Giant Unilamellar Vesicles Membrane Research Articles

Entry of a Six-Residue Antimicrobial Peptide Derived from Lactoferricin B into Single Vesicles and Escherichia coli Cells without Damaging their Membranes.

Lactoferricin B (LfcinB) and shorter versions of this peptide have antimicrobial activity. However, the elementary processes of interactions of these peptides with lipid membranes and bacteria are still not well understood. To elucidate the mechanism of their antimicrobial activity, we investigated the interactions of LfcinB (4-9) (its sequence of RRWQWR) with Escherichia coli cells and giant unilamellar vesicles (GUVs). LfcinB (4-9) and lissamine rhodamine B red-labeled LfcinB (4-9) (Rh-LfcinB (4-9)) did not induce an influx of a membrane-impermeant fluorescent probe, SYTOX green, from the outside of E. coli cells into their cytoplasm, indicating that no damage occurred in their plasma membrane. To examine the activity of LfcinB (4-9) to enter E. coli cytoplasm, we investigated the interaction of Rh-LfcinB (4-9) with single cells of E. coli containing calcein using confocal microscopy. We found that Rh-LfcinB (4-9) entered the cytoplasm without leakage of calcein. Next, we investigated the interactions of Rh-LfcinB (4-9) with single GUVs of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) mixtures containing a fluorescent probe, Alexa Fluor 647 hydrazide (AF647), using the single GUV method. The results indicate that Rh-LfcinB (4-9) outside the GUV translocated through the GUV membrane and entered its lumen without leakage of AF647. Interaction of Rh-LfcinB (4-9) with DNA increased its fluorescence intensity greatly. Therefore, we can conclude that Rh-LfcinB (4-9) can translocate across lipid membrane regions of the plasma membrane of E. coli cells to enter their cytoplasm without leakage of calcein and its antimicrobial activity is not due to damage of their plasma membranes.

Capturing suboptical dynamic structures in lipid bilayer patches formed from free-standing giant unilamellar vesicles.

There is accumulating evidence that the small-scale lateral organization of biological membranes has a crucial role in signaling and trafficking in cells. However, it has been difficult to characterize these features with existing methods for preparing and analyzing freestanding membranes, because the dynamics occurs below the optical resolution possible with these protocols. We have developed a protocol that permits the imaging of lipid nanodomains and lateral protein organization in membranes of giant unilamellar vesicles (GUVs). Freestanding GUVs are transferred onto a mica support, and after treatment with magnesium chloride, they collapse to form planar lipid bilayer (PLB) patches. Rapid GUV collapse onto the mica preserves the lateral organization of freestanding membranes and thus makes it possible to image 'snapshots' of GUVs up to nanometer resolution by high-resolution microscopy. The method has been applied to classical lipid raft mixtures in which suboptical domain fluctuations have been imaged in both the liquid-ordered and liquid-disordered membrane phases. High-resolution scanning by atomic force microscopy (AFM) of membranes composed of binary and ternary lipid mixtures reconstituted with Na+/K+-ATPase (NKA) has revealed the spatial distribution and orientations of individual proteins, as well as details of membrane lateral structure. Immunolabeling followed by confocal microscopy can also provide information about the spatial distribution of proteins. The protocol opens up a new avenue for quantitative biophysical studies of suboptical dynamic structures in biomembranes, which are local and short-lived. Preparation of GUVs, PLB patches and their imaging takes <24 h.

Study on the Application of Electric Field to Giant Vesicles Comprised of 1,2-Dilauroyl-Sn-Glycero-3-Phosphatidylcholine Using Optical Fluorescence Microscopy

The influence of alternating electric field (AC) in the structure and dynamics of giant unilamellar vesicles (GUVs) comprised of 1,2-dilauroyl-sn-glycero-3-phosphatidylcholine (DLPC) is reported. The investigations were conducted by using optical fluorescence microscopy as the method of analysis. The lipid membrane of the DLPC GUVs at the fluid phase can be deformed and they migrate towards the electrodes under AC electric field. Nevertheless, membrane disruption or vesicle fusion was never noticed. The addition of concentrated glucose solution influences the osmotic pressure of the system leading to the formation of filaments at the outer region of the GUVs. These long flexible cylinders do not retract spontaneously. However, the application of AC electric field (20 V/mm, 20 Hz) enables the filaments to be retracted back to the GUVs membrane at a calculated speed of 0.18 µm.s-1

Copper ATPase CopA from Escherichia coli: Quantitative Correlation between ATPase Activity and Vectorial Copper Transport.

