Formation and maintenance of tubular membrane projections require mechanical force, but their elongation and shortening do not require additional force

Living cells develop their own characteristic shapes depending on their physiological functions, and their morphologies are based on the mechanical characteristics of the cytoskeleton and of membranes. To investigate the role of lipid membranes in morphogenesis, we constructed a simple system that can manipulate liposomes and measure the forces required to transform their shapes. Two polystyrene beads (1 microm in diameter) were encapsulated in giant liposomes and were manipulated using double-beam laser tweezers. Without any specific interaction between the lipid membrane and beads, mechanical forces could be applied to the liposome membrane from the inside. Spherical liposomes transformed into a lemon shape with increasing tension, and tubular membrane projections were subsequently generated in the tips at either end. This process is similar to the liposomal transformation caused by elongation of encapsulated cytoskeletons. In the elongation stage of lemon-shaped liposomes, the force required for the transformation became larger as the end-to-end length increased. Just before the tubular membrane was generated, the force reached the maximum strength (approximately 11 pN). However, immediately after the tubular membrane developed, the force suddenly decreased and was maintained at a constant strength (approximately 4 pN) that was independent of further tube elongation or shortening, even though there was no excess membrane reservoir as occurs in living cells. When the tube length was shortened to approximately 2 microm, the liposome reversed to a lemon shape and the force temporarily increased (to approximately 7 pN). These results indicate that the simple application of mechanical force is sufficient to form a protrusion in a membrane, that a critical force and length is needed to form and to maintain the protrusion, and suggest that the lipid bilayer itself has the ability to buffer the membrane tension.     

Freeze-thaw immobilization of liposomes in chromatographic gel beads: evaluation by confocal microscopy and effects of freezing rate

Biological membranes immobilized in chromatographic gel beads constitute a multifunctional affinity matrix. Membrane protein-solute interactions and drug partitioning into the lipid bilayers can conveniently be studied. By the use of confocal laser-scanning microscopy (CLSM) the distribution of immobilized model membranes in the beads has been visualized for the first time. Freeze-thaw-immobilized liposomes in Superdex 200 gel beads were situated in a thick shell surrounding a liposome-free core. The amount of phospholipids immobilized by freeze-thawing was dependent on the temperature in the cooling bath and the type of test tube used. A bath temperature of -25 degrees C gave higher immobilization yield than freezing at -75 or -8 degrees C did. Freeze-thawing in the presence of liposomes did not affect the gel bead shape or the refractive index homogeneity of the agarose network of the beads, as shown by confocal microscopy.     

Transfer of cytochrome b 5 and NADPH cytochrome c reductase between membranes

NADPH-cytochrome c reductase also reduces cytochrome b 5. The reduction is very slow when the proteins are in solution or bound to different membranes. Only when both proteins share a common membrane, is cytochrome b 5 reduced rapidly by NADPH. The difference in reaction rates indicates recombination on a common membrane of cytochrome b 5 and NADPH reductase originally bound to different vesicles. The recombination of the two proteins occurs with a variety of biological membranes (previously enriched with either reductase or cytochrome b 5) as well as with liposomes. We explain this process as protein transfer rather than vesicle fusion for several reasons: 1. The vesicles do not alter shape or size during incubation. 2. The rate of this process corresponds to the rate of incorporation of the single proteins into liposomes carrying the 'complementary' protein. 3. The exchange of proteins between biological membranes and liposomes occupied by protein does not change the density of either membrane. Protein transfer between membranes appears to be limited to those proteins which had spontaneously recombined with a preformed membrane. In contrast, proteins incorporated into liposomes by means of a detergent were not transferred, nor were endogenous cytochrome b 5 and NADPH-cytochrome c reductase transferred from microsomes to Golgi membranes or lipid vesicles. We conclude that the endogenous proteins and proteins incorporated in the presence of a detergent are linked to the membrane in another manner than the same proteins which had been inserted into a preformed membrane.     

