Spatiotemporal Organization Research Articles

Spatiotemporal organization and correlation of tip-focused exocytosis and endocytosis in regulating pollen tube tip growth

Pollen tube polar growth is a key cellular process during plant fertilization and is regulated by tip-focused exocytosis and endocytosis. However, the spatiotemporal dynamics and localizations of apical exocytosis and endocytosis in the tip region are still a matter of debate. Here, we use a refined spinning-disk confocal microscope coupled with fluorescence recovery after photobleaching for sustained live imaging and quantitative analysis of rapid vesicular activities in growing pollen tube tips. We traced and analyzed the occurrence site of exocytic plasma membrane-targeting of Arabidopsis secretory carrier membrane protein 4 and its subsequent endocytosis in tobacco pollen tube tips. We demonstrated that the pollen tube apex is the site for both vesicle polar exocytic fusion and endocytosis to take place. In addition, we disrupted either tip-focused exocytosis or endocytosis and found that their dynamic activities are closely correlated with one another basing on the spatial organization of actin fringe. Collectively, our findings attempt to propose a new exocytosis and endocytosis-coordinated yin-yang working model underlying the apical membrane organization and dynamics during pollen tube tip growth.

The Role of Liquid-Liquid Phase Separation in Actin Polymerization.

To date, it has been shown that the phenomenon of liquid-liquid phase separation (LLPS) underlies many seemingly completely different cellular processes. This provided a new idea of the spatiotemporal organization of the cell. The new paradigm makes it possible to provide answers to many long-standing, but still unresolved questions facing the researcher. In particular, spatiotemporal regulation of the assembly/disassembly of the cytoskeleton, including the formation of actin filaments, becomes clearer. To date, it has been shown that coacervates of actin-binding proteins that arise during the phase separation of the liquid-liquid type can integrate G-actin and thereby increase its concentration to initiate polymerization. It has also been shown that the activity intensification of actin-binding proteins that control actin polymerization, such as N-WASP and Arp2/3, can be caused by their integration into liquid droplet coacervates formed by signaling proteins on the inner side of the cell membrane.

Event Building Algorithm in a Distributed Stream Processing Data Acquisition Platform: D-Matrix

Event building is an important task for data acquisition (DAQ) systems in high-energy physics experiments. D-Matrix is designed as a generic distributed stream processing DAQ platform, containing hardware description language (HDL)-based modules and software modules. In the D-Matrix system, the data transfer is abstracted into the form of a stream, and the event building is implemented through a cascade of various reusable stream processing modules. These stream processing modules will determine their work modes automatically based on a set of attributes, which describe the abstracted data stream characteristics, such as spatiotemporal organization and granularity. This article introduces the event building architecture, the physics data structure abstraction, and the algorithmic implementation of specific event building modules in the D-Matrix system. An application of the D-Matrix event building architecture in the CSR external-target experiment is presented in the end.

Physical determinants of multiphase organisation in multi-component protein/RNA condensates

The formation of biomolecular condensates via phase separation achieves spatiotemporal organisation within the cell by creating intracellular microenvironments that contribute to the regulation of chemical reactions. Internal subcompartmentalisation of biomolecules to form multiphase architectures has been observed in a number of biomolecular condensates, where immiscible liquid-like phases with different compositions coexist within the same compartment. Understanding the biophysical mechanisms that drive the formation of multiphase condensates is highly desirable since these condensates are responsible for mediating many fundamental biological processes. Here, we couple a genetic algorithm to our residue-resolution coarse-grained model, Mpipi, to evolve two-component protein mixtures towards increasing multiphasicity (i.e., demixing into two compositionally distinct liquid-like phases). Given a target protein, our method allows us to predict a partner protein that supports multiphasic compartmentalisation and investigate the underlying thermodynamics of the multiphasicity. We find that the balance of homo- and heterotypic interaction energies between the two components is important for controlling multiphase organisation: a large difference between homotypic interactions of the two components favours demixing, and the strength of heterotypic interactions should be small but comparable to that of the weaker homotypic interactions for multilayered condensates to form. Our approach for designing multilayered condensates achieves this balance of interaction energies by optimising the composition and patterning of the protein sequences. Lastly, since many multiphase condensates in cells contain RNA, we extend our approach to include RNA as a third component and investigate the role of protein-RNA interactions in stabilising condensates and multilayered architectures.

