Neuroligin-2 dependent conformational activation of collybistin reconstituted in supported hybrid membranes.

The assembly of the postsynaptic transmitter sensing machinery at inhibitory nerve cell synapses requires the intimate interplay between cell adhesion proteins, scaffold and adaptor proteins, and γ-aminobutyric acid (GABA) or glycine receptors. We developed an in vitro membrane system to reconstitute this process, to identify the essential protein components, and to define their mechanism of action, with a specific focus on the mechanism by which the cytosolic C terminus of the synaptic cell adhesion protein Neuroligin-2 alters the conformation of the adaptor protein Collybistin-2 and thereby controls Collybistin-2-interactions with phosphoinositides (PtdInsPs) in the plasma membrane. Supported hybrid membranes doped with different PtdInsPs and 1,2-dioleoyl-sn-glycero-3-{[N-(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl} nickel salt (DGS-NTA(Ni)) to allow for the specific adsorption of the His6-tagged intracellular domain of Neuroligin-2 (His-cytNL2) were prepared on hydrophobically functionalized silicon dioxide substrates via vesicle spreading. Two different collybistin variants, the WT protein (CB2SH3) and a mutant that adopts an intrinsically 'open' and activated conformation (CB2SH3/W24A-E262A), were bound to supported membranes in the absence or presence of His-cytNL2. The corresponding binding data, obtained by reflectometric interference spectroscopy, show that the interaction of the C terminus of Neuroligin-2 with Collybistin-2 induces a conformational change in Collybistin-2 that promotes its interaction with distinct membrane PtdInsPs.

How arginine derivatives alter the stability of lipid membranes: dissecting the roles of side chains, backbone and termini.

Arginine (R)-rich peptides constitute the most relevant class of cell-penetrating peptides and other membrane-active peptides that can translocate across the cell membrane or generate defects in lipid bilayers such as water-filled pores. The mode of action of R-rich peptides remains a topic of controversy, mainly because a quantitative and energetic understanding of arginine effects on membrane stability is lacking. Here, we explore the ability of several oligo-arginines R[Formula: see text] and of an arginine side chain mimic R[Formula: see text] to induce pore formation in lipid bilayers employing MD simulations, free-energy calculations, breakthrough force spectroscopy and leakage assays. Our experiments reveal that R[Formula: see text] but not R[Formula: see text] reduces the line tension of a membrane with anionic lipids. While R[Formula: see text] peptides form a layer on top of a partly negatively charged lipid bilayer, R[Formula: see text] leads to its disintegration. Complementary, our simulations show R[Formula: see text] causes membrane thinning and area per lipid increase beside lowering the pore nucleation free energy. Model polyarginine R[Formula: see text] similarly promoted pore formation in simulations, but without overall bilayer destabilization. We conclude that while the guanidine moiety is intrinsically membrane-disruptive, poly-arginines favor pore formation in negatively charged membranes via a different mechanism. Pore formation by R-rich peptides seems to be counteracted by lipids with PC headgroups. We found that long R[Formula: see text] and R[Formula: see text] but not short R[Formula: see text] reduce the free energy of nucleating a pore. In short R[Formula: see text], the substantial effect of the charged termini prevent their membrane activity, rationalizing why only longer [Formula: see text] are membrane-active.

Viscoelasticity of Native and Artificial Actin Cortices Assessed by Nanoindentation Experiments.

Cell cortices are responsible for the resilience and morphological dynamics of cells. Measuring their mechanical properties is impeded by contributions from other filament types, organelles, and the crowded cytoplasm. We established a versatile concept for the precise assessment of cortical viscoelasticity based on force cycle experiments paired with continuum mechanics. Apical cell membranes of confluent MDCK II cells were deposited on porous substrates and locally deformed. Force cycles could be described with a time-dependent area compressibility modulus obeying the same power law as employed for whole cells. The reduced fluidity of apical cell membranes compared to living cells could partially be restored by reactivating myosin motors. A comparison with artificial minimal actin cortices (MACs) reveals lower stiffness and higher fluidity attributed to missing cross-links in MACs.