P-glycoprotein inserted in planar lipid bilayers formed by liposomes opened on amorphous carbon and Langmuir-Blodgett monolayer

The insertion of proteins into planar lipid layers is of outstanding interest as the resulting films are suitable for the investigation of protein structure and aggregation in a lipid environment and/or the development of biotechnological applications as biosensors. In this study, purified P-glycoprotein (P-gp), a membrane drug pump, was incorporated in model membranes deposited on solid supports according to the method by Puu and Gustafson, Biochim. Biophys. Acta 1327 (1997) 149-161. The models were formed by a double lipid layer obtained by opening P-gp-containing liposomes onto two hydrophobic supports: amorphous carbon films and Langmuir-Blodgett (L-B) lipid monolayers, which were then observed by transmission electron microscopy and atomic force microscopy, respectively. Before the opening of liposomes, the P-gp structure and functionality were verified by circular dichroism spectroscopy and enzymatic assay. Our micrographs showed that liposomes containing P-gp fuse to the substrates more easily than plain liposomes, which keep their rounded shape. This suggests that the protein plays an essential role in the fusion of liposomes. To localize P-gp, the immunogold labeling of two externally exposed protein epitopes was carried out. Both imaging techniques confirmed that P-gp was successfully incorporated in the model membranes and that the two epitopes preserved the reactivity with specific mAbs, after sample preparation. Model membranes obtained on L-B monolayer incorporated few molecules with respect to those incorporated in the model membrane deposited onto amorphous carbon, probably because of the different mechanism of proteoliposome opening. Finally, all particles appeared as isolated units, suggesting that P-gp molecules were present as monomers.     

P-glycoprotein inserted in planar lipid bilayers formed by liposomes opened on amorphous carbon and Langmuir-Blodgett monolayer

The insertion of proteins into planar lipid layers is of outstanding interest as the resulting films are suitable for the investigation of protein structure and aggregation in a lipid environment and/or the development of biotechnological applications as biosensors. In this study, purified P-glycoprotein (P-gp), a membrane drug pump, was incorporated in model membranes deposited on solid supports according to the method by Puu and Gustafson, Biochim. Biophys. Acta 1327 (1997) 149-161. The models were formed by a double lipid layer obtained by opening P-gp-containing liposomes onto two hydrophobic supports: amorphous carbon films and Langmuir-Blodgett (L-B) lipid monolayers, which were then observed by transmission electron microscopy and atomic force microscopy, respectively. Before the opening of liposomes, the P-gp structure and functionality were verified by circular dichroism spectroscopy and enzymatic assay. Our micrographs showed that liposomes containing P-gp fuse to the substrates more easily than plain liposomes, which keep their rounded shape. This suggests that the protein plays an essential role in the fusion of liposomes. To localize P-gp, the immunogold labeling of two externally exposed protein epitopes was carried out. Both imaging techniques confirmed that P-gp was successfully incorporated in the model membranes and that the two epitopes preserved the reactivity with specific mAbs, after sample preparation. Model membranes obtained on L-B monolayer incorporated few molecules with respect to those incorporated in the model membrane deposited onto amorphous carbon, probably because of the different mechanism of proteoliposome opening. Finally, all particles appeared as isolated units, suggesting that P-gp molecules were present as monomers.     

Mechanics of Biomimetic Liposomes Encapsulating an Actin Shell

Cell-shape changes are insured by a thin, dynamic, cortical layer of cytoskeleton underneath the plasma membrane. How this thin cortical structure impacts the mechanical properties of the whole cell is not fully understood. Here, we study the mechanics of liposomes or giant unilamellar vesicles, when a biomimetic actin cortex is grown at the inner layer of the lipid membrane via actin-nucleation-promoting factors. Using a hydrodynamic tube-pulling technique, we show that tube dynamics is clearly affected by the presence of an actin shell anchored to the lipid bilayer. The same force pulls much shorter tubes in the presence of the actin shell compared to bare membranes. However, in both cases, we observe that the dynamics of tube extrusion has two distinct features characteristic of viscoelastic materials: rapid elastic elongation, followed by a slower elongation phase at a constant rate. We interpret the initial elastic regime by an increase of membrane tension due to the loss of lipids into the tube. Tube length is considerably shorter for cortex liposomes at comparable pulling forces, resulting in a higher spring constant. The presence of the actin shell seems to restrict lipid mobility, as is observed in the corral effect in cells. The viscous regime for bare liposomes corresponds to a leakout of the internal liquid at constant membrane tension. The presence of the actin shell leads to a larger friction coefficient. As the tube is pulled from a patchy surface, membrane tension increases locally, leading to a Marangoni flow of lipids. As a conclusion, the presence of an actin shell is revealed by its action that alters membrane mechanics.     