Dynamic communication between TRPC3 and IP3R shape Ca2+ signaling signatures.

Physical and functional coupling between TRPC channels and IP3 receptors (IP3R) has been proposed as a pivotal mechanism in cellular Ca2+ signaling. Here we set out to explore the impact of TRPC3-IP3R communication at the level of spatiotemporal organisation of Ca2+ signaling. The low affinity Ca2+ sensor R-GECO1.2 was fused as a reporter to the C-terminus of TRPC3 and the channel function of this construct was modified specifically by point mutations. Cytoplasmic Ca2+ changes at the TRPC3 channels expressed in HEK293 cells originated from both the entry of Ca2+ through the TRPC channel and Ca2+ mobilization via IP3R. Local Ca2+ changes within the TRPC3 signaling domain were predominantly biphasic with IP3R-dependent initial Ca2+ transients, while exclusively monophasic signals were recorded when all three IP3R isoforms were lacking. Abrogation of Ca2+ entry through TRPC3 by mutational impairment of Ca2+ permeability (E630Q), cation permeation (E630K), or DAG sensitivity (G652A), promoted local Ca2+ oscillations. Ca2+ signals at E630Q, E630K, and G652A channels featured initial Ca2+ transients along with oscillatory activity. By contrast, oscillations and initial Ca2+ transients, were virtually lacking, when the TRPC3 channels were sensitized by subthreshold stimulation of phospholipase C. TIRF imaging provided evidence for dynamic colocalization of TRPC3 and IP3R. Our results suggest that TRPC3-mediated Ca2+ entry controls IP3R activity at ER-PM junctions to shape Ca2+ signaling signatures and enable specificity of downstream signaling.

Individual activator and repressor transcription factors act as modulators of global chromatin mobility.

Eukaryotic gene regulation is governed by the spatiotemporal organization of chromatin structures1,2. Alteration in chromatin structures is controlled by activator and repressor transcription factors (TFs) that lead to decreased or increased compaction (chromatin opening and closing, respectively)3-5. While it is known that this activity induces local changes in chromatin mobility, whether larger-scale chromatin structures respond dynamically to the activity of specific TFs remains unclear. Here we asked how TFs with opposite effects on modulating local chromatin accessibility impact nucleus-wide chromatin dynamics in living cells. We combine High-resolution Diffusion mapping (Hi-D)6 and Dense Flow reConstruction and Correlation (DFCC)7,8 to obtain an imaging-based, nanometer-scale analysis of local diffusion processes and long-range coordinated movements of both chromatin and TFs. We show that individual activator and repressor TFs increase nucleus-wide chromatin mobility and induce opposite coherent chromatin motions at the micron scale. Our quantitative analysis thus reveals the broad and differential impact of TFs opening and closing chromatin in nucleus-wide chromatin dynamics. This establishes the impact of TFs in shaping long-range chromatin mobility and provides a biophysical understanding of how global chromatin structure responds dynamically to TFs in living cells.

A universal code for phase separation of prion-like low complexity domains: Insights from quantitative computer simulations.

Biomolecular phase separation is a critical mechanism that contributes to intracellular spatiotemporal organization via the formation of biomolecular condensates. In addition to their widespread implications in aggregation-related disorders, prion-like low complexity domains (PLCDs) have been shown to exhibit demixing behavior and their aberrant condensation has been linked to disease. Here, we investigate the relationship between amino acid sequence and phase behaviour of the PLCDs exploring an unprecedented set of 140 different protein sequences. To this end, we perform direct coexistence molecular dynamics simulations of our residue-resolution coarse-grained protein model, which we have shown accurately predicts quantitative phase diagrams. For variants where experimental phase behaviors have been characterized, we show that our predictions are in excellent agreement with the experimental results. Analysis of the full simulation set of 140 variants reveals that the identity and patterning of sticker residues (e.g., Y, F) are crucial to define the phase behaviour of PLCDs. Notably, aromatic and charged residues have context-dependent effects on the critical temperature of PLCD condensation. Regarding spacer residues (e.g., S, G, N, T), PLCDs condensates are remarkably stable in front of mutations of glycine and serine but are more sensitive to mutations of asparagine and glutamine. Remarkably, these results extend to the entire family of PLCDs tested and, therefore, reveal universal codes for PLCDs phase behavior. Furthermore, the findings here help shed light on the functions of PLCDs and aid in the design of modifications to counter their aberrant phase separation.