Fusion Pore Formation Observed during SNARE-Mediated Vesicle Fusion with Pore-Spanning Membranes.

Planar pore-spanning membranes (PSMs) have been shown to be a versatile tool to resolve elementary steps of the neuronal fusion process. However, in previous studies, we monitored only lipid mixing between fusing large unilamellar vesicles and PSMs and did not gather information about the formation of fusion pores. To address this important step of the fusion process, we entrapped sulforhodamine B at self-quenching concentrations into large unilamellar vesicles containing the v-SNARE synaptobrevin 2, which were docked and fused with lipid-labeled PSMs containing the t-SNARE acceptor complex ΔN49 prepared on gold-coated porous silicon substrates. By dual-color spinning disk fluorescence microscopy with a time resolution of ∼20 ms, we could unambiguously distinguish between bursting vesicles, which was only rarely observed (<0.01%), and fusion pore formation. From the time-resolved dual-color fluorescence time traces, we were able to identify different fusion pathways, including remaining three-dimensional postfusion structures with released content and transient openings and closings of the fusion pores. Our results on fusion pore formation and lipid diffusion from the PSM into the fusing vesicle let us conclude that the content release, i.e., fusion pore formation after the merger of the two lipid membranes occurs almost simultaneously.

Leaflet-Dependent Distribution of PtdIns[4,5]P2 in Supported Model Membranes.

Supported planar lipid bilayers (SLBs) prepared by spreading of unilamellar vesicles on hydrophilic substrates such as silicon dioxide are frequently used to investigate lipid-protein interactions by means of surface-sensitive methods. In recent years, the receptor lipid phosphatidylinositol-4,5-bisphosphate (PtdIns[4,5]P2) became particularly important as a significant number of proteins bind to this lipid at the inner leaflet of the plasma membrane. Here, we investigated how the lipid PtdIns[4,5]P2 distributes between the two leaflets of an SLB on SiO2 surfaces. We prepared SLBs on SiO2 by spreading small unilamellar vesicles and quantified the adsorption of PtdIns[4,5]P2 binding proteins providing information about the accessibility of PtdIns[4,5]P2. We compared protein binding to PtdIns[4,5]P2 in SLBs with that in lipid monolayers on a 1,1,1-trimethyl-N-(trimethylsilyl)silanamine-functionalized SiO2 surface using reflectometric interference spectroscopy and atomic force microscopy. Our results clearly demonstrate that the accessibility of PtdIns[4,5]P2 for protein binding is reduced in SLBs compared to that in supported hybrid membranes, which is discussed in terms of PtdIns[4,5]P2 distribution in the two leaflets of SLBs.

Phase separation in pore-spanning membranes induced by differences in surface adhesion.

Lipid domains in plasma membranes act as molecular sorting platforms for e.g., signalling processes. In model membranes, such as freestanding or supported bilayers, some lipid domains with defined chemical composition, lipid packing and physical behaviour can be reproduced. However, in vivo, the plasma membrane experiences a proteinaceous scaffold underneath, which can sort, compartmentalize and recruit components within the membrane. The influence of such scaffolds on the phase behaviour of lipid membranes has been barely studied. Here, we investigated the partial attachment of a membrane to a support and its influence on the phase behaviour using pore-spanning membranes (PSMs). PSMs were prepared on SiOx=1-2 functionalized silicon substrates with 1.2 µm-sized pores by spreading giant unilamellar vesicles (GUVs) composed of DOPC/sphingomyelin (1 : 1) with different cholesterol concentrations. Using two different fluorophores, PSMs were visualized by fluorescence microscopy allowing us to distinguish between different membrane phases, a gel (lß), a liquid ordered (lo), and a liquid disordered (ld) phase. At low cholesterol concentrations, coexistence of lß and ld was found, while at higher cholesterol concentrations, coexistence of lo and ld was predominant. Below the mixing temperature, determined by temperature scans, the more ordered phase was always found in the freestanding PSMs, whereas the ld-phase was present in the supported PSMs. We attribute this lipid sorting to a stronger adhesion of the ld-phase lipids to the underlying scaffold. The difference in adhesion alters the phase behaviour from a nominal DOPC/sphingomyelin (1 : 1) mixture to a DOPC/sphingomyelin (1 : 2-1 : 4) mixture compared to phase diagrams obtained from GUVs highlighting the importance of differential adhesive surfaces on lipid domain formation.

