Giant membrane vesicles (GUVs) and giant plasma membrane vesicles (GPMVs) are valuable models for studying the properties of cellular membranes. We analyzed experimental data on vesicle shapes in three-dimensional space to estimate their reduced volumes, focusing on osmotic deflation and membrane asymmetry. Shape changes in GPMVs illustrate how osmolarity influences the membrane structure in the absence of the cytoskeleton or other cellular organelles. By examining the experimentally observed shapes and their corresponding reduced volumes, we compared GPMV shapes to theoretical predictions for simple phospholipid vesicles, utilizing the area-difference elasticity and spontaneous curvature models. We mapped DOPC GUVs using the area-difference elasticity model and applied the spontaneous curvature model to map DOPC: cholesterol GUVs and GPMVs. The reported experiments showcase advanced methods that provide valuable biophysical insights, demonstrating that the GPMV shape observed in the experiments and their reduced volume can be mapped onto the same shape diagram as red blood cells (RBCs) and vesicles composed of phospholipids. This finding offers new perspectives in the field.

Giant membrane vesicles (GUVs) and Giant plasma membrane vesicles (GPMVs) are used as models to study membrane properties. We conducted a comparative study to examine how reducing the volume of vesicles with different lipid compositions, solution symmetries, solution asymmetries, and membrane charges affects their morphology. We used three-dimensional visualization techniques to study the shape of the vesicles. Although the vesicles may not be perfectly spherical, they exhibit some fluctuations in their shape. To understand these variations, we used confocal image stacks for visualization. Our experimental observations show that the membrane ' s charge influences the deflation of the GUVs in the presence of trans-bilayer sugar asymmetries. The lipid bilayers of our GUVs have a uniform distribution of lipids in both leaflets, indicating no asymmetry in lipid composition. We induce trans-bilayer asymmetries by exposing each leaflet of the bilayer to different solution compositions. We also estimated and compared the deformation of GPMV extracted from HEK-293 cells with trans-bilayer buffer asymmetries and inherent leaflet compositional asymmetry with biomimetic membranes.

Small GTP-binding (G) proteins of the Rabs, Arfs, and Arls (Arf-like) family mediate the recruitment of their effectors to subcellular membrane-bound compartments, which in turn mediates vesicle budding, motility, and tethering. Here, we report that Tectonin-β-propeller repeat containing protein 2 (TECPR2), a protein mutated in a form of hereditary sensory and autonomic neuropathy (HSAN), is an effector of early endosomal Rab protein, Rab5, and interacts with Rab5 via its C-terminal TECPR repeats. The HSAN-associated TECPR2 (R1336W) missense variant was deficient in Rab5-binding and, consequently, in membrane recruitment. TECPR2-depletion led to perinuclear collapse of recycling endosomes and increased overlap of sorting and degradative subdomain markers on early endosomes. Consistent with a possible role in endocytic recycling, we observed impaired recycling and increased lysosomal degradation of α5β1 integrin receptors upon TECPR2 knockdown. TECPR2 regulates the early endosomal localization of the cargo adaptor for β1 integrins, SNX17, and its-associated protein complex WASH, which mediates actin polymerization on early endosomes. Finally, TECPR2 depletion in the zebrafish model resulted in delayed motility and changes in the neuromuscular junction. Our study supports an early endosomal role for TECPR2 in cargo recycling and provides insights into how its loss-of-function results in a neurodegenerative genetic disorder. ### Competing Interest Statement The authors have declared no competing interest.

Soluble alpha-amylases play an important role in the catabolism of polysaccharides. In this work, we show that the malt α -amylase can interact with the lipid membrane and further alter its mechanical properties. Vesicle fluctuation spectroscopy is used for quantitative measurement of the membrane bending rigidity of phosphatidylcholines lipid vesicles from the shape fluctuation based on the whole contour of Giant Unilamellar Vesicles (GUVs). The bending rigidity of the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine lipid vesicles in water increases significantly with the presence of 0.14 micromolar alpha-amylase (AA) in the exterior solution. It appears that the enzyme present in the external solution interacts with the outer layer of the bilayer membrane, leading to an asymmetry of the solution on either side of the bilayer membrane and altering its elasticity. At AA concentration of 1.5 micromolars and above, changes in the morphology of the GUV membrane are observed. The interaction between AA in the external solution and the external leaflet causes the bilayer membrane to curve spontaneously, leading to the formation of outbuds, giving a positive spontaneous curvature of C0 ≤ 0.05 μm-1 at ≈ 1 mg / ml of the AA concentration. We validate and characterize its concentration-dependent role in stabilizing the membrane curvature. Our findings indicate that the involvement of the enzyme, depending on the concentration, can have a considerable effect on the mechanical characteristics of the membrane.

