Proximity proteomics provides a new resource for exploring the function of Afadin and the complexity of cell-cell adherens junctions

Biology Open, Journal Year: 2025, Volume and Issue: 14(2)

Published: Jan. 30, 2025

The network of proteins at the interface between cell-cell adherens junctions and actomyosin cytoskeleton provides robust yet dynamic connections that facilitate cell shape change motility. While this was initially thought to be a simple linear connection via classic cadherins their associated catenins, we now have come appreciate many more are involved, providing robustness mechanosensitivity. Defining full set in remains key objective our field. Proximity proteomics means define these networks. Mammalian Afadin its Drosophila homolog Canoe parts protein network, facilitating diverse changes during gastrulation other events embryonic morphogenesis. Here report results several proximity screens, defining neighborhood both N- C-termini mammalian premier epithelial model, MDCK cells. We compare with previous screens done types, efforts junctional proteins. These reveal value multiple neighbors offer interesting insights into overlap composition different junctions.

Language: Английский

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Programming cellular condensates for living materials using an intrinsically disordered protein display platform

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: March 3, 2025

Abstract Self-organization of multicellular systems is vital for building structure in living system but remains underexplored engineered materials. We developed iDP 2 , a platform enabling high-density display intrinsically disordered proteins on the surface E. coli using CsgF as surface-bound anchor protein to which other domains can be fused. Display depends lack intrinsic and enable multivalent weak interactions that drive environmental responsiveness sequence-programmable self-segregation. allow programming material properties like viscoelasticity, stability, offering method rationally control cell-cell interactions. This approach could applied engineer self-organizing tissues or materials with customized properties.

Language: Английский

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The mechanobiology of biomolecular condensates

Biophysics Reviews, Journal Year: 2025, Volume and Issue: 6(1)

Published: March 1, 2025

The central goal of mechanobiology is to understand how the mechanical forces and material properties organelles, cells, tissues influence biological processes functions. Since first description biomolecular condensates, it was hypothesized that they obtain are tuned their functions inside cells. Thus, represent an intriguing playground for mechanobiology. idea condensates exhibit diverse adaptive highlights need different states respond external whether these responses linked physiological roles within cell. For example, liquids buffer dissipate, while solids store transmit stress, relaxation time a viscoelastic can act as frequency filter. Hence, liquid-solid transition condensate in force transmission pathway determine signals transduced in-between affecting differentiation, neuronal network dynamics, behavior stimuli. Here, we review our current understanding molecular drivers rigidity phase transitions set forth complex cellular environment. We will then summarize technical advancements were necessary insights into rich fascinating finally, highlight recent examples connection specific Our provide comprehensive summary field on cells harness regulate mechanics achieve

Language: Английский

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Chemically active wetting

Proceedings of the National Academy of Sciences, Journal Year: 2025, Volume and Issue: 122(15)

Published: April 9, 2025

Wetting of liquid droplets on passive surfaces is ubiquitous in our daily lives, and the governing physical laws are well understood. When become active, however, wetting remain elusive. Here, we propose chemically active as a class systems where surface due to binding process that maintained away from equilibrium. We derive corresponding nonequilibrium thermodynamic theory show fundamentally changes behavior, leading steady, states with droplet shapes reminiscent pancake or mushroom. The origin such anomalous can be explained by mapping electrostatics, pairs sinks sources correspond electrostatic dipoles along triple line. This an example more general analogy, localized chemical activity gives rise multipole field potential. underlying physics relevant for cells, droplet-forming proteins bind membranes accompanied turnover biological fuels.

Language: Английский

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Repression of Connexin26 Hemichannel activity protects the Barrier Function of Respiratory Airway Epithelial Cells against LPS-induced Alteration

Published: April 11, 2025

In respiratory airway epithelial cells, lipopolysaccharide (LPS) treatment induced an enhancement of connexin 26 (Cx26) hemichannel activity shown by dye uptake experiments after siRNA-mediated knock-down Cx26. This effect was already observed at infection relevant concentrations (≤ 10 ng/mL LPS) and involved tumor necrosis factor (TNF)-α- Ca²⁺-dependent signaling. High (1 µg/mL reduced the transepithelial electrical resistance (TEER) Calu-3 cells 35% within application time 3 h followed a recovery. Parallel to barrier alteration, tight junction organization rate (TiJOR) claudin-4 (CLDN4) 75% h. After TEER recovery, CLDN4 TiJOR stayed reduced. Low (10 required three times repeated for reduction 30%. The small molecule CVB4-57, newly published as potential inhibitor Cx26 hemichannels, mitigated effects LPS on function. Molecular docking studies revealed interaction between CVB4-57 thereby reducing its activity. We conclude that LPS-related acts like “molecular scar” weakens lung epithelium, which could be attenuated agents targeting hemichannels.