Size and mobility of lipid domains tuned by geometrical constraints.

In the plasma membrane of eukaryotic cells, proteins and lipids are organized in clusters, the latter ones often called lipid domains or "lipid rafts." Recent findings highlight the dynamic nature of such domains and the key role of membrane geometry and spatial boundaries. In this study, we used porous substrates with different pore radii to address precisely the extent of the geometric constraint, permitting us to modulate and investigate the size and mobility of lipid domains in phase-separated continuous pore-spanning membranes (PSMs). Fluorescence video microscopy revealed two types of liquid-ordered (lo) domains in the freestanding parts of the PSMs: (i) immobile domains that were attached to the pore rims and (ii) mobile, round-shaped lo domains within the center of the PSMs. Analysis of the diffusion of the mobile lo domains by video microscopy and particle tracking showed that the domains' mobility is slowed down by orders of magnitude compared with the unrestricted case. We attribute the reduced mobility to the geometric confinement of the PSM, because the drag force is increased substantially due to hydrodynamic effects generated by the presence of these boundaries. Our system can serve as an experimental test bed for diffusion of 2D objects in confined geometry. The impact of hydrodynamics on the mobility of enclosed lipid domains can have great implications for the formation and lateral transport of signaling platforms.

SNARE-Mediated Fusion of Single Chromaffin Granules with Pore-Spanning Membranes.

Pore-spanning membranes (PSMs) composed of supported membrane parts as well as freestanding membrane parts are shown to be very versatile to investigate SNARE-mediated fusion on the single-particle level. They provide a planar geometry readily accessible by confocal fluorescence microscopy, which enabled us for the first time, to our knowledge, to investigate the fusion of individual natural secretory granules (i.e., chromaffin granules (CGs)) on the single-particle level by two-color fluorescence microscopy in a time-resolved manner. The t-SNARE acceptor complex ΔN49 was reconstituted into PSMs containing 2 mol % 1,2-dipalmitoyl-sn-glycero-3-phosphatidylinositol-4,5-bisphosphate and Atto488-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, and CGs were fluorescently labeled with 2-((1E,3E)-5-((Z)-3,3-dimethyl-1-octadecylindolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-1-octadecyl-3H-indol-1-ium perchlorate. We compared the dynamics of docked and hemifused CGs as well as their fusion efficacy and kinetics with the results obtained for synthetic synaptobrevin 2-doped vesicles fusing with PSMs of the same composition. Whereas the synthetic vesicles were fully immobile on supported PSMs, docked as well as hemifused CGs were mobile on both PSM parts, which suggests that this system resembles more closely the natural situation. The fusion process of CGs proceeded through three-dimensional post-lipid-mixing structures, which were readily resolved on the gold-covered pore rims of the PSMs and which are discussed in the context of intermediate states observed in live cells.

Reconstituting the formation of hierarchically porous silica patterns using diatom biomolecules.

