PI(4,5)P2 Clustering and Its Impact on Biological Functions.

Located at the inner leaflet of the plasma membrane (PM), phosphatidyl-inositol 4,5-bisphosphate [PI(4,5)P2] composes only 1-2 mol% of total PM lipids. With its synthesis and turnover both spatially and temporally regulated, PI(4,5)P2 recruits and interacts with hundreds of cellular proteins to support a broad spectrum of cellular functions. Several factors contribute to the versatile and dynamic distribution of PI(4,5)P2 in membranes. Physiological multivalent cations such as Ca2+ and Mg2+ can bridge between PI(4,5)P2 headgroups, forming nanoscopic PI(4,5)P2-cation clusters. The distinct lipid environment surrounding PI(4,5)P2 affects the degree of PI(4,5)P2 clustering. In addition, diverse cellular proteins interacting with PI(4,5)P2 can further regulate PI(4,5)P2 lateral distribution and accessibility. This review summarizes the current understanding of PI(4,5)P2 behavior in both cells and model membranes, with emphasis on both multivalent cation- and protein-induced PI(4,5)P2 clustering. Understanding the nature of spatially separated pools of PI(4,5)P2 is fundamental to cell biology.

Physical Principles of Membrane Shape Regulation by the Glycocalyx.

Cells bend their plasma membranes into highly curved forms to interact with the local environment, but how shape generation is regulated is not fully resolved. Here, we report a synergy between shape-generating processes in the cell interior and the external organization and composition of the cell-surface glycocalyx. Mucin biopolymers and long-chain polysaccharides within the glycocalyx can generate entropic forces that favor or disfavor the projection of spherical and finger-like extensions from the cell surface. A polymer brush model of the glycocalyx successfully predicts the effects of polymer size and cell-surface density on membrane morphologies. Specific glycocalyx compositions can also induce plasma membrane instabilities to generate more exotic undulating and pearled membrane structures and drive secretion of extracellular vesicles. Together, our results suggest a fundamental role for the glycocalyx in regulating curved membrane features that serve in communication between cells and with the extracellular matrix.

Nano-scale domains in the plasma membrane are like macroscopic domains in asymmetric bilayers.

Unfavorable lipid-lipid pairwise interactions between HiTm and LowTm lipids drive liquid-disordered (Ld) + liquid-ordered (Lo) phase separation. Large size of phase domains is opposed by lipid dipole repulsions, which are more significant compared with the pairwise interactions for naturally abundant LowTm lipids such as palmitoyl oleoyl phosphatidylcholine. During the nano-to-macro domain size transition, no lipid phase transition occurs, and measured properties of Ld + Lo nanodomains are found to be essentially the same as those of macrodomains. Use of macrodomains in mixtures to model cell plasma membranes (PM) is helpful, enabling study by optical microscopy. Use of asymmetric giant unilamellar vesicles to model a PM reveals that ordered phase domains in one leaflet induce ordered domains in an otherwise uniform phase in the apposing leaflet that models a cytoplasmic leaflet. Because macro and nano phase properties are so similar, we conclude that a cell PM that has nano-scale Ld + Lo phase domains in the exoplasmic leaflet is likely to induce nano-scale ordered domains in the cytoplasmic leaflet.

An Unexpected Driving Force for Lipid Order Appears in Asymmetric Lipid Bilayers.

An ordered phase in one leaflet of an asymmetric bilayer can induce a precisely superimposed induced order domain in the apposed leaflet. Order is induced in such simple lipid compositions as dioleoylphosphatidylcholine/cholesterol (DOPC)/chol) which is expected to be a uniform and disordered lipid mixture. Dye partitioning can be used to label and identify coexisting liquid-disordered (Ld), liquid-ordered (Lo), or gel-ordered (Lß) molecules in a phase-separated leaflet. In the other leaflet of an asymmetric bilayer, dye partitioning also labels and identifies any induced order domains created by an Lo or gel phase domain in the apposed leaflet as well as the state of disorder of the lipid surrounding the induced ordered region. We explore a molecular level mechanism by which a disorder-prone uniform mixture of DOPC/chol = 0.8/0.2 would spontaneously separate into ordered regions coexisting with disordered regions. A redistribution of cholesterol seems to take place in the regions apposed to the ordered phase. The precision of the superposition of Lo or gel domains with their induced order domains implies a strong energy penalty that would be incurred if order/disorder interfaces were to form at the bilayer midplane. We conclude that the energy penalty for Lo/Ld or gel/Ld contact in the bilayer midplane is sufficient to drive disorderly DOPC/chol into an ordered state that reduces unfavorable order-disorder contacts at the bilayer midplane interface.