The Phenomenon of Sacrality in the "Liturgical" Symphony of Andrey Eshpay

The focus of this article is the Sixth "Liturgical" Symphony of Andrei Yakovlevich Eshpai, one of the brightest representatives of the national and world musical culture of our time. The composition is considered in the perspective of sacrality, which fully determines its artistic and constructive patterns and dramatic perspective. The scientific novelty of the work is due to the subject of research itself, which was first used in relation to this work. To study the phenomenon of sacrality, it was necessary to use an interdisciplinary approach, which includes methods of system-structural, comparative, semiotic, hermeneutic analysis. Close attention was paid to considering the properties of musical texture as the main indicator of the spatiotemporal organization of musical material. The connotative-denotative properties of the musical syntax turned out to be analytically significant. No less important was the role of intertextual interactions that determine the language mechanisms of the text and reveal the plan of its content. It was established that the phenomenon of sacrality, responding to the aesthetic attitudes of the master, is implemented comprehensively - as a conjugation of philosophical, mythological and spiritual-religious discourses, each of which has its own system of signs. The functioning of sign systems at the syntagmatic and paradigmatic levels of the text lead to linguistic differences in the instrumental and choral sections of the symphony, reveal phenomena of semantic multiplicity, polychronicity, ensuring the end-to-end formation and development of the liturgical idea. As a result of such interactions, the genre space of the symphony is formed, which is a synthetic, universal, communicative concept.

Spatiotemporal Metabolic Liver Zonation and Consequences on Pathophysiology

Hepatocytes are the main workers in the hepatic factory, managing metabolism of nutrients and xenobiotics, production and recycling of proteins, and glucose and lipid homeostasis. Division of labor between hepatocytes is critical to coordinate complex complementary or opposing multistep processes, similar to distributed tasks at an assembly line. This so-called metabolic zonation has both spatial and temporal components. Spatial distribution of metabolic function in hepatocytes of different lobular zones is necessary to perform complex sequential multistep metabolic processes and to assign metabolic tasks to the right environment. Moreover, temporal control of metabolic processes is critical to align required metabolic processes to the feeding and fasting cycles. Disruption of this complex spatiotemporal hepatic organization impairs key metabolic processes with both local and systemic consequences. Many metabolic diseases, such as nonalcoholic steatohepatitis and diabetes, are associated with impaired metabolic liver zonation. Recent technological advances shed new light on the spatiotemporal gene expression networks controlling liver function and how their deregulation may be involved in a large variety of diseases. We summarize the current knowledge about spatiotemporal metabolic liver zonation and consequences on liver pathobiology.

Liaisons dangereuses: Intrinsic Disorder in Cellular Proteins Recruited to Viral Infection-Related Biocondensates.

Liquid-liquid phase separation (LLPS) is responsible for the formation of so-called membrane-less organelles (MLOs) that are essential for the spatio-temporal organization of the cell. Intrinsically disordered proteins (IDPs) or regions (IDRs), either alone or in conjunction with nucleic acids, are involved in the formation of these intracellular condensates. Notably, viruses exploit LLPS at their own benefit to form viral replication compartments. Beyond giving rise to biomolecular condensates, viral proteins are also known to partition into cellular MLOs, thus raising the question as to whether these cellular phase-separating proteins are drivers of LLPS or behave as clients/regulators. Here, we focus on a set of eukaryotic proteins that are either sequestered in viral factories or colocalize with viral proteins within cellular MLOs, with the primary goal of gathering organized, predicted, and experimental information on these proteins, which constitute promising targets for innovative antiviral strategies. Using various computational approaches, we thoroughly investigated their disorder content and inherent propensity to undergo LLPS, along with their biological functions and interactivity networks. Results show that these proteins are on average, though to varying degrees, enriched in disorder, with their propensity for phase separation being correlated, as expected, with their disorder content. A trend, which awaits further validation, tends to emerge whereby the most disordered proteins serve as drivers, while more ordered cellular proteins tend instead to be clients of viral factories. In light of their high disorder content and their annotated LLPS behavior, most proteins in our data set are drivers or co-drivers of molecular condensation, foreshadowing a key role of these cellular proteins in the scaffolding of viral infection-related MLOs.

RAS nanoclusters are cell surface transducers that convert extracellular stimuli to intracellular signalling.

