallization from a melt, provided that the limited size the islands can grow into is correctly considered. In consequence, after an incubation time period that depends on the growth temperature, islands nucleate and cover the substrate surface with time. The activation energy of island nucleation is 0.84±0.13eV. The coverage with islands depends only on the undercooling and is independent of the cooling rate in case near equilibrium growth conditions are maintained. In these cases the islands have the shape of truncated pyramids with four {111}– side facets and a base width λ that only depends on the misfit f (λ ∝ 1/f2). Deviations from the equilibrium growth stage at high growth rates (thus higher growth driving forces) result in the formation of a higher density of smaller islands with smaller facet angles. At higher growth rates, some kinetic influences begin to appear indicated by the additional appearance of shallower pyramids with four {115}– facet side faces.

The initiation of crystal growth in glasses

1970 ◽  
Vol 37 (291) ◽  
pp. 741-758 ◽  
Author(s):  
P. S. Rogers
Keyword(s):  
Phase Separation ◽  
Crystal Growth ◽  
Nucleation And Growth ◽  
Nucleation Theory ◽  
Classical Nucleation Theory ◽  
Nucleating Agents ◽  
Metastable Liquid ◽  
The Third ◽  
Kinetics Of ◽  
Liquid Liquid Phase Separation

SummaryThe application of classical nucleation theory to the initiation of crystal growth in glasses is discussed. Its application to experimental results obtained for the rate of nucleation in three types of glass, one showing nucleation separately from crystal growth, another showing simultaneous nucleation and growth, and the third showing crystal growth after metastable liquid/liquid phase separation, is then described. Recent work on the kinetics of unmixing in glasses is outlined. The influence of so-called ‘nucleating agents’ in the glasses described appears to be exerted through changes in the anionic structure.

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