Plants require dynamic mechanisms of cellular organization to continuously adapt to fluctuating environmental conditions. Biomolecular condensation, which refers to nonstoichiometric assembly of macromolecules into membraneless compartments via phase separation, emerges as an essential regulatory strategy for plant development and stress responses. Unlike membrane-bound organelles, condensates can dynamically assemble/disassemble in response to different environmental cues. Increasing evidence indicates that versatile plant condensates associate with cellular membranes to regulate processes such as signal transduction, intracellular trafficking, organelle biogenesis, and gene expression. In this review, we summarize recent progress on representative condensates in association with the plant endomembrane system. We also highlight methodological advances, from multiscale in vivo characterization to in vitro reconstitution, which enable quantitative dissection of membrane-condensate interactions, with an emphasis on the biophysical principles governing them.

Photoswitchable lipids enable optical control of membrane area, mechanics and phase behavior, offering a platform to study stimuli-responsive biomimetic systems. Here we ask how the spatial organization of coexisting membrane phases governs photoinduced mechanical responses. We incorporate the photoswitch azobenzene-phosphatidylcholine (azoPC) and dipalmitoylphosphatidylcholine (DPPC) into gel-fluid phase-separated giant unilamellar vesicles (GUVs). Using differential scanning calorimetry and temperature-controlled confocal microscopy, we quantify phase transitions and visualize domain dynamics. Dispersed domains produce global GUV crumpling upon UV-light-induced trans-to-cis isomerization of azoPC, whereas coarsened fluid domains locally confine deformation to budding regions of the GUVs; both responses are reversed by blue light. Temperature-controlled imaging reveals that the gel-fluid transition in GUVs is considerably broader than the calorimetric profile suggests, with coexisting phases detectable well above the calorimetry peak transition temperature. Well above the transition temperature, i.e. in the fully melted membrane, UV irradiation unexpectedly induces reversible nucleation of gel-like flower domains, consistent with an increased transition temperature in the cis azoPC state due to lipid packing incompatibility with DPPC. Membrane domain architecture thus dictates the spatial distribution of photoinduced remodeling. More broadly, photoswitchable lipids can reversibly switch membrane phase equilibria as well as morphology, pointing to potential implications for the design of stimuli-responsive synthetic membrane systems and soft actuators.

Phase separation is a fundamental principle of cellular organization that typically leads to two coexisting phases: a dense one, where intermolecular interactions are stronger and molecules are more tightly packed, and a dilute, less packed phase, with weaker interactions and lower molecular concentration. This process drives the formation of both lateral membrane domains (rafts) and liquid-like protein and nucleic acid condensates. This Review explores the dynamic interplay between biomolecular condensates and membrane lipid domains and how phase separation occurs in the three-dimensional (bulk) cellular interior and at two-dimensional membrane interfaces. We examine how membranes act as platforms influencing condensate formation and function and, conversely, how condensates modulate membrane properties and organization. By highlighting diverse examples from cell signalling, tight-junction assembly and stress responses, we emphasize how these coupled interactions are crucial for cellular organization, function and fitness.

Though extracellular vesicles (EVs) contain much of the cellular machinery required for actively remodeling extracellular matrix (ECM), they are mostly appreciated for their roles in reprogramming cell proxies. Using a bottom-up biomimetic system, we show that breast cancer cell-derived EVs at the nanoscale can play an active role in collagen I matrix formation at the microscale. EVs nucleate new fibrils, recruiting collagen molecules from solution and enhancing fibril growth and network formation, resulting in more densely packed matrices with significantly increased storage and loss moduli. These effects are specific to EV membrane composition and cannot be reproduced using trypsinized EVs, synthetic liposomes, or mechanically extruded plasma membrane material. EVs become integrated into the fibril structures that they help form, reminiscent of matrix vesicles found within tissues. This represents a plausible way by which EVs are deposited into the ECM, becoming signaling cues for resident cells.

Lipid membrane fusion is a fundamental process underlying numerous biological functions in both physiological and pathological conditions. Understanding the physico-chemical mechanisms governing fusion, from the minimal molecular and structural requirements to the factors regulating its progression, is essential for elucidating complex biological phenomena and developing new biomedical and synthetic biology strategies. In this review, we adopt an interdisciplinary perspective to analyze how lipid composition, environmental conditions, and fusogenic agents influence the different stages of the fusion process, from membrane docking to fusion pore formation. We critically examine the main in vitro membrane models, discussing their advantages and limitations, and integrate experimental results with contributions from molecular simulations, which have allowed us to resolve the fusion intermediates and the underlying energy landscape at the nanoscale level. Particular emphasis is placed on synthetic fusogenic agents, including peptides, nucleic acids, nanoparticles, and polymers, highlighting their mechanisms of action and possibilities for rational design. Finally, we discuss emerging applications of membrane fusion in synthetic biology and biomedicine, with a focus on biomimetic systems, controlled drug delivery, and fusogenic lipid particles that promote endosomal escape. Overall, this review aims to provide a unifying conceptual framework linking fundamental principles of membrane fusion with advanced technological applications.