Mutations of rat sarcoma virus (RAS) oncogenes (HRAS, KRAS and NRAS) can contribute to the development of cancers and genetic disorders (RASopathies). The spatiotemporal organization of RAS is an important property that warrants further investigation. In order to function, wild-type or oncogenic mutants of RAS must be localized to the inner leaflet of the plasma membrane (PM), which is driven by interactions between their C-terminal membrane-anchoring domains and PM lipids. The isoform-specific RAS-lipid interactions promote the formation of nanoclusters on the PM. As main sites for effector recruitment, these nanoclusters are biologically important. Since the spatial distribution of lipids is sensitive to changing environments, such as mechanical and electrical perturbations, RAS nanoclusters act as transducers to convert external stimuli to intracellular mitogenic signalling. As such, effective inhibition of RAS oncogenesis requires consideration of the complex interplay between RAS nanoclusters and various cell surface and extracellular stimuli. In this review, we discuss in detail how, by sorting specific lipids in the PM, RAS nanoclusters act as transducers to convert external stimuli into intracellular signalling.

Effective simulations of interacting active droplets

Droplets form a cornerstone of the spatiotemporal organization of biomolecules in cells. These droplets are controlled using physical processes like chemical reactions and imposed gradients, which are costly to simulate using traditional approaches, like solving the Cahn–Hilliard equation. To overcome this challenge, we here present an alternative, efficient method. The main idea is to focus on the relevant degrees of freedom, like droplet positions and sizes. We derive dynamical equations for these quantities using approximate analytical solutions obtained from a sharp interface limit and linearized equations in the bulk phases. We verify our method against fully-resolved simulations and show that it can describe interacting droplets under the influence of chemical reactions and external gradients using only a fraction of the computational costs of traditional methods. Our method can be extended to include other processes in the future and will thus serve as a relevant platform for understanding the dynamics of droplets in cells.

Plasma membrane topography governs the 3D dynamic localization of IgM B cell antigen receptor clusters

B lymphocytes recognize bacterial or viral antigens via different classes of the B cell antigen receptor (BCR). Protrusive structures termed microvilli cover lymphocyte surfaces, and are thought to perform sensory functions in screening antigen‐bearing surfaces. Here, we have used lattice light‐sheet microscopy in combination with tailored custom‐built 4D image analysis to study the cell‐surface topography of B cells of the Ramos Burkitt's Lymphoma line and the spatiotemporal organization of the IgM‐BCR. Ramos B‐cell surfaces were found to form dynamic networks of elevated ridges bridging individual microvilli. A fraction of membrane‐localized IgM‐BCR was found in clusters, which were mainly associated with the ridges and the microvilli. The dynamic ridge‐network organization and the IgM‐BCR cluster mobility were linked, and both were controlled by Arp2/3 complex activity. Our results suggest that dynamic topographical features of the cell surface govern the localization and transport of IgM‐BCR clusters to facilitate antigen screening by B cells.

Human Being and Recreational Topology in Russian culture of Second Half of the Twentieth Century

The article examines the peculiarities of influence on the formation and development of the spatiotemporal organization of human being of topological structures which are around the individual and external to him and characterizing the concrete historical stage of the development of the Russian culture of the second half of the twentieth century. The methodology and theoretical basis is the chronotopological concept by A.A. Ukhtomsky and M.M. Bakhtin, asserting the immanent interconnectedness of time, space and human, as well as the theory of social chronotope and topological philosophizing. Time and space of human being are understood as a chronotopological structure, which includes a system of topological and related temporal practices, immanent to the individual and developing together with him throughout his life. As a spatial structure that has been formed in culture, recreational topology is studied, which is a closed, well-groomed and isolated from the surrounding space or locus, which is the result of realized human topological practices for the arrangement and care of space or place of residence, work, recreation, scientific research, conservation and demonstration of cultural heritage. Getting into such a closed equipped space, radically different from the surrounding alienated unsettled public space, a person begins to perceive it as a topological standard, consciously or unconsciously transferring and reproducing it in his own individual chronotopology, in the sphere of his personal world and microchronotope of consciousness. If the recreational space is associated with a personally significant event in human life, it acquires the ethical significance for the individual, becomes existentially filled for him, acquires unity with his existence.