The genetically-controlled formation of complex-shaped inorganic materials by living organisms is an intriguing phenomenon. It illustrates our incomplete understanding of biological morphogenesis and demonstrates the feasibility of ecologically benign routes for materials technology. Amorphous SiO2 (silica) is taxonomically the most widespread biomineral, with diatoms, a large group of single-celled microalgae, being the most prolific producers. Silica is the main component of diatom cell walls, which exhibit species-specific patterns of pores that are hierarchically arranged and endow the material with advantageous properties. Despite recent advances in characterizing diatom biomolecules involved in biosilica morphogenesis, the mechanism of this process has remained controversial. Here we describe the in vitro synthesis of diatom-like, porous silica patterns using organic components that were isolated from biosilica of the diatom Cyclotella cryptica. The synthesis relies on the synergism of soluble biomolecules (long-chain polyamines and proteins) with an insoluble nanopatterned organic matrix. Biochemical dissection of the process revealed that the long-chain polyamines rather than the proteins are essential for efficient in vitro synthesis of the hierarchically porous silica patterns. Our results support the organic matrix hypothesis for morphogenesis of diatom biosilica and introduce organic matrices from diatoms as a new tool for the synthesis of meso- to microporous inorganic materials.

Continuous Pore-Spanning Lipid Bilayers on Silicon Oxide-Coated Porous Substrates.

A number of techniques has been developed and analyzed in recent years to generate pore-spanning membranes (PSMs). While quite a number of methods rely on nanoporous substrates, only a few use micrometer-sized pores to be able to individually resolve suspending membranes by means of fluorescence microscopy. To be able to produce PSMs on pores that are micrometer in size, an orthogonal functionalization strategy resulting in a hydrophilic surface is highly desirable. Here, we report on a method to prepare PSMs based on the evaporation of a thin layer of silicon monoxide on top of the porous substrate. PM-IRRAS experiments demonstrate that the final surface is composed of SiOx with 1 < x < 2. The hydrophilic surface turned out to be well suited to spread giant unilamellar vesicles forming PSMs. As the method does not rely on a gold coating as frequently used for orthogonal functionalization, fluorescence micrographs provide information not only from the freestanding membrane areas but also from the supported ones. The observation of the entire PSM area enabled us to observe phase-separation in these membranes on the freestanding and supported parts as well as protein binding and possible lipid reorganization of the membranes induced by binding of the protein Shiga toxin.

Voltage Dependence of Conformational Dynamics and Subconducting States of VDAC-1.

The voltage-dependent anion channel 1 (VDAC-1) is an important protein of the outer mitochondrial membrane that transports energy metabolites and is involved in apoptosis. The available structures of VDAC proteins show a wide ß-stranded barrel pore, with its N-terminal α-helix (N-α) bound to its interior. Electrophysiology experiments revealed that voltage, its polarity, and membrane composition modulate VDAC currents. Experiments with VDAC-1 mutants identified amino acids that regulate the gating process. However, the mechanisms for how these factors regulate VDAC-1, and which changes they trigger in the channel, are still unknown. In this study, molecular dynamics simulations and single-channel experiments of VDAC-1 show agreement for the current-voltage relationships of an "open" channel and they also show several subconducting transient states that are more cation selective in the simulations. We observed voltage-dependent asymmetric distortions of the VDAC-1 barrel and the displacement of particular charged amino acids. We constructed conformational models of the protein voltage response and the pore changes that consistently explain the protein conformations observed at opposite voltage polarities, either in phosphatidylethanolamine or phosphatidylcholine membranes. The submicrosecond VDAC-1 voltage response shows intrinsic structural changes that explain the role of key gating amino acids and support some of the current gating hypotheses. These voltage-dependent protein changes include asymmetric barrel distortion, its interaction with the membrane, and significant displacement of N-α amino acids.

Mode of Ezrin-Membrane Interaction as a Function of PIP2 Binding and Pseudophosphorylation.