Cu-ATPases are membrane copper transporters present in all kingdoms of life. They play a central role in Cu homeostasis by pumping Cu ions across cell membranes with energy derived from ATP hydrolysis. In this work, the Cu-ATPase CopA from Escherichia coli was expressed and purified in fully functional form and demonstrated to bind Cu(I) with subfemtomolar affinity. It was incorporated into the lipid membrane of giant unilamellar vesicles (GUVs) whose dimensions match those of eukaryotic cells. An 1H NMR approach provided a quantitative ATPase activity assay for the enzyme either dissolved in detergent or embedded in GUV membranes. The activity varied with the Cu(I) availability in an optimized assay solution for either environment, demonstrating a direct correlation between ATPase activity and Cu(I) transport. Quantitative analysis of the Cu content trapped by the GUVs is consistent with a Cu:ATP turnover ratio of 1.

Phosphatidylserine lipids and membrane order precisely regulate the activity of Polybia-MP1 peptide

Polybia-MP1 (IDWKKLLDAAKQIL-NH2) is a lytic peptide from the Brazilian wasp venom with known anti-cancer properties. Previous evidence indicates that phosphatidylserine (PS) lipids are relevant for the lytic activity of MP1. In agreement with this requirement, phosphatidylserine lipids are translocated to the outer leaflet of cells, and are available for MP1 binding, depending on the presence of liquid-ordered domains. Here, we investigated the effect of PS on MP1 activity when this lipid is reconstituted in membranes of giant or large liposomes with different lipid-phase states. By monitoring the membrane and soluble luminal content of giant unilamellar vesicles (GUVs), using fluorescence confocal microscopy, we were able to determine that MP1 has a pore-forming activity at the membrane level. Liquid-ordered domains, which were phase-separated within the membrane of GUVs, influenced the pore-forming activity of MP1. Experiments evaluating the membrane-binding and lytic activity of MP1 on large unilamellar vesicles (LUVs), with the same lipid composition as GUVs, demonstrated that there was synergy between liquid-ordered domains and PS, which enhanced both activities. Based on our findings, we propose that the physicochemical properties of cancer cell membranes, which possess a much higher concentration of PS than normal cells, renders them susceptible to MP1 binding and lytic pore formation. These results can be correlated with MP1's potent and selective anti-cancer activity and pave the way for future research to develop cancer therapies that harness and exploit the properties of MP1.

Formation of Large Hypericin Aggregates in Giant Unilamellar Vesicles—Experiments and Modeling

The incorporation of hypericin (Hyp) from aqueous solutions into giant unilamellar vesicle (GUV) membranes has been studied experimentally and by means of kinetic Monte Carlo modeling. The time evolution of Hyp fluorescence originating from Hyp monomers dissolved in the GUV membrane has been recorded by confocal microscopy and while trapping individual GUVs in optical tweezers. It was shown that after reaching a maximum, the fluorescence intensity gradually decreased toward longer times. Formation of oversized Hyp clusters has been observed on the GUV surface at prolonged time. A simplified kinetic Monte Carlo model is presented to follow the aggregation/dissociation processes of Hyp molecules in the membrane. The simulation results reproduced the basic experimental observations: the scaling of the characteristic fluorescence decay time with the vesicle diameter and the buildup of large Hyp clusters in the GUV membrane.

Effects of Mechanical Properties of Lipid Bilayers on the Entry of Cell-Penetrating Peptides into Single Vesicles.

The translocation of cell-penetrating peptides (CPPs) through plasma membranes of living cells is an important physiological phenomenon in biomembranes. To reveal the mechanism underlying the translocation of a CPP, transportan 10 (TP10), through lipid bilayers, we examined the effects of the mechanical properties of lipid bilayers on the entry of carboxyfluorescein (CF)-labeled TP10 (CF-TP10) into a giant unilamellar vesicle (GUV) using the single GUV method. First, we examined the effect of lateral tension in membranes on the entry of CF-TP10 into single GUVs comprising a mixture of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) (2/8). CF-TP10 entered the GUV lumen before the membrane permeation of Alexa Fluor 647 hydrazide (AF647) from the GUV and thus before pore formation in the membrane. The fraction of entry of CF-TP10 before pore formation and the rate of membrane rupture increased with tension. The CF-TP10-induced fractional change in the membrane area increased continuously with time until membrane rupture, but it increased more slowly than did the CF-TP10 concentration in the GUV membrane. A high mole fraction of cholesterol inhibited the entry of CF-TP10 into single GUVs by suppressing the translocation of CF-TP10 from the external to the internal monolayer, although higher concentrations of CF-TP10 induced the formation of pores through which CF-TP10 rapidly translocated. Suppression of the translocation of CF-TP10 by cholesterol can be reasonably explained by the large line tension of a prepore. We discussed the role of mechanical properties in membranes on the entry of CF-TP10 into single GUVs and proposed a hypothesis of the mechanism that CF-TP10 translocates across a bilayer through transient hydrophilic prepores in the membrane.