Giant membrane vesicles (GUVs) and giant plasma membrane vesicles (GPMVs)are useful models for studying cellular membrane properties. Our research analyzed the reduced volume of vesicles made from phospholipid and 10% cholesterol to investigate transbilayer sugar asymmetries. We found that GPMVs have an average reduced volume of (0.88 \pm 0.06) with buffer asymmetry of 323 mM, lower than the (0.92 \pm 0.08) observed for DOPC: cholesterol vesicles with sucrose/glucose asymmetry of 390 mM. GUVs with different sugars inside and outside were more deflated, demonstrating a greater volume reduction than those with the same sugar inside and out. We applied the area-difference elasticity (ADE) model to map GPMVs and used the spontaneous curvature (SC) model to analyze DOPC: cholesterol GUVs, extracting spontaneous curvature based on their reduced volume. ### Competing Interest Statement The authors have declared no competing interest.

Tip links are seen under microscopes as double-helical tetrameric complexes of long nonclassical cadherins, cadherin-23 and protocadherin-15. The twisted filamentous structure enables tip links to regulate mechanotransduction in hearing and balance. While the molecular details of the double-helical protocadherin-15 cis dimers have been deciphered, a similar conformation of cadherin-23 is still elusive. In a search of cadherin-23 cis dimers, we performed photoinduced cross-linking of unmodified proteins in solution and on lipid membranes and observed no trace of cadherin-23 cis dimers. Reportedly, tip links are dynamic connections, assembling and disassembling in seconds. Using lipid vesicles, we measured significantly slower aggregations between cis dimers of tip link cadherins than via dimer-monomer interactions, indicating that the trans interactions between two cis dimers may possess steric restraints and defer reassociations. Reconnections of tip links are thus kinetically most desired between protocadherin-15 cis dimers and cadherin-23 monomers. Here we propose that the helical geometry of tip links is induced by protocadherin-15 cis dimers, while cadherin-23 remains single before tip linking.

Cis and trans-interactions among cadherins secure multicellularity. While the molecular structure of trans-interactions of cadherins is well understood, work to identify the molecular cues that spread the cis-interactions two-dimensionally is still ongoing. Here, we report that transient, weak, yet multivalent, and spatially distributed hydrophobic interactions that are involved in liquid-liquid phase separations of biomolecules in solution, alone can drive the lateral-clustering of cadherin-23 on a membrane. No specific cis-dimer interactions are required for the lateral clustering. In cells, the cis-clustering accelerates cell-cell adhesion and, thus, contributes to cell-adhesion kinetics along with strengthening the junction. Although the physiological connection of cis-clustering with rapid adhesion is yet to be explored, we speculate that the over-expression of cadherin-23 in M2-macrophages may facilitate faster attachments to circulatory tumor cells during metastasis.

In the lyotropic phase of lipids with excess water, multilamellar tubules (MLTs) grow from defects. A phenomenological model for the stability of MLTs is developed that is universal and independent of the underlying growth mechanisms of MLTs. The stability of MLTs implies that they are in hydrostatic equilibrium and stable as elastic objects that have compression and bending elasticity. The results show that even with solvent pressure differences of 0.1 atm, the density profile is not significantly altered, so suggesting the stability is due to the trapped solvent. The results are of sufficient value in relation to lamellar stability models and may have implications beyond the described MLT models, especially in other models of membrane systems.

The catalytic action of invertase generates bilayer asymmetry that stabilises membrane curvature. The driving mechanism for the generation of membrane curvature by invertase is investigated using giant unilamellar vesicles (GUVs). The invertase cleaves the sucrose in the exterior compartment, thereby creating a sugar asymmetry across the bilayer membrane that is measured for GUV membranes consisting of the lipid Dioleoylphosphatidylcholine (DOPC). Finally, the advantage of this method to control membrane curvature and to stabilize multi-sphere morphologies is demonstrated. The GUV system in the presence of invertase is beneficial as a tool to generate multiple on-demand compartments with more extended stability after the enzymatic activity has established the asymmetry.

