Controlling Physical and Biochemical Parameters of Actin Nucleation Using a Patterned Model Lipid Membrane.

Self-assembly of nanoscale actin cytoskeletal proteins into filamentous networks requires organizing actin nucleation areas on the plasma membrane through recruiting actin nucleators and nucleation-promoting factors (NPFs) to the areas. To investigate impacts of the nucleation geometry on actin network assembly, we localized NPF or nucleator on defined micropatterns of laterally mobile lipid bilayers confined in a framework of a polymerized lipid bilayer. We demonstrated that actin network assembly in purified protein mixtures was confined on NPF- or nucleator-localized fluid bilayers. By controlling the shape and size of nucleation areas as well as the density and types of localized NPFs and nucleators, we showed that these parameters regulate actin network architectures. Actin network assembly in Xenopus egg extracts was also spatially controlled by patterning bilayers containing phosphatidylinositol 4,5-bisphoshate (PI(4,5)P2), an essential lipid signaling mediator. Therefore, the system provides a promising platform to investigate the physical and biochemical principles for actin network assembly.

Nanofluidic Model Membrane for the Single-Molecule Observation of Membrane Proteins.

Membrane proteins play essential roles in the cell, and they constitute one of the most important targets of drugs. Studying membrane proteins in a controlled model membrane environment can provide unambiguous, quantitative information on their molecular properties and functions. However, reconstituting membrane proteins in a model system poses formidable technological challenges. Here, we developed a novel model membrane platform for highly sensitive observation of membrane proteins by combining a micropatterned lipid membrane and a nanofluidic channel. A micropatterned model membrane was generated by lithographically integrating a polymerized lipid bilayer and a natural (fluid) lipid bilayer. A nanofluidic channel having a defined thickness was formed between the fluid bilayer and a polydimethylsiloxane (PDMS) slab by attaching the polymeric bilayer and PDMS slab using an adhesion layer composed of silica nanoparticles that are coated with a biocompatible polymer brush. As we reconstituted rhodopsin (Rh), a G-protein-coupled receptor (GPCR), from a detergent-solubilized state into the fluid bilayer, only successfully reconstituted Rh molecules diffused laterally in the lipid bilayer and migrated into the nanogap junction, where they could be observed with a vastly improved signal-to-background ratio. The nanogap junction effectively separates the sites of reconstitution and observation and provides a novel platform for studying the molecular properties and functions of membrane proteins at the single-molecular level.

Model Lipid Membranes Assembled from Natural Plant Thylakoids into 2D Microarray Patterns as a Platform to Assess the Organization and Photophysics of Light-Harvesting Proteins.

Natural photosynthetic "thylakoid" membranes found in green plants contain a large network of light-harvesting (LH) protein complexes. Rearrangement of this photosynthetic machinery, laterally within stacked membranes called "grana", alters protein-protein interactions leading to changes in the energy balance within the system. Preparation of an experimentally accessible model system that allows the detailed investigation of these complex interactions can be achieved by interfacing thylakoid membranes and synthetic lipids into a template comprised of polymerized lipids in a 2D microarray pattern on glass surfaces. This paper uses this system to interrogate the behavior of LH proteins at the micro- and nanoscale and assesses the efficacy of this model. A combination of fluorescence lifetime imaging and atomic force microscopy reveals the differences in photophysical state and lateral organization between native thylakoid and hybrid membranes, the mechanism of LH protein incorporation into the developing hybrid membranes, and the nanoscale structure of the system. The resulting model system within each corral is a high-quality supported lipid bilayer that incorporates laterally mobile LH proteins. Photosynthetic activity is assessed in the hybrid membranes versus proteoliposomes, revealing that commonly used photochemical assays to test the electron transfer activity of photosystem II may actually produce false-positive results.

Photosynthetic Model Membranes of Natural Plant Thylakoid Embedded in a Patterned Polymeric Lipid Bilayer.

Thylakoid membranes in the chloroplast of plants, algae, and cyanobacteria are the powerhouse of photosynthesis, capturing solar energy and converting it into chemical energy. Although their structures and functions have been extensively studied, the intrinsically heterogeneous and dynamic nature of the membrane structures is still not fully understood. Investigating native thylakoid membranes in vivo is difficult due to their small size and limited external access to the chloroplast interior, while the bottom-up approaches based on model systems have been hampered by the sheer complexity of the native membrane. Here, we try to fill the gap by reconstituting the whole thylakoid membrane into a patterned substrate-supported planer bilayer. A mixture of thylakoid membrane purified from spinach leaves and synthetic phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) vesicles spontaneously formed a laterally continuous and fluid two-dimensional (2D) membrane in the scaffold of the patterned polymeric bilayer. Chlorophyll fluorescence arising from photosystem II (PSII) recovered after photobleaching, suggesting that the membrane components are laterally mobile. The reversible changes of chlorophyll fluorescence in the presence of the electron acceptors and/or inhibitors indicated that the electron transfer activity of PSII was retained. Furthermore, we confirmed the electron transfer activity of photosystem I (PSI) by observing the generation of nicotinamide adenine dinucleotide phosphate (NADPH) in the presence of water-soluble ferredoxin and ferredoxin-NADP+ reductase. The lateral mobility of membrane-bound molecules and the functional reconstitution of major photosystems provide evidence that our hybrid thylakoid membranes could be an excellent experimental platform to study the 2D molecular organization and machinery of photosynthesis.