Ezrin, a protein of the ezrin, radixin, moesin (ERM) family, provides a regulated linkage between the plasma membrane and the cytoskeleton. The hallmark of this linkage is the activation of ezrin by phosphatidylinositol-4,5-bisphosphate (PIP2) binding and a threonine phosphorylation at position 567. To analyze the influence of these activating factors on the organization of ezrin on lipid membranes and the proposed concomitant oligomer-monomer transition, we made use of supported lipid bilayers in conjunction with atomic force microscopy and fluorescence microscopy. Bilayers doped with either PIP2 as the natural receptor lipid of ezrin or a Ni-nitrilotriacetic acid-equipped lipid to bind the proteins via their His6-tags to the lipid membrane were used to bind two different ezrin variants: ezrin wild-type and ezrin T567D mimicking the phosphorylated state. Using a combination of reflectometric interference spectroscopy, atomic force microscopy, and Förster resonance energy transfer experiments, we show that only the ezrin T567D mutant, upon binding to PIP2-containing bilayers, undergoes a remarkable conformational change, which we attribute to an opening of the conformation resulting in monomeric protein on the lipid bilayer.

Phosphatidylinositol 4,5-bisphosphate alters the number of attachment sites between ezrin and actin filaments: a colloidal probe study.

Direct linkage between the plasma membrane and the actin cytoskeleton is controlled by the protein ezrin, a member of the ezrin-radixin-moesin protein family. To function as a membrane-cytoskeleton linker, ezrin needs to be activated in a process that involves binding of ezrin to phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphorylation of a conserved threonine residue. Here, we used colloidal probe microscopy to quantitatively analyze the interaction between ezrin and F-actin as a function of these activating factors. We show that the measured individual unbinding forces between ezrin and F-actin are independent of the activating parameters, in the range of approximately 50 piconewtons. However, the cumulative adhesion energy greatly increases in the presence of PIP2 demonstrating that a larger number of bonds between ezrin and F-actin has formed. In contrast, the phosphorylation state, represented by phosphor-mimetic mutants of ezrin, only plays a minor role in the activation process. These results are in line with in vivo experiments demonstrating that an increase in PIP2 concentration recruits more ezrin to the apical plasma membrane of polarized cells and significantly increases the membrane tension serving as a measure of the adhesion sites between the plasma membrane and the F-actin network.

Combining reflectometry and fluorescence microscopy: an assay for the investigation of leakage processes across lipid membranes.

The passage of solutes across a lipid membrane plays a central role in many cellular processes. However, the investigation of transport processes remains a serious challenge in pharmaceutical research, particularly the transport of uncharged cargo. While translocation reactions of ions across cell membranes is commonly measured with the patch-clamp, an equally powerful screening method for the transport of uncharged compounds is still lacking. A combined setup for reflectometric interference spectroscopy (RIfS) and fluorescence microscopy measurements is presented that allows one to investigate the passive exchange of uncharged compounds across a free-standing membrane. Pore-spanning lipid membranes were prepared by spreading giant 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) vesicles on porous anodic aluminum oxide (AAO) membranes, creating sealed attoliter-sized compartments. The time-resolved leakage of different dye molecules (pyranine and crystal violet) as well as avidin through melittin induced membrane pores and defects was investigated.

Modulating the lateral tension of solvent-free pore-spanning membranes.

