Thermodynamics of wetting, prewetting and surface phase transitions with surface binding

In living cells, protein-rich condensates can wet the cell membrane and surfaces of membrane-bound organelles. Interestingly, many phase-separating proteins also bind to membranes leading to a molecular layer of bound molecules. Here we investigate how binding to membranes affects wetting, prewetting and surface phase transitions. We derive a thermodynamic theory for a three-dimensional bulk in the presence of a two-dimensional, flat membrane. At phase coexistence, we find that membrane binding facilitates complete wetting and thus lowers the wetting angle. Moreover, below the saturation concentration, binding facilitates the formation of a thick layer at the membrane and thereby shifts the prewetting phase transition far below the saturation concentration. The distinction between bound and unbound molecules near the surface leads to a large variety of surface states and complex surface phase diagrams with a rich topology of phase transitions. Our work suggests that surface phase transitions combined with molecular binding represent a versatile mechanism to control the formation of protein-rich domains at intra-cellular surfaces

Theory of Wetting Dynamics with Surface Binding

Biomolecules, such as proteins and RNAs, can phase separate in the cytoplasm of cells to form biological condensates. Such condensates are liquid-like droplets that can wet biological surfaces such as membranes. Many molecules that can participate in phase separation can also reversibly bind to membrane surfaces. When a droplet wets such a surface, these molecules can diffuse both inside the droplet or in the bound state on the surface. How the interplay between surface binding and surface diffusion affects the wetting kinetics is not well understood. Here, we derive the governing equations using non-equilibrium thermodynamics by relating the diffusive fluxes and forces at the surface coupled to the bulk. We use our theory to study the spreading kinetics in the presence of surface binding and find that binding speeds up wetting by nucleating a droplet inside the surface. Our results are relevant both to artificial systems and to condensates in cells. They suggest that the wetting of droplets in living cells could be regulated by two-dimensional droplets in the surface-bound layer changing the binding affinity to biological surfaces

ZO-1 Guides Tight Junction Assembly and Epithelial Morphogenesis via Cytoskeletal Tension-Dependent and -Independent Functions

Formation and maintenance of tissue barriers require the coordination of cell mechanics and cell–cell junction assembly. Here, we combined methods to modulate ECM stiffness and to measure mechanical forces on adhesion complexes to investigate how tight junctions regulate cell mechanics and epithelial morphogenesis. We found that depletion of the tight junction adaptor ZO-1 disrupted junction assembly and morphogenesis in an ECM stiffness-dependent manner and led to a stiffness-dependant reorganisation of active myosin. Both junction formation and morphogenesis were rescued by inhibition of actomyosin contractility. ZO-1 depletion also impacted mechanical tension at cell-matrix and E-cadherin-based cell–cell adhesions. The effect on E-cadherin also depended on ECM stiffness and correlated with effects of ECM stiffness on actin cytoskeleton organisation. However, ZO-1 knockout also revealed tension-independent functions of ZO-1. ZO-1-deficient cells could assemble functional barriers at low tension, but their tight junctions remained corrupted with strongly reduced and discontinuous recruitment of junctional components. Our results thus reveal that reciprocal regulation between ZO-1 and cell mechanics controls tight junction assembly and epithelial morphogenesis, and that, in a second, tension-independent step, ZO-1 is required to assemble morphologically and structurally fully assembled and functionally normal tight junctions

STED-FLCS:An Advanced Tool to Reveal Spatiotemporal Heterogeneity of Molecular Membrane Dynamics