Alteration of Membrane Physicochemical Properties by Two Factors for Membrane Protein Integration.

After a nascent chain of a membrane protein emerges from the ribosomal tunnel, the protein is integrated into the cell membrane. This process is controlled by a series of proteinaceous molecular devices, such as signal recognition particles and Sec translocons. In addition to these proteins, we discovered two endogenous components regulating membrane protein integration in the inner membrane of Escherichia coli. The integration is blocked by diacylglycerol (DAG), whereas the blocking is relieved by a glycolipid named membrane protein integrase (MPIase). Here, we investigated the influence of these integration-blocking and integration-promoting factors on the physicochemical properties of membrane lipids via solid-state NMR and fluorescence measurements. These factors did not have destructive effects on membrane morphology because the membrane maintained its lamellar structure and did not fuse in the presence of DAG and/or MPIase at their effective concentrations. We next focused on membrane flexibility. DAG did not affect the mobility of the membrane surface, whereas the sugar chain in MPIase was highly mobile and enhanced the flexibility of membrane lipid headgroups. Comparison with a synthetic MPIase analog revealed the effects of the long sugar chain on membrane properties. The acyl chain order inside the membrane was increased by DAG, whereas the increase was cancelled by the addition of MPIase. MPIase also loosened the membrane lipid packing. Focusing on the transbilayer movement, MPIase reduced the rapid flip-flop motion of DAG. On the other hand, MPIase could not compensate for the diminished lateral diffusion by DAG. These results suggest that by manipulating the membrane lipids dynamics, DAG inhibits the protein from contacting the inner membrane, whereas the flexible long sugar chain of MPIase increases the opportunity for interaction between the membrane and the protein, leading to membrane integration of the newly formed protein.

Self-Spreading of Phospholipid Bilayer in a Patterned Framework of Polymeric Bilayer.

Phospholipid bilayers spontaneously spread on a hydrophilic substrate such as glass in aqueous solution due to the energetic gain of surface wetting. This process (self-spreading) was utilized to form a patterned model biological membrane containing reconstituted membrane proteins. A mechanically stable framework of a polymerized lipid bilayer was first generated by the lithographic polymerization of a diacetylene phospholipid. Then, natural lipid membranes (fluid bilayers) were introduced into the channels between polymeric bilayers by the self-spreading from a phospholipid reservoir. The spreading velocity could be fitted into a slope of -0.5 in a double logarithmic plot versus time due to the balance between the spreading force and resistive drag. The preformed polymeric bilayer accelerated the spreading by the energetic gain of covering hydrophobic edges with a lipid bilayer. At the same time, the domains of the polymeric bilayer obstructed spreading, and the spreading velocity linearly decreased with their fractional coverage. Above the critical coverage of ca. 50%, self-spreading was completely blocked (percolation threshold) and the fluid bilayer was confined in the polymer-free regions. Nonspecific adsorption of lipids onto the surface of polymeric bilayers was negligible, which enabled a heightened signal-to-background ratio in the reconstitution and observation of membrane proteins. Self-spread bilayers had a higher density of lipids than those formed by the spontaneous rupture of vesicles (vesicle fusion), presumably due to the continual supply of lipid molecules from the reservoir. These features give the self-spreading important advantages for preparing patterned model membranes with reconstituted membrane proteins.

Nanofluidic Biosensor Created by Bonding Patterned Model Cell Membrane and Silicone Elastomer with Silica Nanoparticles.