Membrane lipids: where they are and how they behave.

Throughout the biological world, a 30 A hydrophobic film typically delimits the environments that serve as the margin between life and death for individual cells. Biochemical and biophysical findings have provided a detailed model of the composition and structure of membranes, which includes levels of dynamic organization both across the lipid bilayer (lipid asymmetry) and in the lateral dimension (lipid domains) of membranes. How do cells apply anabolic and catabolic enzymes, translocases and transporters, plus the intrinsic physical phase behaviour of lipids and their interactions with membrane proteins, to create the unique compositions and multiple functionalities of their individual membranes?

Asymmetric Bilayers by Hemifusion: Method and Leaflet Behaviors.

We describe a new method to prepare asymmetric giant unilamellar vesicles (aGUVs) via hemifusion. Hemifusion of giant unilamellar vesicles and a supported lipid bilayer, triggered by calcium, promotes the lipid exchange of the fused outer leaflets mediated by lipid diffusion. We used different fluorescent dyes to monitor the inner and the outer leaflets of the unsupported aGUVs. We confirmed that almost all newly exchanged lipids in the aGUVs are found in the outer leaflet of these asymmetric vesicles. In addition, we test the stability of the aGUVs formed by hemifusion in preserving their contents during the procedure. For aGUVs prepared from the hemifusion of giant unilamellar vesicles composed of 1,2-distearoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phosphocholine/cholesterol = 0.39/0.39/0.22 and a supported lipid bilayer of 1,2-dioleoyl-sn-glycero-3-phosphocholine/cholesterol = 0.8/0.2, we observed the exchanged lipids to alter the bilayer properties. To access the physical and chemical properties of the asymmetric bilayer, we monitored the dye partition coefficients of individual leaflets and the generalized polarization of the fluorescence probe 6-dodecanoyl-2-[ N-methyl-N-(carboxymethyl)amino] naphthalene, a sensor for the lipid packing/order of its surroundings. For a high percentage of lipid exchange (>70%), the dye partition indicates induced-disordered and induced-ordered domains. The induced domains have distinct lipid packing/order compared to the symmetric liquid-disordered and liquid-ordered domains.

Gramicidin Increases Lipid Flip-Flop in Symmetric and Asymmetric Lipid Vesicles.

Unlike most transmembrane proteins, phospholipids can migrate from one leaflet of the membrane to the other. Because this spontaneous lipid translocation (flip-flop) tends to be very slow, cells facilitate the process with enzymes that catalyze the transmembrane movement and thereby regulate the transbilayer lipid distribution. Nonenzymatic membrane-spanning proteins with unrelated primary functions have also been found to accelerate lipid flip-flop in a nonspecific manner and by various hypothesized mechanisms. Using deuterated phospholipids, we examined the acceleration of flip-flop by gramicidin channels, which have well-defined structures and known functions, features that make them ideal candidates for probing the protein-membrane interactions underlying lipid flip-flop. To study compositionally and isotopically asymmetric proteoliposomes containing gramicidin, we expanded a recently developed protocol for the preparation and characterization of lipid-only asymmetric vesicles. Channel incorporation, conformation, and function were examined with small angle x-ray scattering, circular dichroism, and a stopped-flow spectrofluorometric assay, respectively. As a measure of lipid scrambling, we used differential scanning calorimetry to monitor the effect of gramicidin on the melting transition temperatures of the two bilayer leaflets. The two calorimetric peaks of the individual leaflets merged into a single peak over time, suggestive of scrambling, and the effect of the channel on the transbilayer lipid distribution in both symmetric 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and asymmetric 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles was quantified from proton NMR measurements. Our results show that gramicidin increases lipid flip-flop in a complex, concentration-dependent manner. To determine the molecular mechanism of the process, we used molecular dynamics simulations and further computational analysis of the trajectories to estimate the extent of membrane deformation. Together, the experimental and computational approaches were found to constitute an effective means for studying the effects of transmembrane proteins on lipid distribution in both symmetric and asymmetric model membranes.