Membrane fusion is essential for signaling, cargo delivery, and synthetic cell growth, yet its mechanical consequences remain poorly defined. How fusion-driven membrane growth can be sustained without compromising compartment stability remains an unresolved challenge. Here, we established a minimal reconstituted system where content-loaded small liposomes fuse with single cell-sized giant unilamellar vesicles (GUVs), combining micropipette delivery, electrodeformation, and live imaging. Fusion outcomes were quantified through lipid and content mixing assays, GUV electrodeformation to track area and tension, and phase contrast imaging to monitor leakage. GUVs incorporated lipids and cargo from hundreds of thousands of vesicles at unprecedented efficiency rates, enabling substantial growth. However, accumulation of leaflet asymmetries induced curvature and tension, driving budding, rupture and leakage. Hemifusion amplified these destabilizing effects. Lipid number asymmetries emerge as a dominant mechanical cost of fusion, highlighting how cells may regulate these processes and guiding the design of therapeutic delivery systems and synthetic cells capable of robust and stable growth.

This chapter summarizes a spectrum of phenomena observed on model membranes exposed to electric fields. The considered model membrane system is giant unilamellar vesicles with sizes in the range of tens of microns. Because of their large size, the response of the membrane to electric fields can be directly visualized under the microscope. The membrane behavior is exemplified by several types of responses: First, the vesicles undergo morphological changes and adopt prolate, oblate, or spherocylindrical shapes. In general, the vesicle morphology depends on the conductivity conditions of the immersion and encapsulated solutions, and in the case of alternating fields – on the field frequency. Second, after switching the electric field off, these shapes can relax back to a sphere. The relaxation times depend on the initial membrane tension and on the reached transmembrane potential. Third, the vesicles can undergo topological changes such as formation of pores and, in the case of vesicles in contact, fusion. All these processes depend on the material characteristics of the membrane such as mechanical (bending rigidity and stretching elasticity), rheological (membrane shear surface viscosity), and electrical (capacitance) properties of the lipid bilayer. This chapter gives an overview of these properties and their dependence on the membrane phase state, and presents approaches for directly assessing them using giant unilamellar vesicles.

In the late 20th century, calcium took on the identity of an independent fusogen, when it was found to induce fusion of anionic large unilamellar vesicles (LUVs), yet its ability to drive fusion in cell-sized membranes remains poorly understood. Here, we directly quantify calcium-mediated fusion of giant unilamellar vesicles (GUVs) using a microfluidic trapping platform combined with confocal microscopy, enabling simultaneous measurement of lipid mixing, content mixing, and fusion outcomes across hundreds of single vesicles. We systematically map fusion efficiency as a function of calcium concentration, membrane composition, and mechanically imposed tension. We find that calcium-induced fusion of GUVs in the absence of proteins is remarkably fickle and composition sensitive, as the vesicles need to be sufficiently unstable to allow the opening of the fusion pore, yet stable enough to prevent bursting and collapse. Negatively charged GUVs containing high fractions of DOPE exhibit the highest fusogenic responsiveness, whereas other compositions undergo extensive lipid mixing without pore formation. Increasing membrane tension can shift this balance and promote full fusion, revealing a narrow parameter space in which calcium acts as an effective protein-free fusogen for cell-sized membranes. These findings clarify long-standing discrepancies between LUV-and GUV-based calcium fusion assays and provide quantitative design rules for employing calcium as a fusogen in synthetic biology and membrane-reconstitution studies, where controlled membrane growth, vesicle-vesicle fusion, and module integration are central to building and sustaining artificial cells.