Temporalities Beyond Transition: Form, Genre, and Contemporary Trans Novels

ABSTRACT: Popular narratives about trans identity traditionally rely on a metaphorical understanding of trans embodiment as a linear and unidirectional journey. This paper discusses how this temporality is questioned, reshaped, interrupted, and sidestepped in five recent novels by trans and non-binary authors: Torrey Peters’s Detransition, Baby (2021), Andrea Lawlor’s Paul Takes the Form of a Mortal Girl (2017), Juno Dawson’s Wonderland (2020), Rivers Solomon’s An Unkindness of Ghosts (2017), and Alison Rumfitt’s Tell Me I’m Worthless (2021). The spatiotemporal organization of these novels negotiates the linearities that are conventionally employed in relation to trans stories, as each text engages with the conventions of specific genres—from the Bildungsroman to the picaresque, from science fiction to “chick lit,” from young adult literature to horror—in order to move beyond the structure of the personal transition journey.

The Phenomenon of Sacrality in Andrei Eshpai's "Liturgical" Symphony

This article focuses on the Sixth "Liturgical" Symphony of Andrei Yakovlevich Eshpai, one of the brightest representatives of the national and world musical culture of our time. The composition is considered from the perspective of sacrality, which fully determines its artistic and constructive patterns and dramatic perspective. The scientific novelty of the work is due to the subject of research itself, which was first used in relation to this work. To study the phenomenon of sacrality, it was necessary to use an interdisciplinary approach, including system-structural, comparative, semiotic, and hermeneutic analysis methods. Close attention was paid to considering the properties of musical texture as the main indicator of the spatiotemporal organization of musical material. The connotative and denotative properties of the musical syntax turned out to be analytically significant. No less important was the role of intertextual interactions that determine the text's language mechanisms and reveal its content’s plan. It was established that the phenomenon of sacrality, responding to the aesthetic attitudes of the master, is implemented comprehensively—as a conjugation of philosophical, mythological, and spiritual-religious discourses, each of which has its own system of signs. The functioning of sign systems at the syntagmatic and paradigmatic levels of the text lead to linguistic differences in the instrumental and choral sections of the symphony, revealing phenomena of semantic multiplicity, polychronicity, ensuring the end-to-end formation and development of the liturgical idea. As a result of such interactions, the genre space of the symphony is formed, which is a synthetic, universal, communicative concept.

Cell-free layer development and spatial organization of healthy and rigid red blood cells in a microfluidic bifurcation.

Bifurcations and branches in the microcirculation dramatically affect blood flow as they determine the spatiotemporal organization of red blood cells (RBCs). Such changes in vessel geometries can further influence the formation of a cell-free layer (CFL) close to the vessel walls. Biophysical cell properties, such as their deformability, which is impaired in various diseases, are often thought to impact blood flow and affect the distribution of flowing RBCs. This study investigates the flow behavior of healthy and artificially hardened RBCs in a bifurcating microfluidic T-junction. We determine the RBC distribution across the channel width at multiple positions before and after the bifurcation. Thus, we reveal distinct focusing profiles in the feeding mother channel for rigid and healthy RBCs that dramatically impact the cell organization in the successive daughter channels. Moreover, we experimentally show how the characteristic asymmetric CFLs in the daughter vessels develop along their flow direction. Complimentary numerical simulations indicate that the buildup of the CFL is faster for healthy than for rigid RBCs. Our results provide fundamental knowledge to understand the partitioning of rigid RBC as a model of cells with pathologically impaired deformability in complex in vitro networks.

Особенности эстетической рефлексии в мемуарно-автобиографической прозе А.Н. Бенуа