The plasma membrane of animal cells is attached to the cytoskeleton, which significantly contributes to the lateral tension of the membrane. Lateral membrane tension has been shown to be an important physical regulator of cellular processes such as cell motility and morphology as well as exo- and endocytosis. Here, we report on lipid bilayers spanning highly ordered pore arrays, where we can control the lateral membrane tension by chemically varying the surface functionalization of the porous substrate. Surface functionalization was achieved by a gold coating on top of the pore rims of the hexagonal array of pores in silicon nitride substrates with pore radii of 600 nm followed by subsequent incubation with various n-propanolic mixtures of 6-mercapto-1-hexanol (6MH) and O-cholesteryl N-(8'-mercapto-3',6'-dioxaoctyl)carbamate (CPEO3). Pore-spanning membranes composed of 1,2-diphytanoyl-sn-glycero-3-phosphocholine were prepared by spreading giant unilamellar vesicles on these functionalized porous silicon nitride substrates. Different mixtures of 6MH and CPEO3 provided self-assembled monolayers (SAMs) with different compositions as analyzed by contact angle and PM-IRRAS measurements. Site specific force-indentation experiments on the pore-spanning membranes attached to the different SAMs revealed a clear dependence of the amount of CPEO3 in the monolayer on the lateral membrane tension. While bilayers on pure 6MH monolayers show an average lateral membrane tension of 1.4 mN m(-1), a mixed monolayer of CPEO3 and 6MH obtained from a solution with 9.1 mol % CPEO3 exhibits a lateral tension of 5.0 mN m(-1). From contact angle and PM-IRRAS results, the mole fraction of CPEO3 in solution can be roughly translated into a CPEO3 surface concentration of 40 mol %. Our results clearly demonstrate that the free energy difference between the supported and freestanding part of the membrane depends on the chemical composition of the SAM, which controls the lateral membrane tension.

Permeabilization assay for antimicrobial peptides based on pore-spanning lipid membranes on nanoporous alumina.

Screening tools to study antimicrobial peptides (AMPs) with the aim to optimize therapeutic delivery vectors require automated and parallelized sampling based on chip technology. Here, we present the development of a chip-based assay that allows for the investigation of the action of AMPs on planar lipid membranes in a time-resolved manner by fluorescence readout. Anodic aluminum oxide (AAO) composed of cylindrical pores with a diameter of 70 nm and a thickness of up to 10 µm was used as a support to generate pore-spanning lipid bilayers from giant unilamellar vesicle spreading, which resulted in large continuous membrane patches sealing the pores. Because AAO is optically transparent, fluid single lipid bilayers and the underlying pore cavities can be readily observed by three-dimensional confocal laser scanning microscopy (CLSM). To assay the membrane permeabilizing activity of the AMPs, the translocation of the water-soluble dyes into the AAO cavities and the fluorescence of the sulforhodamine 101 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanol-l-amine triethylammonium salt (Texas Red DHPE)-labeled lipid membrane were observed by CLSM in a time-resolved manner as a function of the AMP concentration. The effect of two different AMPs, magainin-2 and melittin, was investigated, showing that the concentrations required for membrane permeabilization and the kinetics of the dye entrance differ significantly. Our results are discussed in light of the proposed permeabilization models of the two AMPs. The presented data demonstrate the potential of this setup for the development of an on-chip screening platform for AMPs.

Driving a planar model system into the 3(rd) dimension: generation and control of curved pore-spanning membrane arrays.

The generation of a regular array of micrometre-sized pore-spanning membranes that protrude from the underlying surface as a function of osmotic pressure is reported. Giant unilamellar vesicles are spread onto non-functionalized Si/SiO(2) substrates containing a highly ordered array of cavities with pore diameters of 850 nm, an interpore distance of 4 µm and a pore depth of 10 µm. The shape of the resulting pore-spanning membranes is controlled by applying an osmotic pressure difference between the bulk solution and the femtoliter-sized cavity underneath each membrane. By applying Young-Laplace's law assuming moderate lateral membrane tensions, the response of the membranes to the osmotic pressure difference can be theoretically well described. Protruded pore-spanning membranes containing the receptor lipid PIP(2) specifically bind the ENTH domain of epsin resulting in an enlargement of the protrusions and disappearance as a result of ENTH-domain induced defects in the membranes. These results are discussed in the context of an ENTH-domain induced reduction of lateral membrane tension and formation of defects as a result of helix insertion of the protein in the bilayer.

Bioinspired Membrane Interfaces: Controlling Actomyosin Architecture and Contractility.