Cell-penetrating peptides (CPPs) with a cationic-hydrophobic character are recognized as carriers for delivering various therapeutics and diagnostic agents across cell membranes and into the cells. Among the most studied CPPs, nona-arginine (R9) exhibits superior penetration compared to nona-lysine (K9), suggesting that the penetration ability depends not only on charge, distribution and concentration of peptides but also on the lipid membrane composition. However, for heptapeptides composed of arginine (R), lysine (K) and phenylalanine (F) residues, which show some CPPs properties, these interactions remain unexplored. This study sheds light on the adsorption of R5F2/K5F2 on model prokaryotic (PRO) and eukaryotic (EU) lipid membranes containing a zwitterionic lipid (phosphatidylcholine; PC) and an anionic lipid (either phosphatidylglycerol, PG, in the PRO model, or phosphatidylserine, PS in EU) at the 90:10 molar ratio. Using differential scanning calorimetry (DSC) and temperature-dependent UV-Vis spectroscopy, we observed peptide-induced changes in the interfacial water layer that affect the fluidity and rigidity of lipid bilayers. The distinct adsorption behavior of R5F2/K5F2 on PRO and EU lipid bilayers revealed the changes in lipid packing and hydrocarbon chain conformations as exclusively peptide-dependent features. The peptide-induced formation of vacancies in the non-polar bilayer part is consistent with partial leakage observed in giant unilamellar vesicles. The synchronized arrangement could represent a mechanism for the concerted translocation of CPPs, along with their potential cargo across the lipid membrane. This study provides new insights into the peptide-lipid interactions underlying CPPs functionality. ### Competing Interest Statement The authors have declared no competing interest.

Biomolecular condensates play a central role in cellular processes by interacting with membranes driving wetting transitions and inducing mutual remodeling. While condensates are known to locally alter membrane properties such as lipid packing and hydration, it remains unclear how membrane composition and phase state in turn affect condensate affinity. Here, we show that it is not only the membrane phase itself, but rather the degree of lipid packing that determines the condensate affinity for membranes. Increasing lipid chain length, saturation, or cholesterol content, enhances lipid packing, thereby decreasing condensate interaction. This regulatory mechanism is consistent across various condensate-membrane systems, highlighting the critical role of the membrane interface. In addition, protein adsorption promotes extensive membrane remodeling, including the formation of tubes and double-membrane sheets. Our findings reveal a mechanism by which membrane composition fine-tunes condensate wetting, highlighting its potential impact on cellular functions and organelle interactions.

Intrinsically disordered proteins and polypeptides can undergo liquid-liquid phase separation (LLPS) to form condensates/coacervates, which play numerous regulatory roles in the cell. Recently, the relevance of such LLPS occurring in the vicinity of membranes has been brought to light by several experimental studies. Membrane-adsorbed condensates are crucial for biomolecular localization, and in some cases, phase separation of proteins at the membrane surface induces significant changes in membrane morphology. A detailed microscopic understanding of the mechanisms behind these observations remains incomplete. Here we combine experiments and molecular simulations to unravel structural and dynamic features of the coacervate/membrane interface across scales. We study poly-Lysine/poly-Aspartate (K10/D10) coacervates as a prototype of phase-separated condensates with different unilamellar liposomes. Using a multiscale characterization approach that combines confocal microscopy, hyperspectral imaging, fluorescence recovery after photobleaching, and two complementary coarse-grained approaches, we show that the membrane-condensate affinity can be tuned by the anionic lipid content and quantified through the intrinsic contact angle – a material property derived from system geometry – both in vitro and in silico . We find that the membrane region in contact with the condensate displays a nearly two-fold reduced fluidity compared to the bare membrane. This is attributed to orientational ordering of lipid tails, resulting in decreased area per lipid. Moreover, we observed local lipid de-mixing induced by the coacervate adsorption. This study provides an effective framework for integrating experiment and computation to characterize the properties of coacervate/membrane interfaces that are critical to the functional impacts of these interactions. ### Competing Interest Statement The authors have declared no competing interest. NSF, CHE-2154804 European Unions Horizon Europe research Marie Skłodowska-Curie, 101168939 Alexander von Humboldt Foundation, https://ror.org/012kf4317

Biomolecular condensates are cellular organelles that form via liquid-liquid phase separation of proteins and nucleic acids. The functional role of condensates is tightly coupled to their material properties such as viscosity and hydrophobicity, which serve as phenomenological markers of cellular state in health and disease. These properties depend critically on condensate composition and water content. However, available approaches to determine condensate composition typically rely on invasive procedures that can disrupt or destroy the tested sample. Here, we introduce Raman spectroscopy coupled with spectral phasor analysis as an in situ, label-free approach to resolve the chemical profile and molecular concentrations within aqueous polymer solutions and within the dense and dilute phases of biomolecular condensate systems. In addition to quantifying the protein and water volume fractions, our method yields a precise readout of client molecule partitioning inside condensates. Across a wide range of condensate systems, we find that the dense phase remains overwhelmingly water-rich, even in condensates that exhibit low apparent dielectric constants. By explicitly accounting for protein backbone contributions to the Raman spectrum, we assess the contribution of "solid-like" hydrogen-bonded water resulting from protein hydration and find that the overwhelming majority of water molecules within condensates largely retain bulk "liquid-like" vibrational properties. Finally, by combining Raman-based compositional analysis with environment-sensitive fluorescent probes, we investigate the microscopic determinants of condensate hydrophobicity. We show that condensate hydrophobicity emerges from a combined contribution of the macromolecular structural features and water partitioning inside condensates, rather than from water content or hydrogen bonding alone.