Multivalent Cation-Bridged PI(4,5)P2 Clusters Form at Very Low Concentrations.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 or PIP2), is a key component of the inner leaflet of the plasma membrane in eukaryotic cells. In model membranes, PIP2 has been reported to form clusters, but whether these locally different conditions could give rise to distinct pools of unclustered and clustered PIP2 is unclear. By use of both fluorescence self-quenching and Förster resonance energy transfer assays, we have discovered that PIP2 self-associates at remarkably low concentrations starting below 0.05 mol% of total lipids. Formation of these clusters was dependent on physiological divalent metal ions, such as Ca2+, Mg2+, Zn2+, or trivalent ions Fe3+ and Al3+. Formation of PIP2 clusters was also headgroup-specific, being largely independent of the type of acyl chain. The similarly labeled phospholipids phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol exhibited no such clustering. However, six phosphoinositide species coclustered with PIP2. The degree of PIP2 cation clustering was significantly influenced by the composition of the surrounding lipids, with cholesterol and phosphatidylinositol enhancing this behavior. We propose that PIP2 cation-bridged cluster formation, which might be similar to micelle formation, can be used as a physical model for what could be distinct pools of PIP2 in biological membranes. To our knowledge, this study provides the first evidence of PIP2 forming clusters at such low concentrations. The property of PIP2 to form such clusters at such extremely low concentrations in model membranes reveals, to our knowledge, a new behavior of PIP2 proposed to occur in cells, in which local multivalent metal ions, lipid compositions, and various binding proteins could greatly influence PIP2 properties. In turn, these different pools of PIP2 could further regulate cellular events.

FRET Detects the Size of Nanodomains for Coexisting Liquid-Disordered and Liquid-Ordered Phases.

Biomembranes with as few as three lipid components can form coexisting liquid-disordered (Ld) and liquid-ordered (Lo) phases. In the coexistence region of Ld and Lo phases, the lipid mixtures 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/chol or brain sphingomyelin (bSM)/DOPC/chol form micron-scale domains that are easily visualized with light microscopy. Although large domains are not observed in the mixtures DSPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/chol and bSM/POPC/chol, lateral heterogeneity is nevertheless detected using techniques with nanometer-scale spatial resolution. We propose a simple and accessible method to measure domain sizes below optical resolution (∼200 nm). We measured nanodomain size for the latter two mixtures by combining experimental Förster resonance energy transfer data with a Monte-Carlo-based analysis. We found a domain radius of 7.5-10 nm for DSPC/POPC/chol, similar to values obtained previously by neutron scattering, and ∼5 nm for bSM/POPC/chol, slightly smaller than measurable by neutron scattering. These analyses also detect the domain-size transition that is observed by fluorescence microscopy in the four-component lipid mixture bSM/DOPC/POPC/chol. Accurate measurements of fluorescent-probe partition coefficients are especially important for the analysis; therefore, we exploit three different methods to measure the partition coefficient of fluorescent molecules between Ld and Lo phases.

Membrane Bending Moduli of Coexisting Liquid Phases Containing Transmembrane Peptide.

A number of highly curved membranes in vivo, such as epithelial cell microvilli, have the relatively high sphingolipid content associated with "raft-like" composition. Given the much lower bending energy measured for bilayers with "nonraft" low sphingomyelin and low cholesterol content, observing high curvature for presumably more rigid compositions seems counterintuitive. To understand this behavior, we measured membrane rigidity by fluctuation analysis of giant unilamellar vesicles. We found that including a transmembrane helical GWALP peptide increases the membrane bending modulus of the liquid-disordered (Ld) phase. We observed this increase at both low-cholesterol fraction and higher, more physiological cholesterol fraction. We find that simplified, commonly used Ld and liquid-ordered (Lo) phases are not representative of those that coexist. When Ld and Lo phases coexist, GWALP peptide favors the Ld phase with a partition coefficient of 3-10 depending on mixture composition. In model membranes at high cholesterol fractions, Ld phases with GWALP have greater bending moduli than the Lo phase that would coexist.

Cholesterol Promotes Protein Binding by Affecting Membrane Electrostatics and Solvation Properties.