Avidin–biotin-immobilized liposome column for chromatographic fluorescence on-line analysis of solute–membrane interactions

Unilamellar liposomes with entrapped fluorescent dye calcein were stably immobilized in gel beads by avidin–biotin-binding. The immobilized liposomes remained extremely stable upon storage and chromatographic runs. The immobilized calcein-entrapped liposomes were utilized for fluorescent analysis of solute–membrane interactions, which in some cases are too weak to be detected by chromatographic retardation. A liposome column was used as a sensitive probe to detect the interactions of membranes with pharmaceutical drugs, peptides and proteins. Retardation of the solutes was monitored using a UV detector. Perturbation of the membranes, reflected as leakage of the entrapped calcein by some of the solutes, can thus be detected on-line using a flow-fluorescent detector. For the amphiphilic drugs or synthetic peptides, perturbation of membranes became more pronounced when the retardation (hydrophobicity) of the molecules increased. On the other hand, in the case of positively-charged peptides, polylysine, or partially denatured bovine carbonic anhydrase, significant dye leakage from the liposomes was observed although the retardation was hardly to be measured. Weak protein–membrane interactions can thus be assumed from the large leakage of calcein from the liposomes. This provides additional useful information for solute–membrane interactions, as perturbation of the membranes was also indicated by avidin–biotin-immobilized liposome chromatography (ILC).     

Partitioning of triphenylalkylphosphonium homologues in gel bead-immobilized liposomes: chromatographic measurement of their membrane partition coefficients

Unilamellar liposomes of small or large size, SUVs and LUVs, respectively, were stably immobilized in the highly hydrophilic Sepharose 4B or Sephacryl S-1000 gel beads as a membrane stationary phase for immobilized liposome chromatography (ILC). Lipophilic cations of triphenylmethylphosphonium and tetraphenylphosphonium (TPP+) have been used as probes of the membrane potential of cells. Interaction of TPP+ and triphenylalkylphosphonium homologues with the immobilized liposomal membranes was shown by their elution profiles on both zonal and frontal ILC. Retardation of the lipophilic cations on the liposome gel bed was increased as the hydrophobicity of the cations increased, indicating the partitioning of lipophilic cations into the hydrocarbon region of the membranes. The cations did not retard on the Sepharose or Sephacryl gel bed without liposomes, confirming that the cations only interact with the immobilized liposomes. Effects of the solute concentration, flow rate, and gel-matrix substance on the ILC were studied. The stationary phase volume of the liposomal membranes was calculated from the volume of a phospholipid molecule and the amount of the immobilized phospholipid, which allowed us to determine the membrane partition coefficient (KLM) for the lipophilic cations distributed between the aqueous mobile and membrane stationary phases. The values of KLM were generally increased with the hydrophobicity of the solutes increased, and were higher for the SUVs than for the LUVs. The ILC method described here can be applied to measure membrane partition coefficients for other lipophilic solutes (e.g., drugs).     

Atomic force microscopy and light scattering of small unilamellar actin-containing liposomes

Three-dimensional networks of filamentous actin (F-actin) encapsulated inside phosphatidylcholine liposomes are currently being used in an effort to model the cytoskeleton and plasma membrane of eukaryotic cells. In this article, unilamellar lipid vesicles consisting of egg yolk-derived phosphatidylcholine encapsulating monomeric actin (G-actin) were made via extrusion in low ionic strength buffer (G-buffer). Vesicle shape and structure in these dispersions was studied using a combination of fluid-tapping atomic force microscopy, and multiangle static light scattering. After subjecting the liposome dispersion to high ionic strength polymerization buffer (F-buffer) containing K(+) ions, atomic force microscopy imaging and light scattering of these liposomes indicated the formation of specialized structures, including an overall liposome structure transformation from spherical to torus, disk-shaped geometries and tubular assemblies. Several atomic force microscopy control measurements were made to ascertain that the specialized structures formed were not due to free G-actin and F-actin self-assembling on the sample surface, plain liposomes exposed to G- and F-buffer, or liposomes encapsulating G-actin. Liposomes encapsulating G-actin assumed mostly thin disk shapes and some large irregularly shaped aggregates. In contrast, liposomes encapsulating polymerized actin assumed mostly torus or disk shapes along with some high aspect ratio tubular structures.     