Measuring Cellular Reactions upon Particle Approach by Photonic Force Microscopy

The locally changing mobility of a particle in the vicinity of the plasma membrane of a living cell is relevant to medicine and biology, since the viscous drag γ changes significantly with the distance to the interface, thereby triggering mechanosensitive proteins located at the cell surface and influencing the molecular processes of particle binding and endocytosis.In this study we use photonic force microscopy to investigate changes in the diffusive behavior of an optically trapped 1µm polystyrene bead, which approaches the membrane of different biological cells. The bead's temporal fluctuations are tracked interferometrically in 3D with nanometer precision and on a broad spectral bandwidth up to 2 MHz.The autocorrelation of the bead's motion reveals the friction coefficient γ(d) as a function of bead-membrane distance d. The viscous modulus G‘‘(ω,d) and the elastic modulus G’(ω,d) are obtained by analyzing the fluctuation data via the Kramers-Kronig-Relations and give additional access to the frequency-resolved viscous and elastic parameters γ(ω,d) and κ(ω,d), respectively.We investigated the hydrodynamic coupling lengths Λ(d) for different cell types (J774, HT29, MDCK) and a giant unilamellar vesicle (GUV), the latter consisting only of a lipid bilayer and lacking cytoskeleton and molecular motors. All coupling lengths perpendicular to living cell membranes are significantly longer than those of a GUV, leading to strongly enlarged and direction-dependent diffusion times for particle motion close to the membrane. Also, the maximum viscous drag close to cell membranes is significantly larger than at the GUV membrane, giving rise to the conclusion that the composition of the cell interior influences the hydrodynamic interaction. By analyzing the microrheological quantities G(ω,d), we found evidence of a viscoelastic interaction of the bead with the cell coat, which is unique to HT29 cells and additionally regulates the momentum transfer to the cell surface and its mechanosensitive receptors.

Experimental Estimation of Membrane Tension Induced by Osmotic Pressure

Osmotic pressure (Π) induces the stretching of plasma membranes of cells or lipid membranes of vesicles, which plays various roles in physiological functions. However, there have been no experimental estimations of the membrane tension of vesicles upon exposure to Π. In this report, we estimated experimentally the lateral tension of the membranes of giant unilamellar vesicles (GUVs) when they were transferred into a hypotonic solution. First, we investigated the effect of Π on the rate constant, kp, of constant-tension (σex)-induced rupture of dioleoylphosphatidylcholine (DOPC)-GUVs using the method developed by us recently. We obtained the σex dependence of kp in GUVs under Π and by comparing this result with that in the absence of Π, we estimated the tension of the membrane due to Π at the swelling equilibrium, σosmeq. Next, we measured the volume change of DOPC-GUVs under small Π. The experimentally obtained values of σosmeq and the volume change agreed with their theoretical values within the limits of the experimental errors. Finally, we investigated the characteristics of the Π-induced pore formation in GUVs. The σosmeq corresponding to the threshold Π at which pore formation is induced is similar to the threshold tension of the σex-induced rupture. The time course of the radius change of GUVs in the Π-induced pore formation depends on the total membrane tension, σt; for small σt, the radius increased with time to an equilibrium one, which remained constant for a long time until pore formation, but for large σt, the radius increased with time and pore formation occurred before the swelling equilibrium was reached. Based on these results, we discussed the σosmeq and the Π-induced pore formation in lipid membranes.

Effects of Lipid Composition on the Entry of Cell-Penetrating Peptide Oligoarginine into Single Vesicles.