Binding of the retroviral structural protein Gag to the cellular plasma membrane is mediated by the protein's matrix (MA) domain. Prominent among MA-PM interactions is electrostatic attraction between the positively charged MA domain and the negatively charged plasma membrane inner leaflet. Previously, we reported that membrane association of HIV-1 Gag, as well as purified Rous sarcoma virus (RSV) MA and Gag, depends strongly on the presence of acidic lipids and is enhanced by cholesterol (Chol). The mechanism underlying this enhancement was unclear. Here, using a broad set of in vitro and in silico techniques we addressed molecular mechanisms of association between RSV MA and model membranes, and investigated how Chol enhances this association. In neutron scattering experiments with liposomes in the presence or absence of Chol, MA preferentially interacted with preexisting POPS-rich clusters formed by nonideal lipid mixing, binding peripherally to the lipid headgroups with minimal perturbation to the bilayer structure. Molecular dynamics simulations showed a stronger MA-bilayer interaction in the presence of Chol, and a large Chol-driven increase in lipid packing and membrane surface charge density. Although in vitro MA-liposome association is influenced by disparate variables, including ionic strength and concentrations of Chol and charged lipids, continuum electrostatic theory revealed an underlying dependence on membrane surface potential. Together, these results conclusively show that Chol affects RSV MA-membrane association by making the electrostatic potential at the membrane surface more negative, while decreasing the penalty for lipid headgroup desolvation. The presented approach can be applied to other viral and nonviral proteins.

Line Tension Controls Liquid-Disordered + Liquid-Ordered Domain Size Transition in Lipid Bilayers.

To better understand animal cell plasma membranes, we studied simplified models, namely four-component lipid bilayer mixtures. Here we describe the domain size transition in the region of coexisting liquid-disordered (Ld) + liquid-ordered (Lo) phases. This transition occurs abruptly in composition space with domains increasing in size by two orders of magnitude, from tens of nanometers to microns. We measured the line tension between coexisting Ld and Lo domains close to the domain size transition for a variety of lipid mixtures, finding that in every case the transition occurs at a line tension of ∼0.3 pN. A computational model incorporating line tension and dipole repulsion indicated that even small changes in line tension can result in domains growing in size by several orders of magnitude, consistent with experimental observations. We find that other properties of the coexisting Ld and Lo phases do not change significantly in the vicinity of the abrupt domain size transition.

Phase diagram of a polyunsaturated lipid mixture: Brain sphingomyelin/1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine/cholesterol.

Phospholipids having a polyunsaturated acyl chain are abundant in biological membranes, but their behavior in lipid mixtures is difficult to study. Here we elucidate the nature of such mixtures with this report of the first ternary phase diagram containing the polyunsaturated lipid SDPC in mixtures of BSM/SDPC/Chol (brain sphingomyelin/1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine/cholesterol). These mixtures show coexisting macroscopic liquid-disordered (Ld) and liquid-ordered (Lo) phase separation, with phase boundaries determined by FRET and by fluorescence microscopy imaging of giant unilamellar vesicles (GUVs). Surprisingly, SDPC mixes with BSM/Chol similarly to how DOPC and POPC mix with BSM/Chol. Notably, intermediate states are produced within the Ld+Lo liquid-liquid immiscibility region upon addition of fourth component POPC. These mixtures of BSM/SDPC/POPC/Chol exhibit nanoscopic Ld+Lo domains over a very large volume of composition space, possibly because Ld/Lo line tension is not high.

Effects of Membrane Charge and Order on Membrane Binding of the Retroviral Structural Protein Gag.