Applications of phospholipid bilayer nanodiscs in the study of membranes and membrane proteins

Phospholipid bilayer Nanodiscs are novel model membranes derived from high-density lipoprotein particles and have proven to be useful in studies of membrane proteins. Membrane protein enzymology has been hampered by the inherent insolubility of membrane proteins in aqueous environments and has necessitated the use of model membranes such as liposomes and detergent-stabilized micelles. Current model membranes display a polydisperse particle size distribution and can suffer from problems of inconsistency and instability. It is also unclear how well they mimic biological lipid bilayers. In contrast, Nanodiscs, the particle size of which is constrained by a coat of scaffold proteins, are relatively monodisperse, stable model membranes with a "nativelike" lipid bilayer. Nanodiscs have already been used to study a variety of membrane proteins, including cytochrome P450s, seven-transmembrane proteins, and bacterial chemoreceptors. These proteins are simultaneously monomerized, solubilized, and incorporated into the well-defined membrane environment provided by Nanodiscs. Nanodiscs may also provide useful insights into the thermodynamics and biophysics of biological membranes and binding of small molecules to membranes.     

Investigation of mechanisms of synaptic vesicles fusion with acceptor membranes in the model of exocytosis

Fusion of synaptic vesicles with various target membranes was investigated on the cell-free model system that reflects the final step of exocytosis. Plasma membranes, synaptic vesicles and liposomes were used as acceptor membranes. The process of membrane fusion was triggered by Ca2+. We have demonstrated that synaptic vesicles are prone to fuse with liposomes in buffer solution. This process was strongly dependent on ionic force of medium and phospholipid composition of liposomes. Cytosolic proteins of synaptosomes inhibited the fusion of synaptic vesicles with liposomes, while these were required for the fusion of synaptic vesicles with native membrane structures. Trypsinolysis of acceptor membranes markedly inhibited the fusion response. It means protein components of target membrane are necessary for realization of the final step of exocytosis.     

Protein shape and crowding drive domain formation and curvature in biological membranes

Folding, curvature, and domain formation are characteristics of many biological membranes. Yet the mechanisms that drive both curvature and the formation of specialized domains enriched in particular protein complexes are unknown. For this reason, studies in membranes whose shape and organization are known under physiological conditions are of great value. We therefore conducted atomic force microscopy and polarized spectroscopy experiments on membranes of the photosynthetic bacterium Rhodobacter sphaeroides. These membranes are densely populated with peripheral light harvesting (LH2) complexes, physically and functionally connected to dimeric reaction center-light harvesting (RC-LH1-PufX) complexes. Here, we show that even when converting the dimeric RC-LH1-PufX complex into RC-LH1 monomers by deleting the gene encoding PufX, both the appearance of protein domains and the associated membrane curvature are retained. This suggests that a general mechanism may govern membrane organization and shape. Monte Carlo simulations of a membrane model accounting for crowding and protein geometry alone confirm that these features are sufficient to induce domain formation and membrane curvature. Our results suggest that coexisting ordered and fluid domains of like proteins can arise solely from asymmetries in protein size and shape, without the need to invoke specific interactions. Functionally, coexisting domains of different fluidity are of enormous importance to allow for diffusive processes to occur in crowded conditions.     

Labeling phospholipid membranes with lipid mimetic luminescent metal complexes

Lipid-mimetic metallosurfactant based luminophores are promising candidates for labeling phospholipid membranes without altering their biophysical characteristics. The metallosurfactants studied exhibit high structural and physicochemical similarity to phospholipid molecules, designed to incorporate into the membrane structure without the need for covalent attachment to a lipid molecule. In this work, two lipid-mimetic phosphorescent metal complexes are described: [Ru(bpy)₂(dn-bpy)]^2 + and [Ir(ppy)₂(dn-bpy)]⁺ where bpy is 2,2′-bipyridine, dn-bpy is 4,4′-dinonyl-2,2′-bipyridine and ppy is 2-phenylpyridine. Apart from being lipid-mimetic in size, shape and physical properties, both complexes exhibit intense photoluminescence and enhanced photostability compared with conventional organic fluorophores, allowing for prolonged observation. Moreover, the large Stokes shift and long luminescence lifetime associated with these complexes make them more suitable for spectroscopic studies. The complexes are easily incorporated into dimyristoil-phosphatidyl-choline (DMPC) liposomes by mixing in the organic solvent phase. DLS reveals the labeled membranes form liposomes of similar size to that of neat DMPC membrane. Synchrotron Small-Angle X-ray Scattering (SAXS) measurements confirmed that up to 5% of either complex could be incorporated into DMPC membranes without producing any structural changes in the membrane. Fluorescence microscopy reveals that 0.5% label content is sufficient for imaging. Atomic Force Microscopic imaging confirms that liposomes of the labeled bilayers on a mica surface can fuse into a flat lamellar membrane that is morphologically identical to neat lipid membranes. These results demonstrate the potential of such lipid-mimetic luminescent metal complexes as a new class of labels for imaging lipid membranes.     