The cell-penetrating peptide R9, an oligoarginine comprising nine arginines, has been used to transport biological cargos into cells. However, the mechanisms underlying its translocation across membranes remain unclear. In this report, we investigated the entry of carboxyfluorescein (CF)-labeled R9 (CF-R9) into single giant unilamellar vesicles (GUVs) of various lipid compositions and the CF-R9-induced leakage of a fluorescent probe, Alexa Fluor 647 hydrazide (AF647), using a method developed recently by us. First, we investigated the interaction of CF-R9 with dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylcholine (DOPC) GUVs containing AF647 and small DOPG/DOPC vesicles. The fluorescence intensity of the GUV membrane due to CF-R9 (i.e., the rim intensity) increased with time to a steady-state value, and then the fluorescence intensity of the membranes of the small vesicles in the GUV lumen increased without leakage of AF647. This result indicates that CF-R9 entered the GUV lumen from the outside by translocating across the lipid membrane without forming pores through which AF647 could leak. The fraction of entry of CF-R9 at 6 min in the absence of pore formation, Pentry (6 min), increased with an increase in CF-R9 concentration, but the CF-R9 concentration in the lumen was low. We obtained similar results for dilauroyl-PG (DLPG)/ditridecanoyl-PC (DTPC) (2/8) GUVs. The values of Pentry (6 min) of CF-R9 for DLPG/DTPC (2/8) GUVs were larger than those obtained with DOPG/DOPC (2/8) GUVs at the same CF-R9 concentrations. In contrast, a high concentration of CF-R9 induced pores in DLPG/DTPC (4/6) GUVs through which CF-R9 entered the GUV lumen, so the CF-R9 concentration in the lumen was higher. However, CF-R9 could not enter DOPG/DOPC/cholesterol (2/6/4) GUVs. Analysis of the rim intensity showed that CF-R9 was located only in the outer monolayer of the DOPG/DOPC/cholesterol (2/6/4) GUVs. On the basis of analyses of these results, we discuss the elementary processes by which CF-R9 enters GUVs of various lipid compositions.

Brownian Dynamics of Electrostatically Adhering Small Vesicles to a Membrane Surface Induces Domains and Probes Viscosity.

Using single-particle tracking, we investigate the interaction of small unilamellar vesicles (SUVs) that are electrostatically tethered to the freestanding membrane of a giant unilamellar vesicle (GUV). We find that the surface mobility of the GUV-riding SUVs is Brownian, insensitive to the bulk viscosity, vesicle size, and vesicle fluidity but strongly altered by the viscosity of the underlying membrane. Analyzing the diffusional behavior of SUVs within the Saffman-Delbrück model for the dynamics of membrane inclusions supports the notion that the mobility of the small vesicles is coupled to that of dynamically induced lipid clusters within the target GUV membrane. The reversible binding also offers a nonperturbative means for measuring the viscosity of biomembranes, which is an important parameter in cell physiology and function.

High-Speed Single Particle Tracking on Model Lipid Membranes

Interferometric scattering detection microscopy (iSCAT) is a powerful method for single particle tracking (SPT) experiments. Recently, we reported on the use of iSCAT for visualizing the diffusion of gold nanoparticles (GNPs) as small as 5nm attached to lipids in model membranes with nanometer lateral precision and at up to 1 MHz frame rate. Exploiting this high spatial and temporal resolution, we revealed transient nanoscopic confinements of nanometer size on millisecond time scales on supported lipid bilayers (SLBs) [1]. To avoid artifacts caused by the glass-lipid interface, we have extended our studies to giant unilamellar vesicles (GUVs). Here, we demonstrate one of the unique capabilities of iSCAT, namely high axial resolution in tracking the displacement of a nanoparticle. We present three-dimensional trajectories of GNP-labeled lipids and unlabeled virus-like particles diffusing on a GUV membrane [2].[1] C.-L. Hsieh, S. Spindler, J. Ehrig, V. Sandoghdar, J. Phys. Chem. B 118 (2014).[2] S. Spindler, H. Stein, J. Ehrig, V. Sandoghdar, in preparation.