Selective and sensitive detection of specific molecules in a solution containing diverse coexisting molecules is important in many biomedical and environmental applications, including diagnostics and pollutant detection. Here, a nanofluidic biosensor is developed to detect specific target molecules (e.g., toxin proteins) in the presence of nontarget molecules by bonding a patterned model cell membrane and a silicone elastomer (polydimethylsiloxane: PDMS) sheet using surface-modified silica nanoparticles as the adhesive layer. Owing to the uniform size of nanoparticles, a nanometric gap junction is formed between the fluid bilayer and PDMS (nanogap-junction). The thickness of the nanogap-junction is controlled by the size of the silica nanoparticles. Target molecules that specifically bind to the receptor molecules in the fluid bilayer are selectively transported into the nanogap-junction via lateral diffusion through the lipid membrane. A thinner gap formed with smaller nanoparticles can enhance the sensitivity (signal-to-background ratio) more effectively, owing to the suppression of nonspecific penetration of coexisting molecules. Silica nanoparticles also provide excellent mechanical robustness, realizing long-term stability of the gap structure. Nanogap-junction using silica nanoparticles provides a versatile platform for highly selective and sensitive sensing by realizing detection of specific target molecules in a solution containing more concentrated nontarget molecules.

Hybrid Model Membrane Combining Micropatterned Lipid Bilayer and Hydrophilic Polymer Brush.

Substrate-supported planar lipid bilayers (SPBs) are being utilized as a versatile model system of the biological membrane. However, the proximity between the solid support and membrane limits utility of SPBs for the functional analyses of membrane proteins. Here, we present a model membrane that can enlarge the distance between the substrate surface and the membrane by combining a stable scaffold of polymerized lipid bilayer with a hydrophilic polymer brush. A micropatterned SPB was generated by the lithographic polymerization of diacetylene lipids and subsequent incorporation of natural (fluid) lipid bilayers. Hydrophilic polymer brush of poly-2-methacryloyloxyethyl phosphorylcholine (poly(MPC)) was formed on the surface of polymeric bilayer by the in situ atom transfer radical polymerization (ATRP) in aqueous solution, in the presence of embedded fluid lipid bilayers. A model membrane protein (Haloquadratum walsbyi bacteriorhodopsin: HwBR) could be reconstituted into the polymer brush-supported bilayers with significantly reduced immobile molecules. Furthermore, the polymer brush terminals could be functionalized by successively polymerizing MPC and 2-aminoethyl methacrylate (AMA). The reactive amine moiety of poly(AMA) enables to conjugate a wide range of biological molecules and surfaces to the membrane. The combination of micropatterned bilayer and polymer brush mimics the two- and three-dimensional structures of the biological membrane, providing a platform to assay membrane proteins in a truly biomimetic environment.

The Role of the Prod1 Membrane Anchor in Newt Limb Regeneration.

Prod1 is a protein that regulates limb regeneration in salamanders by determining the direction of limb growth. Prod1 is attached to the membrane by a glycosylphosphatidylinositol (GPI) anchor, but the role of membrane anchoring in the limb regeneration process is poorly understood. In this study, we investigated the functional role of the anchoring of Prod1 to the membrane by using its synthetic mimics in combination with solid-state NMR spectroscopy and fluorescent microscopy techniques. Anchoring did not affect the three-dimensional structure of Prod1 but did induce aggregation by aligning the molecules and drastically reducing the molecular motion on the two-dimensional membrane surface. Interestingly, aggregated Prod1 interacted with Prod1 molecules tethered on the surface of opposing membranes, inducing membrane adhesion. Our results strongly suggest that anchoring of the salamander-specific protein Prod1 assists cell adhesion in the limb regeneration process.

Nanometric Gap Structure with a Fluid Lipid Bilayer for the Selective Transport and Detection of Biological Molecules.

The biological membrane is a natural biosensing platform that can detect specific molecules with extremely high sensitivity. We developed a biosensing methodology by combining a model biological membrane and a nanometer-sized gap structure on a glass substrate. The model membrane comprised lithographically patterned polymeric and fluid lipid bilayers. The polymeric bilayer was bonded to a poly(dimethylsiloxane) (PDMS) sheet by using an adhesion layer with a defined thickness (lipid vesicles). Extruded lipid vesicles having a biotin moiety on the surface were used as the adhesion layer in conjunction with the biotin-streptavidin linkage. A gap structure was formed between the fluid bilayer and PDMS (nanogap junction). The thickness of the gap structure was several tens of nanometers, as determined by the thickness of the adhesion layer. The nanogap junction acted as a sensitive biosensing platform. From a mixture of proteins (cholera toxin and albumin), the target protein (cholera toxin) was selectively transported into the gap by the specific binding to a glycolipid (GM1) in the fluid bilayer and lateral diffusion. The target protein molecules were then detected with an elevated signal-to-noise ratio due to the reduced background noise in the nanometric gap. The combination of selective transport and reduced background noise drastically enhanced the sensitivity toward the target protein. The nanogap junction should have broad biomedical applications by realizing highly selective and sensitive biosensing in samples having diverse coexisting molecules.

Evaluating the Raftophilicity of Rhodopsin Photoreceptor in a Patterned Model Membrane.