Effect of retinol and retinoic acid on permeability,electrical resistance and phase transition of lipid bilayers

Retinol and retinoic acid have been incorporated into the artificial membrane systems, planar bimolecular lipid membranes and liposomes, and their effects on several membrane parameters have been measured. 1. Retinol and retinoic acid increased the permeability of egg lecithin liposomes to K⁺, I^? and glucose when incorporated into the membranes at levels as low as 0.5 membrane mol%. Retinoic acid influenced permeability more than did retinol for each of the solutes tested. 2. Retinol and retinoic acid both decreased the electrical resistance of egg lecithin-planar bimolecular lipid membranes from 0.5 to 8 membrane mol%. Retinoic acid effected a larger change than did retinol. 3. Retinol and retinoic acid increased the permeability of dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine liposomes to water at 1.0 and 3.0 membrane mol%. A larger effect on water permeability was measured for retinoic acid than for retinol. 4. Retinol and retinoic acid at 1.0 and 3.0 membrane mol% were shown to lower the phase-transition temperature of liposomes composed of dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine. Phase-transition temperatures were monitored by abrupt changes in water permeability and liposome size associated with the transition. Retinoic acid lowered the phase-transition temperature of dimyristoylphosphatidylcholine liposomes more than did retinol, while both retinoids had almost the same effect on dipalmitoylphosphatidylcholine liposomes.     

Adhesion characteristics and stability assessment of lectin-modified liposomes for site-specific drug delivery

Carbohydrate moieties of the cellular glycocalyx have been suggested to play an important role in biological recognition processes during pathologic conditions, such as inflammation and cancer. Herein, we describe lectin-modified liposomes which might have potential for site-specific drug delivery during the therapy of such diseases. Specific interactions of plain (i.e., unmodified) and PEGylated, lectin-grafted liposomes with model membranes were investigated under real-time flow conditions using a quartz crystal microbalance. In addition, the morphology of the liposomal systems was assessed by atomic force microscopy. Plain liposomes exhibited only unspecific adhesion to glycolipid membranes and had a tendency to coalesce. The degree of membrane interaction was significantly increased when plain liposomes were modified with the lectin, Concanavalin A. However, vesicle fusion also markedly increased as a result of lectin modification. Additional PEGylation of liposomes reduced unspecific adhesion phenomena, as well as coalescence. Moreover, our studies enabled us to establish quartz crystal microbalance and atomic force microscopy as powerful and complementary methods to characterize adhesion properties of targeted drug delivery systems.     

Cell-sized, supported artificial membranes (pseudocytes): response of precursor cytotoxic T lymphocytes to class I MHC proteins

Novel cell-sized, supported artificial membranes bearing class I antigens have been prepared by a simple dialysis procedure and then used to study the requirements for antigen recognition by precursor cytotoxic T lymphocytes (CTL). The membranes were made by mixing lipid, H-2 antigen, and C18 alkylated 5 microns silica beads in deoxycholate, and dialyzing to remove the detergent. The H-2 antigen-bearing, cell-sized beads, termed pseudocytes (artificial cells), were able to simulate generation of secondary CTL responses with the same specificity as alloantigen-bearing spleen cells. Comparative analyses demonstrated that the size of an antigen-bearing structure, and thus its potential for multivalent interaction, was a critical determinant of effectiveness of antigen recognition, and showed that H-2 antigen was recognized as effectively on cell-sized beads as on allogeneic spleen cells. Generation of a response to antigen on the cell-sized beads was completely dependent on addition of lymphokines to the cultures. Thus, unlike liposomes, H-2 antigen on beads was not available to accessory cells for stimulation of Ia-dependent production of lymphokines by T helper cells. These results, as well as direct observations by microscopy, strongly indicate that antigen is recognized on the surface of the beads. Despite effective stimulation of secondary CTL responses, antigen on beads was completely inactive in stimulating a primary CTL response by naive spleen cells. The results of mixing experiments by using beads and alloantigen-bearing cells or plasma membrane vesicles indicate that the lack of a primary response may result from a requirement for a soluble factor(s) that is not needed for generation of secondary responses. The unique advantages of cell-size supported membranes for studying antigen recognition by T cells are discussed. The beads can be handled and used like antigen-bearing cells in functional assays, while possessing well-defined, readily varied, and easily quantitated composition.     