Language: Английский

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Actin polymerization counteracts prewetting of N-WASP on supported lipid bilayers

Proceedings of the National Academy of Sciences, Journal Year: 2024, Volume and Issue: 121(50)

Published: Dec. 4, 2024

Cortical condensates, transient punctate-like structures rich in actin and the nucleation pathway member Neural Wiskott-Aldrich syndrome protein (N-WASP), form during activation of cortex Caenorhabditis elegans oocyte. Their emergence spontaneous dissolution is linked to a phase separation process driven by chemical kinetics. However, mechanisms that drive onset cortical condensate formation near membranes remain unexplored. Here, using reconstituted assay proteins, we demonstrate key component, N-WASP, can collectively undergo surface condensation on supported lipid bilayers via prewetting transition. Actin partitions into where it polymerizes counteracts N-WASP Taken together, dynamics condensate-assisted appear be controlled balance between surface-assisted polymer-driven dissolution. This opens perspectives for understanding how complex intracellular affected separation.

Language: Английский

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Theory of non-dilute binding and surface phase separation applied to membrane-binding proteins

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 1, 2025

Surface binding and surface phase separation of cytosolic scaffold proteins on lipid membranes are involved in many cellular processes such as cell signaling, adhesion, cortex regulation. However, the interplay between is poorly understood. In this work, we study by deriving a general thermodynamic model applying it to vitro reconstitution experiments membrane-binding tight junction initiation. Our theory extends classical isotherm account for non-dilute heterogeneous conditions where components can separate. Using framework demonstrate how governed interaction strength among membrane-bound their affinity membrane surface. Comparing experiments, show that tuning oligomerization state adhesion receptors controls transition patterning protein ZO1. These findings suggest fundamental role formation junction. More broadly, our work highlights common organizational principle membrane-associated structures living cells.

Language: Английский

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Crb3 and NF2: A dynamic duo that controls assembly of the apical junctions and barrier function via Rho/ROCK signaling

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 15, 2025

The gastrointestinal epithelium serves as a critical barrier separating intestinal lumen contents from the underlying tissue environment. Structure and function of apical junctional complex (AJC), comprising tight adherens junctions, are essential for establishing maintaining polarized functional epithelial barrier. In this study, we investigated mechanisms by which an polarity protein Crumbs homolog 3 (CRB3) regulates AJC assembly in primary murine cells. Using colonic cells (colonoids) derived inducible conditional Crb3 knockdown (Crb3 ERΔIEC ) control mice fl/fl ), demonstrate that loss leads to compromised was associated with hypercontractile perijunctional actomyosin defective AJC. We identified CRB3 associates Band 4.1 family cytoskeletal linker proteins, Merlin (NF2) via FERM (band4.1/ezrin/radixin/moesin) binding domain (FBD) CRB3. Interestingly, NF2 cultured phenocopied effect deletion, supporting coordinated role formation assembly. Moreover, increased active Rho detected assembling junctions Crb3-null inhibition ROCKII myosin II alleviated phenotype, highlighting involvement Rho/ROCK signaling. Additionally, vinculin localization at seen indicates elevated tension junctions. Our findings underscore important regulating contractility ring, mechanical signaling during

Language: Английский

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Perspective: fluorescence lifetime imaging and single-molecule spectroscopy for studying biological condensates

Methods in microscopy, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 23, 2025

Abstract Biological condensates, often formed via liquid-liquid phase separation (LLPS), are membraneless compartments organizing biochemical reactions. Recent advances have shifted the focus from identifying condensates to elucidating their dynamic biological functions, such as buffering concentrations, mediating reactions, and regulating signaling. These critical for cellular processes implicated in diseases like cancer neurodegeneration. Advanced microscopy techniques, including fluorescence lifetime imaging (FLIM), FLIM-FRET, correlation spectroscopy (FCS), enable quantitative, real-time investigations of condensate composition, dynamics, material properties, responses environmental stimuli live cells. This perspective highlights utility time-resolved single-molecule techniques shedding light on interactions with membranes, offering insights into physiology pathology.

Language: Английский

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Repression of Connexin26 Hemichannel activity protects the Barrier Function of Respiratory Airway Epithelial Cells against LPS-induced Alteration

Research Square (Research Square), Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 3, 2025

In respiratory airway epithelial cells lipopolysaccharide (LPS) treatment induced an enhancement of connexin 26 (Cx26) hemichannel activity shown by dye uptake experiments combined with siRNA knock-down Cx26. This effect was already observed at infection relevant concentrations (≤ 10 ng/mL LPS) and involved tumor necrosis factor (TNF)-α- Ca²⁺-dependent signaling. High (1 µg/mL reduced the transepithelial electrical resistance (TEER) Calu-3 35 % within application time 3 h followed a recovery. Parallel to barrier alteration, tight junction organization rate (TiJOR) claudin-4 (CLDN4) 75 h. After TEER recovery, CLDN4 TiJOR stayed reduced. Low (10 required three times repeated for reduction 30 %. The small molecule CVB4-57, newly published as potential inhibitor Cx26 hemichannels, mitigated effects LPS on function. Molecular docking studies revealed interaction between CVB4-57 thereby reducing its activity. We conclude that LPS-related acts like “molecular scar” weakens lung epithelium, which could be attenuated agent targeting hemichannels.

Language: Английский

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