Cargo delivery systems enable the targeted transport of therapeutic agents to specific sites, enhancing treatment efficacy while minimizing systemic side effects. However, many existing systems struggle with achieving precise control over the release timing. Here, a photothermal trigger-responsive delivery platform based on Giant Unilamellar Vesicles (GUVs) functionalized with gold nanorods (GNRs) is described to overcome these challenges. Toward this end, cargo-loaded GUVs are produced using the emulsion transfer method and effective GNR attachment to the vesicle membrane is achieved by functionalizing the GNRs with cholesterol. The near-infrared light (NIR)-mediated cargo release mechanism is described. Importantly, high release efficiency is achieved by optimizing the cholesterol concentration on the GNRs, which is essential for controlling the GNR attachment mechanism to the GUV membrane. Finally, GNR-functionalized ampicillin-loaded GUVs are tested in the presence of E. coli bacteria to demonstrate the platform's functionality. Following NIR-triggered antibiotic release, bacterial growth is inhibited. This showcases the practical potential of the developed trigger-responsive system. Notably, GNR-mediated photothermal heating alone also reduces bacterial viability upon extended illumination, indicating a dual antibacterial mechanism. This versatile and biocompatible system offers a controlled delivery method with broad applicability for therapeutics, diagnostics, and other biomedical applications.

[This corrects the article DOI: 10.1371/journal.ppat.1009455.].

Myelin is a lipid-rich membrane that insulates axons, providing support and ensuring efficient nerve impulse conduction. Disruption of this sheath, or demyelination, impairs neural transmission and underlies symptoms like vision loss and muscle weakness in multiple sclerosis (MS). Despite extensive studies using in vitro and in vivo models, the molecular mechanisms driving demyelination remain incompletely understood. To investigate the role of myelin basic protein (MBP) in membrane stability, we prepared model myelin membranes (MMMs) from lipids expectedly undergoing gel-to-fluid phase transition, mimicking both normal and altered myelin, with and without MBP. Differential scanning calorimetry (DSC) revealed that MBP suppresses the main phase transition in normal MMMs, unlike in modified MMMs. FTIR spectra showed strengthening of van der Waals interactions in normal MMMs with MBP upon heating and opposite effects in the analogous modified MMM system. Additionally, phosphate groups were identified as critical sites for MBP–lipid interactions. Circular dichroism (CD) spectroscopy suggests that MBP adopts helical structures that penetrate the bilayer of normal MMMs. These findings offer new insights into the molecular-level interactions between MBP and myelin membranes, with implications for understanding demyelination in diseases like MS.

Light drives plant life through photosynthesis, a process that takes place in the thylakoid membrane of the chloroplast, an organelle of cyanobacterial origin. The formation of thylakoid membranes within the chloroplast involves the eukaryote-specific factor CHLOROPLAST SEC14 LIKE PROTEIN 1 (CPSFL1), which shares strong sequence homology with the vesicle trafficking regulator SEC14. CSPFL1 is essential for vesicle formation, yet its specific molecular function in this process has remained unclear. In this study, we characterized CSPFL1 functions both in vitro and in vivo. Using a minimal membrane system of giant unilamellar vesicles (GUVs), we show that CPSFL1 alone can induce vesiculation. This process is mediated by lipid binding and membrane deformation, driven by curvature sensing and lipid-protein electrostatics. When expressed in the prokaryote E. coli, the eukaryote-specific CSPFL1 induces membrane curvature and vesicle formation. Plastid CPSFL1 co-purifies with vesicular structures. Lipid compositional analysis of CPSFL1-induced vesicles from bacteria reveals the presence of quinone precursors as cargo, linking CSPFL-mediated vesicle formation to prenylquinone transport. Together, our data suggest that during plant evolution, the eukaryotic vesicle formation system was co-opted for the transport of membrane integral metabolites from the inner envelope to the thylakoid membrane. ### Competing Interest Statement The authors have declared no competing interest.