Adhesion characteristics and stability assessment of lectin-modified liposomes for site-specific drug delivery

Carbohydrate moieties of the cellular glycocalyx have been suggested to play an important role in biological recognition processes during pathologic conditions, such as inflammation and cancer. Herein, we describe lectin-modified liposomes which might have potential for site-specific drug delivery during the therapy of such diseases. Specific interactions of plain (i.e., unmodified) and PEGylated, lectin-grafted liposomes with model membranes were investigated under real-time flow conditions using a quartz crystal microbalance. In addition, the morphology of the liposomal systems was assessed by atomic force microscopy. Plain liposomes exhibited only unspecific adhesion to glycolipid membranes and had a tendency to coalesce. The degree of membrane interaction was significantly increased when plain liposomes were modified with the lectin, Concanavalin A. However, vesicle fusion also markedly increased as a result of lectin modification. Additional PEGylation of liposomes reduced unspecific adhesion phenomena, as well as coalescence. Moreover, our studies enabled us to establish quartz crystal microbalance and atomic force microscopy as powerful and complementary methods to characterize adhesion properties of targeted drug delivery systems.     

Mimicking a cytoskeleton by coupling poly(N-isopropylacrylamide) to the inner leaflet of liposomal membranes: effects of photopolymerization on vesicle shape and polymer architecture

Networks of N-isopropylacrylamide (NIPAM) copolymers, coupled to spherical phospholipid bilayers, are suitable as a model for the study of the interaction between the cytoskeleton and cellular membranes, as well as for promising new drug delivery systems with triggerable drug release properties and improved stability. In this article, we describe a simple preparation technique for liposomes from egg phosphatidyl choline (EPC) encapsulating a cross-linked NIPAMminus signTEGDM copolymer skeleton (tetraethylene glycol dimethacrylate, TEGDM) which is coupled only to the inner monolayer by a novel membrane anchor monomer. Polymerization in the lipid vesicles was initiated at the inner membrane surface by the radical initiator 2,2-diethoxy-acetophenone (DEAP) permeating through the membrane from the outside. The effects of photopolymerization and polymer formation on vesicle shape and membrane integrity were studied by transmission electron microscopy (TEM), cryo-TEM, and atomic force microscopy (AFM). Upon UV irradiation, approximately 100% of the vesicles contained a polymer gel and only occasional changes in the spherical shape of the liposomes were observed. The architecture of the polymer network inside the liposomal compartment was determined by the conditions of the photopolymerization. Composite structures of polymer hollow spheres or solid spheres, respectively, tethered to spherical membrane vesicles were produced. The increased stability of the polymer-tethered lipid bilayers against solubilization by sodium cholate, compared to pure EPC vesicles, was determined by radiolabeling the lipid membrane.     

Membrane activity of a C-reactive protein

C-reactive protein (CRP) from the American horseshoe crab, Limulus polyphemus, exhibits complex membrane activities. Here, we describe the behavior of protein and lipid as CRP interacts with model liposomes and bacterial membranes. Limulus C-reactive protein (L-CRP) forms extended fibrilar structures that encapsulate liposomes in the presence of Ca²⁺. We have observed structures consistent in size and shape with these fibers bound to the surface of Gram-negative bacteria. The membranes of Limulus CRP-treated bacteria exhibit significantly different mechano-elastic properties than those of untreated bacteria. In vitro, bilayer lipids undergo a rigidification and reorganization of small domains. We suggest that these interactions reflect the protein’s role as a primary defense molecule, functioning in the entrapment and killing of potential pathogens.