Daptomycin Binds but does not Translocate across PC:PG Membranes

The lipopeptide daptomycin is a last-resort antimicrobial in multiresistant Staphylococcus aureus infections. Despite its importance, little is understood about the interaction of daptomycin with lipid membranes and the subsequent events leading to antibacterial effects. In this work we asked three fundamental questions: (1) Does daptomycin bind to giant unilamellar vesicles (GUVs) composed of phosphatidylcholine (PC) and phosphatidylglycerol (PG)? (2) Does it cause dye flux across the membrane? (3) Does the lipopeptide itself translocate across the membrane? Fluorescence confocal microscopy was used to visualize the binding of daptomycin to GUVs, dye flux into those vesicles, and peptide translocation across their membranes. First, we found that daptomycin binds to POPC:POPG GUVs in the presence of 0.5, 2.0, and 20 mM Ca2+. Second, we placed the vesicles in a solution containing carboxyfluorescein (CF) to test for influx. However, influx was never observed. Third, GUVs containing inner vesicles were observed to test for the translocation of daptomycin across the outer membrane of the GUVs, and onto the membrane of the inner vesicles. Again, translocation was never observed. However, in solutions containing 2 mM Ca2+ a significant amount (about 50%) of membranes that had bound daptomycin collapsed shortly after binding occurred, resulting in the destruction of the GUVs. Furthermore, at high Ca2+ concentrations (20 mM), the formation of daptomycin-rich clusters on the GUV membrane was observed. At lower concentrations of Ca2+ (2 mM), daptomycin was distributed evenly on the GUV membrane.Supported by NIH Grants GM072507 and AI088567.

Membrane Fusion via Snare Mimetics Spatially Confined to Intramembrane Domains

Membrane fusion is an essential step for many cellular functions. Moreover, cells must be able to confine specific fusion events to certain organelles. Therefore, it is of great interest to demonstrate and study spatially targeted membrane fusion events in vitro. In eukaryotic cells, processes such as neurotransmission, exocytosis and viral infection require membrane fusion and are mediated by rather complex SNARE proteins. Fusion has also been observed using SNARE mimetics such as DNA-lipid conjugates [Chan et al., Biointerphases, 3:FA17, 2008] and lipidated peptides [Pahler et al., Biophysical Journal, 103:2295, 2012]. Here, we demonstrate the fusion of large unilamellar vesicles (LUVs) to a specific location on giant unilamellar vesicles (GUVs). As fusion mediators we use coiled-coil peptide heterodimers or hybridized DNA, both of which are conjugated to lipid anchors. These molecules are spatially confined to phase domains within the GUV membranes. We observe liquid-ordered-specific partitioning for the DNA-lipid conjugate as well as liquid-disordered-specific docking and hemi-fusion events using lipid anchored peptides. This work is pursued within the MaxSynBio consortium which is jointly funded by the Federal Ministry of Education and Research of Germany and the Max Planck Society.

Artificial vesicles as an animal cell model for the study of biological application of non-thermal plasma

Artificial cell-like model systems can provide information which is hard to obtain with real biological cells. Giant unilamellar vesicles (GUV) containing intra-membrane DNA or OH radical-binding molecules are used to visualize the cytolytic activity of OH radicals. Changes in the GUV membrane are observed by microscopy or flow cytometry as performed for animal cells after non-thermal plasma treatment. The experimental data shows that OH radicals can be detected inside the membrane, although the biological effects are not as significant as for H2O2. This artificial model system can provide a systemic means to elucidate the complex interactions between biological materials and non-thermal plasma.

Experimental and simulation studies of polyarginines across the membrane of giant unilamellar vesicles

Cell-penetrating peptides have widespread applications in biomedicine because of their capability to translocate across cell membranes alone or with cargos.

Translocation of 5′ mRNA cap analogue — peptide conjugates across the membranes of giant unilamellar vesicles

Cell-penetrating peptides (CPPs) have been extensively studied because of their ability to deliver various cargo molecules, which are often potential therapeutic agents. However, in most cases, the exact entry mechanism of CPPs is still unknown. In this study, we focused our attention on the membrane permeability sequence (MPS) peptide (AAVALLPAVLLALLAK) conjugated to analogues of a 5′ mRNA cap. This unique RNA structure plays a pivotal role in eukaryotic gene expression and has a large therapeutic application potential. We validated the translocation abilities of conjugates across the membranes of giant unilamellar vesicles (GUVs) composed of POPC lipids by application of fluorescence microscopy. Translocation of the MPS peptide itself was observed in contrast to peptide conjugates containing mono- and dinucleotide cap analogues, indicating that even for such small cargos, passive translocation does not occur. However, membrane permeability was observed in the case of conjugated mononucleotides. Fluorescence lifetime microscopy (FLIM) of the C6-NBD-phospholipid revealed changes in lipid packing induced by a penetrating peptide. Our results support the usefulness of artificial membrane systems applied to elucidate membrane crossing mechanisms.