Tip-link, the gating-spring in hearing, modulates the mechanoelectrotransduction process and protects the hearing sensory machineries from overexcited auditory inputs. Tip-links are formed by two exceptionally long cadherins, cadherin-23 and protocadherin-15. It is proposed that both the cadherins first independently form cis-dimers through lateral interactions and finally, engage in trans-interactions to form the heterotetrameric assembly in tip-links. In fact, the molecular details of the protocadherin cis-dimers are well-understood. However, little structural and molecular details are known for independent cis-dimer of cadherin-23. Using photo-induced cross-linking of unmodified proteins in solution and on lipid-membranes, we observed no trace of cadherin-23 cis-dimers, rather we captured trans-homodimers of cadherin-23. We thus, redefine the tip-link structure where protocadherin-15 remains as cis-dimers, however, two individual cadherin-23 binds to protocadherin-15 cis-dimer for tip linking.

Micromechanics techniques are playing an increasing role in characterization of biomembranes. The mechanical properties of membranes play an important role for a whole range of cellular processes. Lipid-protein biomembranes display lateral heterogeneity, domain formation, and morphological changes at mesoscopic and nanoscopic length scales. An attempt is made to introduce how membrane’s material properties can be measured. Both fluctuation analysis and micro-pipette aspiration experiments have been used to quantify the micromechanics of membranes. The relationship between the structure and function of biomembranes is a critical concern in modern biology. This overview calls for a deeper understanding of how the cell complexity might be related to the mechanical properties of the lipid-protein membrane. Mechanical properties can influence cellular response to processes like adhesion, transport, differentiation, proliferation and migration.

Biological cells are contained by a fluid lipid bilayer (plasma membrane, PM) that allows for large deformations, often exceeding 50% of the apparent initial PM area. Isolated lipids self-organize into membranes, but are prone to rupture at small (<2-4%) area strains, which limits progress for synthetic reconstitution of cellular features. Here, it is shown that by preserving PM structure and composition during isolation from cells, vesicles with cell-like elasticity can be obtained. It is found that these plasma membrane vesicles store significant area in the form of nanotubes in their lumen. These act as lipid reservoirs and are recruited by mechanical tension applied to the outer vesicle membrane. Both in experiment and theory, it is shown that a "superelastic" response emerges from the interplay of lipid domains and membrane curvature. This finding allows for bottom-up engineering of synthetic biomaterials that appear one magnitude softer and with threefold larger deformability than conventional lipid vesicles. These results open a path toward designing superelastic synthetic cells possessing the inherent mechanics of biological cells.

In this paper, experimental results on the swelling, dispersion and disintegration of the lamellae composed of the lipid, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) in contact with excess water are described. Multilamellar structures nucleate and grow at the interface between the pure solvent and the lipid. The system evolves slowly via flow and coalescence of lamellae through a variety of non-equilibrium morphologies demonstrating that their dynamics is complex and non-universal, with no unique kinetic pathway. The tubular structures disintegrate slowly into the bulk water phase, inside the sealed sample chamber.

The proliferation of life on earth is based on the ability of single cells to divide into two daughter cells. During cell division, the plasma membrane undergoes a series of morphological transformations which ultimately lead to membrane fission. Here, we show that analogous remodeling processes can be induced by low densities of proteins bound to the membranes of cell-sized lipid vesicles. Using His-tagged fluorescent proteins, we are able to precisely control the spontaneous curvature of the vesicle membranes. By fine-tuning this curvature, we obtain dumbbell-shaped vesicles with closed membrane necks as well as neck fission and complete vesicle division. Our results demonstrate that the spontaneous curvature generates constriction forces around the membrane necks and that these forces can easily cover the force range found in vivo. Our approach involves only one species of membrane-bound proteins at low densities, thereby providing a simple and extendible module for bottom-up synthetic biology.