The creation of biologically inspired artificial lipid bilayers on planar supports provides a unique platform to study membrane-confined processes in a well-controlled setting. At the plasma membrane of mammalian cells, the linkage of the filamentous (F)-actin network is of pivotal importance leading to cell-specific and dynamic F-actin architectures, which are essential for the cell's shape, mechanical resilience, and biological function. These networks are established through the coordinated action of diverse actin-binding proteins and the presence of the plasma membrane. Here, we established phosphatidylinositol-4,5-bisphosphate (PtdIns[4,5]P2)-doped supported planar lipid bilayers to which contractile actomyosin networks were bound via the membrane-actin linker ezrin. This membrane system, amenable to high-resolution fluorescence microscopy, enabled us to analyze the connectivity and contractility of the actomyosin network. We found that the network architecture and dynamics are not only a function of the PtdIns[4,5]P2 concentration but also depend on the presence of negatively charged phosphatidylserine (PS). PS drives the attached network into a regime, where low but physiologically relevant connectivity to the membrane results in strong contractility of the actomyosin network, emphasizing the importance of the lipid composition of the membrane interface.

From LUVs to GUVs─How to Cover Micrometer-Sized Pores with Membranes.

Pore-spanning membranes (PSMs) are a versatile tool to investigate membrane-confined processes in a bottom-up approach. Pore sizes in the micrometer range are most suited to visualize PSMs using fluorescence microscopy. However, the preparation of these PSMs relies on the spreading of giant unilamellar vesicles (GUVs). GUV production faces several limitations. Thus, alternative ways to generate PSMs starting from large or small unilamellar vesicles that are more reproducibly prepared are highly desirable. Here we describe a method to produce PSMs obtained from large unilamellar vesicles, making use of droplet-stabilized GUVs generated in a microfluidic device. We analyzed the lipid diffusion in the free-standing and supported parts of the PSMs using z-scan fluorescence correlation spectroscopy and fluorescence recovery after photobleaching experiments in combination with finite element simulations. Employing atomic force indentation experiments, we also investigated the mechanical properties of the PSMs. Both lipid diffusion constants and lateral membrane tension were compared to those obtained on PSMs derived from electroformed GUVs, which are known to be solvent- and detergent-free, under otherwise identical conditions. Our results demonstrate that the lipid diffusion, as well as the mechanical properties of the resulting PSMs, is almost unaffected by the GUV formation procedure but depends on the chosen substrate functionalization. With the new method in hand, we were able to reconstitute the syntaxin-1A transmembrane domain in microfluidic GUVs and PSMs, which was visualized by fluorescence microscopy.

Benefits and limitations of porous substrates as biosensors for protein adsorption.

Porous substrates have gained widespread interest for biosensor applications based on molecular recognition. Thus, there is a great demand to systematically investigate the parameters that limit the transport of molecules toward and within the porous matrix as a function of pore geometry. Finite element simulations (FES) and time-resolved optical waveguide spectroscopy (OWS) experiments were used to systematically study the transport of molecules and their binding on the inner surface of a porous material. OWS allowed us to measure the kinetics of protein adsorption within porous anodic aluminum oxide membranes composed of parallel-aligned, cylindrical pores with pore radii of 10-40 nm and pore depths of 0.8-9.6 µm. FES showed that protein adsorption on the inner surface of a porous matrix is almost exclusively governed by the flux into the pores. The pore-interior surface nearly acts as a perfect sink for the macromolecules. Neither diffusion within the pores nor adsorption on the surface are rate limiting steps, except for very low rate constants of adsorption. While adsorption on the pore walls is mainly governed by the stationary flux into the pores, desorption from the inner pore walls involves the rate constants of desorption and adsorption, essentially representing the protein-surface interaction potential. FES captured the essential features of the OWS experiments such as the initial linear slopes of the adsorption kinetics, which are inversely proportional to the pore depth and linearly proportional to protein concentration. We show that protein adsorption kinetics allows for an accurate determination of protein concentration, while desorption kinetics could be used to capture the interaction potential of the macromolecules with the pore walls.