UNLABELLED: The retroviral structural protein Gag binds to the inner leaflet of the plasma membrane (PM), and many cellular proteins do so as well. We used Rous sarcoma virus (RSV) Gag together with membrane sensors to study the principles governing peripheral protein membrane binding, including electrostatics, specific recognition of phospholipid headgroups, sensitivity to phospholipid acyl chain compositions, preference for membrane order, and protein multimerization. We used an in vitro liposome-pelleting assay to test protein membrane binding properties of Gag, the well-characterized MARCKS peptide, a series of fluorescent electrostatic sensor proteins (mNG-KRn), and the specific phosphatidylserine (PS) binding protein Evectin2. RSV Gag and mNG-KRn bound well to membranes with saturated and unsaturated acyl chains, whereas the MARCKS peptide and Evectin2 preferentially bound to membranes with unsaturated acyl chains. To further discriminate whether the primary driving force for Gag membrane binding is electrostatic interactions or preference for membrane order, we measured protein binding to giant unilamellar vesicles (GUVs) containing the same PS concentration in both disordered (Ld) and ordered (Lo) phases. RSV Gag and mNG-KRn membrane association followed membrane charge, independent of membrane order. Consistent with pelleting data, the MARCKS peptide showed preference for the Ld domain. Surprisingly, the PS sensor Evectin2 bound to the PS-rich Ld domain with 10-fold greater affinity than to the PS-rich Lo domain. In summary, we found that RSV Gag shows no preference for membrane order, while proteins with reported membrane-penetrating domains show preference for disordered membranes. IMPORTANCE: Retroviral particles assemble on the PM and bud from infected cells. Our understanding of how Gag interacts with the PM and how different membrane properties contribute to overall Gag assembly is incomplete. This study examined how membrane charge and membrane order influence Gag membrane association. Consistent with previous work on RSV Gag, we report here that electrostatic interactions provide the primary driving force for RSV Gag membrane association. Using phase-separated GUVs with known lipid composition of the Ld and Lo phases, we demonstrate for the first time that RSV Gag is sensitive to membrane charge but not membrane order. In contrast, the cellular protein domain MARCKS and the PS sensor Evectin2 show preference for disordered membranes. We also demonstrate how to define GUV phase composition, which could serve as a tool in future studies of protein membrane interactions.

Subnanometer Structure of an Asymmetric Model Membrane: Interleaflet Coupling Influences Domain Properties.

Cell membranes possess a complex three-dimensional architecture, including nonrandom lipid lateral organization within the plane of a bilayer leaflet, and compositional asymmetry between the two leaflets. As a result, delineating the membrane structure-function relationship has been a highly challenging task. Even in simplified model systems, the interactions between bilayer leaflets are poorly understood, due in part to the difficulty of preparing asymmetric model membranes that are free from the effects of residual organic solvent or osmotic stress. To address these problems, we have modified a technique for preparing asymmetric large unilamellar vesicles (aLUVs) via cyclodextrin-mediated lipid exchange in order to produce tensionless, solvent-free aLUVs suitable for a range of biophysical studies. Leaflet composition and structure were characterized using isotopic labeling strategies, which allowed us to avoid the use of bulky labels. NMR and gas chromatography provided precise quantification of the extent of lipid exchange and bilayer asymmetry, while small-angle neutron scattering (SANS) was used to resolve bilayer structural features with subnanometer resolution. Isotopically asymmetric POPC vesicles were found to have the same bilayer thickness and area per lipid as symmetric POPC vesicles, demonstrating that the modified exchange protocol preserves native bilayer structure. Partial exchange of DPPC into the outer leaflet of POPC vesicles produced chemically asymmetric vesicles with a gel/fluid phase-separated outer leaflet and a uniform, POPC-rich inner leaflet. SANS was able to separately resolve the thicknesses and areas per lipid of coexisting domains, revealing reduced lipid packing density of the outer leaflet DPPC-rich phase compared to typical gel phases. Our finding that a disordered inner leaflet can partially fluidize ordered outer leaflet domains indicates some degree of interleaflet coupling, and invites speculation on a role for bilayer asymmetry in modulating membrane lateral organization.

HIV-1 Gag protein can sense the cholesterol and acyl chain environment in model membranes.

Membrane binding of the HIV-1 group-specific antigen (Gag) structural protein, a critical step in viral assembly at the plasma membrane, is mediated by the myristoylated, highly basic matrix (MA) domain, which interacts with negatively charged lipids in the inner leaflet. According to a popular model, virus particles bud from membrane rafts, microdomains enriched in cholesterol and high-melting phospholipids with higher order than found outside rafts. How Gag might recognize membrane rafts, if they exist in the inner leaflet, is unknown. Using a liposome flotation assay with proteins translated in vitro, we investigated whether Gag can sense the composition of the hydrophobic part of the bilayer, by fixing lipid head group composition and varying hydrophobic properties. In liposomes composed solely of phosphatidylserine and phosphatidylcholine, and with the same overall membrane negative charge, Gag strongly preferred lipids with both acyl chains unsaturated over those with only one chain unsaturated. Adding cholesterol increased Gag binding and led to closer packing of phospholipids. However, higher membrane order, as measured by electron spin resonance, was not correlated with increased Gag binding. Gag proteins from two other retroviruses gave similar results. These liposome binding preferences were qualitatively recapitulated by purified myristoylated HIV-1 MA. Phosphatidylinositol 4,5-bisphosphate and cholesterol enhanced binding in an additive manner. Taken together, these results show that Gag is sensitive both to the acyl chains of phosphatidylserine and to cholesterol concentration and other details of the membrane environment. These observations may help explain how retroviruses acquire a raft-like lipid composition.