We use a microfluidic method to estimate the water permeability coefficient (p) of membranes. As model lipid membranes we employ giant unilamellar vesicles (GUVs) composed of palmitoyloleoyl phosphatidylcholine and cholesterol (10 mol%). We have developed a microfluidic device with multiple chambers to trap GUVs and allow controlled osmotic exchange. Each chamber has a ring-shaped pressure-controlled valve which upon closure allows isolation of the GUVs in a defined volume. Opening the valves leads to a rapid fluid exchange between the trapping region and the microchannel network outside, thus allowing precise control over solution concentration around the GUVs contrary to other experimental approaches for permeability measurements reported in the literature. The area and volume changes of individual vesicles are monitored with confocal microscopy. The solute concentration in the immediate vicinity of the GUVs, and thus the concentration gradient across the membrane, is independently assessed. The data are well fitted by a simple model for water permeability which assumes that the rate of change in volume of a GUV per unit area is linearly proportional to concentration difference with permeability as the proportionality constant. Experiments of GUV osmotic deflation with hypertonic solutions yield the permeability of POPC/cholesterol 9/1 membranes to be p = 15.7 ± 5.5 μm s-1. For comparison, we also show results using two other approaches, which either do not take into account local concentration changes and/or do not resolve the precise vesicle shape. We point out the errors associated with these limitations. Finally, we also demonstrate the applicability of the microfluidic device for studying the dynamics of vesicles under flow.

Simple sugars such as glucose and sucrose are ubiquitous in all organisms. One remarkable property of these small solutes is their ability to protect biomembranes against dehydration damage. This property, which reflects the underlying sugar-lipid interactions, has been intensely studied for lipid bilayers interacting with a single sugar at low hydration. Here, we use giant vesicles to investigate fully hydrated lipid membranes in contact with two sugars, glucose and sucrose. The vesicles were osmotically balanced, with the same total sugar concentration in the interior and exterior aqueous solutions. However, the two solutions differed in their composition: the interior solution contained only sucrose whereas the exterior one contained primarily glucose. This sugar asymmetry generated a striking variety of multispherical or "multi-balloon" vesicle shapes. Each multisphere involved only a single membrane that formed several spherical segments, which were connected by narrow, hourglass-shaped membrane necks. These morphologies revealed that the sugar-lipid interactions generated a significant spontaneous curvature with a magnitude of about 1 μm-1. Such a spontaneous curvature can be generated both by depletion and by adsorption layers of the sugar molecules arising from effectively repulsive and attractive sugar-lipid interactions. All multispherical shapes are stable over a wide range of parameters, with a substantial overlap between the different stability regimes, reflecting the rugged free energy landscape in shape space. One challenge for future studies is to identify pathways within this landscape that allow us to open and close the membrane necks of these shapes in a controlled and reliable manner. We will then be able to apply these multispheres as metamorphic chambers for chemical reactions and nanoparticle growth.

Biological cells are contained by a fluid lipid bilayer (plasma membrane, PM) that allows for large deformations, often exceeding 50% of the initial (or projected) PM area. Biochemically isolated lipids self-organize into membranes, but the extraordinary deformability of the plasma membrane is lost. Pure lipid bilayers are prone to rupture at small (<2-4%) area strains and this limits progress for synthetic reconstitution of cellular features such as migration, phagocytosis and division. Here, we show that by preserving PM structure and composition during isolation from cells, vesicles with cell-like elasticity are obtained. We found that these plasma membrane vesicles store significant area in the form of nanotubes in their lumen. These are recruited by mechanical tension applied to the outer vesicle membrane showing an apparent elastic response. This “super-elastic” response emerges from the interplay of lipid liquid-liquid phase separation and membrane asymmetry. This finding allows for bottom-up engineering of synthetic vesicles that appear over one magnitude softer and with three fold larger deformability than conventional lipid vesicles.

Vesicle fluctuation analysis (VFA) is a simple experimental technique for extracting information about membrane physical properties by analyzing the conformational fluctuations of giant vesicles visible under optical microscopy. Detailed analysis of membrane conformational fluctuations reveal information about material properties of the membrane, in particular about the bending rigidity, but it has also a range of additional applications. VFA provides a unique possibility to analyze the consequences of the cooperative phenomena occurring at mesocopic length scales that are contained within the membrane fluctuations at macroscopic length by testing theoretical predictions of phenomenological models and extracting estimates of model parameters. This chapter presents the background and principles for VFA of quasi-spherical giant unilamellar vesicles (GUVs). The minimal requirements for establishing an experimental facility are discussed along with the key elements of image analysis and software for data treatment. Finally, some applications and future possibilities of VFA are given.