Heterogeneous diffusion dynamics of molecules play an important role in many cellular signaling events, such as of lipids in plasma membrane bioactivity. However, these dynamics can often only be visualized by single-molecule and super-resolution optical microscopy techniques. Using fluorescence lifetime correlation spectroscopy (FLCS, an extension of fluorescence correlation spectroscopy, FCS) on a super-resolution stimulated emission depletion (STED) microscope, we here extend previous observations of nanoscale lipid dynamics in the plasma membrane of living mammalian cells. STED-FLCS allows an improved determination of spatiotemporal heterogeneity in molecular diffusion and interaction dynamics via a novel gated detection scheme, as demonstrated by a comparison between STED-FLCS and previous conventional STED-FCS recordings on fluorescent phosphoglycerolipid and sphingolipid analogues in the plasma membrane of live mammalian cells. The STED-FLCS data indicate that biophysical and biochemical parameters such as the affinity for molecular complexes strongly change over space and time within a few seconds. Drug treatment for cholesterol depletion or actin cytoskeleton depolymerization not only results in the already previously observed decreased affinity for molecular interactions but also in a slight reduction of the spatiotemporal heterogeneity. STED-FLCS specifically demonstrates a significant improvement over previous gated STED-FCS experiments and with its improved spatial and temporal resolution is a novel tool for investigating how heterogeneities of the cellular plasma membrane may regulate biofunctionality

Discovery of anti-inflammatory physiological peptides that promote tissue repair by reinforcing epithelial barrier formation

上皮バリアを形成するペプチドJIPの発見 --JIPは上皮組織修復に貢献する--. 京都大学プレスリリース. 2021-11-18.Epithelial barriers that prevent dehydration and pathogen invasion are established by tight junctions (TJs), and their disruption leads to various inflammatory diseases and tissue destruction. However, a therapeutic strategy to overcome TJ disruption in diseases has not been established because of the lack of clinically applicable TJ-inducing molecules. Here, we found TJ-inducing peptides (JIPs) in mice and humans that corresponded to 35 to 42 residue peptides of the C terminus of alpha 1-antitrypsin (A1AT), an acute-phase anti-inflammatory protein. JIPs were inserted into the plasma membrane of epithelial cells, which promoted TJ formation by directly activating the heterotrimeric G protein G13. In a mouse intestinal epithelial injury model established by dextran sodium sulfate, mouse or human JIP administration restored TJ integrity and strongly prevented colitis. Our study has revealed TJ-inducing anti-inflammatory physiological peptides that play a critical role in tissue repair and proposes a previously unidentified therapeutic strategy for TJ-disrupted diseases

Hydrophobic mismatch sorts SNARE proteins into distinct membrane domains

The clustering of proteins and lipids in distinct microdomains is emerging as an important principle for the spatial patterning of biological membranes. Such domain formation can be the result of hydrophobic and ionic interactions with membrane lipids as well as of specific protein–protein interactions. Here using plasma membrane-resident SNARE proteins as model, we show that hydrophobic mismatch between the length of transmembrane domains (TMDs) and the thickness of the lipid membrane suffices to induce clustering of proteins. Even when the TMDs differ in length by only a single residue, hydrophobic mismatch can segregate structurally closely homologous membrane proteins in distinct membrane domains. Domain formation is further fine-tuned by interactions with polyanionic phosphoinositides and homo and heterotypic protein interactions. Our findings demonstrate that hydrophobic mismatch contributes to the structural organization of membranes

Simultaneous optical and electrical recordings in horizontal lipid bilayers: Membrane dynamics and protein interactions

In this thesis the deployment of a methodological combination of two single molecule techniques, the planar bilayer technique and fluorescence fluctuation spectroscopy, is presented. The newly devised electro-optical setup will serve as a sophisticated model system for electrical excitable biological membranes. The expectation on a combined electro-optical setup is to be able to correlate the function of membrane channels (electrical activity) with its structural properties (fluorescence assays). The thesis is grouped into four chapters: A general introduction, providing the biological and methodological background, is followed by two studies on the application of the electro- optical setup in the field of membrane biophysics. In the first study the electrical and diffusion properties of planar bilayer membranes made of simple and ternary lipid mixtures are characterized. Additionally, the influence of temperature dependent lipid phase separation on the electrical activity of the ion channel gramicidin A is studied. The second study addresses the conformational changes of the pore-forming toxin Colicin A during membrane binding and ion channel formation. Finally, the potentials and the limitation of the presented setup are discussed