Phase behavior and domain size in sphingomyelin-containing lipid bilayers.

Membrane raft size measurements are crucial to understanding the stability and functionality of rafts in cells. The challenge of accurately measuring raft size is evidenced by the disparate reports of domain sizes, which range from nanometers to microns for the ternary model membrane system sphingomyelin (SM)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/cholesterol (Chol). Using Förster resonance energy transfer (FRET) and differential scanning calorimetry (DSC), we established phase diagrams for porcine brain SM (bSM)/dioleoyl-sn-glycero-3-phosphocholine (DOPC)/Chol and bSM/POPC/Chol at 15 and 25°C. By combining two techniques with different spatial sensitivities, namely FRET and small-angle neutron scattering (SANS), we have significantly narrowed the uncertainty in domain size estimates for bSM/POPC/Chol mixtures. Compositional trends in FRET data revealed coexisting domains at 15 and 25°C for both mixtures, while SANS measurements detected no domain formation for bSM/POPC/Chol. Together these results indicate that liquid domains in bSM/POPC/Chol are between 2 and 7nm in radius at 25°C: that is, domains must be on the order of the 2-6nm Förster distance of the FRET probes, but smaller than the ~7nm minimum cluster size detectable with SANS. However, for palmitoyl SM (PSM)/POPC/Chol at a similar composition, SANS detected coexisting liquid domains. This increase in domain size upon replacing the natural SM component (which consists of a mixture of chain lengths) with synthetic PSM, suggests a role for SM chain length in modulating raft size in vivo.

Phase diagram of a 4-component lipid mixture: DSPC/DOPC/POPC/chol.

We report the first 4-component phase diagram for the lipid bilayer mixture, DSPC/DOPC/POPC/chol (distearoylphosphatidylcholine/dioleoylphosphatidylcholine/1-palmitoyl, 2-oleoylphosphatidylcholine/cholesterol). This phase diagram, which has macroscopic Ld+Lo phase domains, clearly shows that all phase boundaries determined for the 3-component mixture containing DOPC transition smoothly into the boundaries for the 3-component mixture containing POPC, which has nanoscopic phase domains of Ld+Lo. Our studies start from two published ternary phase diagrams, and show how these can be combined into a quaternary phase diagram by study of a few hundred samples of intermediate compositions.

Toward a better raft model: modulated phases in the four-component bilayer, DSPC/DOPC/POPC/CHOL.

The liquid-liquid (Ld + Lo) coexistence region within a distearoyl-phosphatidylcholine/dioleoyl-phosphatidylcholine/palmitoyl-oleoyl-phosphatidylcholine/cholesterol (DSPC/DOPC/POPC/CHOL) mixture displays a nanoscopic-to-macroscopic transition of phase domains as POPC is replaced by DOPC. Previously, we showed that the transition goes through a modulated phase regime during this replacement, in which patterned liquid phase morphologies are observed on giant unilamellar vesicles (GUVs). Here, we describe a more detailed investigation of the modulated phase regime along two different thermodynamic tielines within the Ld + Lo region of this four-component mixture. Using fluorescence microscopy of GUVs, we found that the modulated phase regime occurs at relatively narrow DOPC/(DOPC+POPC) ratios. This modulated phase window shifts to higher values of DOPC/(DOPC+POPC) when CHOL concentration is increased, and coexisting phases become closer in properties. Monte Carlo simulations reproduced the patterns observed on GUVs, using a competing interactions model of line tension and curvature energies. Sufficiently low line tension and high bending moduli are required to generate stable modulated phases. Altogether, our studies indicate that by tuning the lipid composition, both the domain size and morphology can be altered drastically within a narrow composition space. This lends insight into a possible mechanism whereby cells can reorganize plasma membrane compartmentalization simply by tuning the local membrane composition or line tension.