The aim of the study is to determine the specifics of the chronotope of St. Petersburg in the memoir-autobiographical work Moi Vospominaniya [My Memories] (1934-1960) of the Russian emigrant artist Alexandre Benois and to identify the relationships between the aesthetic settings of his paintings and their reflection in his perception of the city in the verbal text. The work uses an integrated approach based on historical and literary, comparative-typological, phenomenological and cultural methods. Moi Vospominaniya by Benois is considered as an egotext about art, in which the author represents the concept of his own creativity. This work has been studied in the aspect of implementing the idea of synthesizing the arts through the interaction of pictorial and verbal artistic imagery, relevant to the culture of the turn of the 20th century. Moi Vospominaniya is characterized by a multi-level structure of the spatio-temporal organization of the narrative: the chronotope is embodied both in real-historical, “open” time and in an imaginary space, subjective “closed” time, the space of art, the mystical chronotope. The space in the text is modeled by art, but represents the real world, restored by the author's memory. Sometimes elements of another being dangerous to humans penetrate into it: the mystical atmosphere of Oranienbaum and Pavlovsk, white nights, the oneiric chronotope - terrible prophetic dreams, life-giving pictures. The author concludes that in Benois' memoir-autobiographical narration the images of the artistic space are modeled, in which elements characteristic of his pictorial and theatrical-decorative works, the combination of realistic and fantastic beginnings are realized. The space-time of St. Petersburg in the text is polysemantic, it incorporates various cultural and spiritual phenomena. The real and the surreal mutually influence and condition each other, the conscious and unconscious spheres of life and art are intertwined. Hoaxes in the narrative are exposed to cultural monuments, artifacts, family relics, symbolizing the connection of the life of the autobiographical hero with previous eras, the merging of dreams, fantasies and reality, conveying his attitude. By turning to the art of the past, the alarming symptoms of the future are discerned: dispersal of anxiety and fear in the narrative becomes evidence of the hero's fears of the invasion of something hostile, a sign of human doom, the death of a peaceful way of life, old life, culture, a forerunner of new disasters. Mystical and everyday life exist together not only in the life of the hero himself, but also of his contemporaries, the author pays special attention to this, foreseeing a negative and dangerous trend in the overconcentration of infernal manifestations (growing religious mystical searches, the spread of sectarianism in various layers of Russian society).

Fine-grained, nonlinear registration of live cell movies reveals spatiotemporal organization of diffuse molecular processes.

We present an application of nonlinear image registration to align in microscopy time lapse sequences for every frame the cell outline and interior with the outline and interior of the same cell in a reference frame. The registration relies on a subcellular fiducial marker, a cell motion mask, and a topological regularization that enforces diffeomorphism on the registration without significant loss of granularity. This allows spatiotemporal analysis of extremely noisy and diffuse molecular processes across the entire cell. We validate the registration method for different fiducial markers by measuring the intensity differences between predicted and original time lapse sequences of Actin cytoskeleton images and by uncovering zones of spatially organized GEF- and GTPase signaling dynamics visualized by FRET-based activity biosensors in MDA-MB-231 cells. We then demonstrate applications of the registration method in conjunction with stochastic time-series analysis. We describe distinct zones of locally coherent dynamics of the cytoplasmic protein Profilin in U2OS cells. Further analysis of the Profilin dynamics revealed strong relationships with Actin cytoskeleton reorganization during cell symmetry-breaking and polarization. This study thus provides a framework for extracting information to explore functional interactions between cell morphodynamics, protein distributions, and signaling in cells undergoing continuous shape changes. Matlab code implementing the proposed registration method is available at https://github.com/DanuserLab/Mask-Regularized-Diffeomorphic-Cell-Registration.

Tie-Line Analysis Reveals Interactions Driving Heteromolecular Condensate Formation

Phase separation of biomolecules gives rise to membraneless organelles that contribute to the spatiotemporal organization of the cell. In most cases, such biomolecular condensates contain multiple components, but the manner in which interactions between components control the stability of condensates have remained challenging to elucidate. Here, we develop an approach to determine tie-line gradients in ternary biomolecular phase-separation systems based on measurements of the dilute phase concentration of only one component. We show that the sign of the tie-line gradient is related to the cross-interaction energy between the polymers in the system and discriminates between associative and segregative phase separation. Using this approach, we study the interaction between protein fused in sarcoma (FUS) and polyethylene glycol (PEG) polymer chains and measure positive tie-line gradients. Our results show that PEG drives phase separation through an associative interaction with FUS, other than through acting as an inert crowder. We further study the interaction between poly(A) ribonucleic acid (RNA) (3.0±0.5 kDa [kilodalton]) and the protein G3BP1, and using the tie-line gradient as a reporter for the stoichiometry of polymers in the condensate, we determine a G3BP1-to-poly(A) RNA molar ratio of 1∶4 in the dense phase. Our framework for measuring tie-line gradients opens up a route for the characterization of interaction types and compositions in ternary phase-separation systems.Received 31 May 2022Revised 24 October 2022Accepted 1 December 2022DOI:https://doi.org/10.1103/PhysRevX.12.041038Published by the American Physical Society under the terms of the Creative Commons Attribution 4.0 International license. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI.Published by the American Physical SocietyPhysics Subject Headings (PhySH)Research AreasLiquid-liquid phase transitionProtein-protein interactionsTechniquesConfocal imagingBiological Physics