The modified fluorescence based vesicle fluctuation spectroscopy technique for determination of lipid bilayer bending properties

Lipid bilayer is the main constitutive element of biological membrane, which confines intracellular space. The mechanical properties of biological membranes may be characterized by various parameters including membrane stiffness or membrane bending rigidity, which can be measured using flicker noise spectroscopy. The flicker noise spectroscopy exploits the spontaneous thermal undulations of the membrane. The method is based on the quantitative analysis of a series of microscopic images captured during thermal membrane fluctuations. Thus, measured bending rigidity coefficient depends on the image quality as well as the selection of computational tools for image processing and mathematical model used. In this work scanning and spinning disc confocal microscopies were used to visualize fluctuating membranes of giant unilamellar vesicles. The bending rigidity coefficient was calculated for different acquisition modes, using different fluorescent probes and different image processing methods. It was shown that both imaging approaches gave similar bending coefficient values regardless of acquisition time. Using the developed methodology the effect of fluorescent probe type and aqueous phase composition on the value of the membrane bending rigidity coefficient was measured. Specifically it was found that the bending rigidity coefficient of DOPC bilayer in water is smaller than that determined for POPC membrane. It has been found that the POPC and DOPC bending rigidities coefficient in sucrose solution was lower than that in water. Fluorescence imaging makes possible the quantitative analysis of membrane mechanical properties of inhomogeneous membrane.

Preparing giant unilamellar vesicles (GUVs) of complex lipid mixtures on demand: Mixing small unilamellar vesicles of compositionally heterogeneous mixtures

Giant unilamellar vesicles (GUVs) are simple model membrane systems of cell-size, which are instrumental to study the function of more complex biological membranes involving heterogeneities in lipid composition, shape, mechanical properties, and chemical properties. We have devised a method that makes it possible to prepare a uniform sample of ternary GUVs of a prescribed composition and heterogeneity by mixing different populations of small unilamellar vesicles (SUVs). The validity of the protocol has been demonstrated by applying it to ternary lipid mixture of DOPC, DPPC, and cholesterol by mixing small unilamellar vesicles (SUVs) of two different populations and with different lipid compositions. The compositional homogeneity among GUVs resulting from SUV mixing is quantified by measuring the area fraction of the liquid ordered–liquid disordered phases in giant vesicles and is found to be comparable to that in GUVs of the prescribed composition produced from hydration of dried lipids mixed in organic solvent. Our method opens up the possibility to quickly increase and manipulate the complexity of GUV membranes in a controlled manner at physiological buffer and temperature conditions. The new protocol will permit quantitative biophysical studies of a whole new class of well-defined model membrane systems of a complexity that resembles biological membranes with rafts.

Introducing a fluorescence-based standard to quantify protein partitioning into membranes

The affinity of peripheral membrane proteins for a lipid bilayer can be described using the partition coefficient (KP). Although several methods to determine KP are known, all possess limitations. To address some of these issues, we developed both: a versatile method based on single molecule detection and fluorescence imaging for determining KP, and a simple measurement standard employing hexahistidine-tagged enhanced green fluorescent protein (eGFP-His6) and free standing membranes of giant unilamellar vesicles (GUVs) functionalized with NTA(Ni) lipids as binding sites. To ensure intrinsic control, our method features two measurement modes. In the single molecule mode, fluorescence correlation spectroscopy (FCS) is applied to quantify free and membrane associated protein concentrations at equilibrium and calculate KP. In the imaging mode, confocal fluorescence images of GUVs are recorded and analyzed with semi-automated software to extract protein mean concentrations used to derive KP. Both modes were compared by determining the affinity of our standard, resulting in equivalent KP values. As observed in other systems, eGFP-His6 affinity for membranes containing increasing amounts of NTA(Ni) lipids rises in a stronger-than-linear fashion. We compared our dual approach with a FCS-based assay that uses large unilamellar vesicles (LUVs), which however fails to capture the stronger-than-linear trend for our NTA(Ni)-His6 standard. Hence, we determined the KP of the MARCKS effector domain with our FCS approach on GUVs, whose results are consistent with previously published data using LUVs. We finally provide a practical manual on how to measure KP and understand it in terms of molecules per lipid surface.