Lipid rafts in the cell membrane are believed to affect various membrane functions, including the signaling by G-protein coupled receptors (GPCRs). However, the regulatory roles of lipid rafts on GPCRs' functions are still poorly understood, partially owing to the lack of the methods to quantitatively evaluate the affinity of membrane proteins to lipid raft (raftophilicity). Here, we describe a methodology to gauge the raftophilicity of a representative GPCR in vertebrate photoreceptor, i.e., rhodopsin (Rh), and its cognate G protein transducin (Gt) by using a patterned model membrane. We generated a substrate-supported planar lipid bilayer that has patterned regions of liquid-ordered (Lo) and liquid-disordered (Ld) membrane domains. We reconstituted Rh and Gt into the patterned membrane and observed their lateral distribution and diffusion. Mobile and functional Rh molecules could be reconstituted through the rapid dilution of solubilized Rh, by optimizing the reconstitution conditions including the chamber design, protein/detergent concentrations, and solution mixing. We determined the partition and diffusion coefficients of Rh and Gt in the Lo-rich and Ld-rich regions. Both Rh and Gt were predominantly localized in the Ld phase, suggesting their low affinity to lipid rafts. Patterned model membrane offers a robust and scalable platform for systematically and quantitatively studying the functional roles of lipid rafts in biological membranes including retinal disk membranes.

Uptake of raft components into amyloid ß-peptide aggregates and membrane damage.

Amyloid aggregation and deposition of amyloid ß-peptide (Aß) are pathologic characteristics of Alzheimer's disease (AD). Recent reports have shown that the association of Aß with membranes containing ganglioside GM1 (GM1) plays a pivotal role in amyloid deposition and the pathogenesis of AD. However, the molecular interactions responsible for membrane damage associated with Aß deposition are not fully understood. In this study, we microscopically observed amyloid aggregation of Aß in the presence of lipid vesicles and on a substrate-supported planar membrane containing raft components and GM1. The experimental system enabled us to observe lipid-associated aggregation of Aß, uptake of the raft components into Aß aggregates, and relevant membrane damage. The results indicate that uptake of raft components from the membrane into Aß deposits induces macroscopic heterogeneity of the membrane structure.

Oligomerization and aggregation of NAP-22 with several metal ions.

Metal ions participate in various biochemical processes such as electron transport chain, gene transcription, and enzymatic reactions. Furthermore, the aggregation promoting effect of several metal ions on neuronal proteins such as prion, tau, Aß peptide, and α-synuclein, has been reported. NAP-22 (also called BASP1 or CAP-23) is a neuron-enriched calmodulin-binding protein and one of the major proteins in the detergent-resistant membrane microdomain fraction of the neuronal cell membrane. Previously, we showed oligomer formation of NAP-22 in the presence of several phospholipids and fatty acids. In this study, we found the aggregation of NAP-22 by FeCl2, FeCl3, and AlCl3 using native-PAGE. Oligomer or aggregate formation of NAP-22 by ZnCl2 or CuSO4 was shown with SDS-PAGE after cross-linking with glutaraldehyde. Morphological analysis with electron microscopy revealed the formation of large aggregates composed of small annular oligomers in the presence of FeCl3, AlCl3, or CuSO4. In case of FeCl2 or ZnCl2, instead of large aggregates, scattered annular and globular oligomers were observed. Interestingly, metal ion induced aggregation of NAP-22 was inhibited by several coenzymes such as NADP+, NADPH, or thiamine pyrophosphate. Since NAP-22 is highly expressed in the presynaptic region of the synapse, this result suggests the participation of metal ions not only on the protein and membrane dynamics at the presynaptic region, but also on the metabolic regulation though the interaction with coenzymes.

Microarrays of phospholipid bilayers generated by inkjet printing.

We report an efficient and reproducible method to generate a microarray of model biological membranes on a solid substrate by applying the inkjet printing technology. Although inkjet printing is currently widely used for industrial fabrication processes, including biological materials, printing lipid membranes remains technically challenging due to the hydrophobic nature of droplets and instability of the lipid bilayer structure against dehydration. In the present study, we printed lipids onto a glass substrate covered with a micropatterned membrane of a polymeric phospholipid bilayer. Polymeric bilayers were formed by the lithographic photopolymerization of a diacetylene-containing phospholipid, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DiynePC). After removal of nonpolymerized DiynePC with a detergent solution, natural lipid membranes were incorporated into the polymer-free regions (corrals) by using an electric-field-based inkjet printing device that can eject subfemtoliter volume droplets. To avoid rapid dehydration and destabilization, we preprinted an aqueous solution containing agarose and trehalose onto the corrals and subsequently printed lipid suspensions ("two-step-printing method"). After rinsing, stable lipid bilayer membranes were formed in the corrals. The bilayers were continuous and fluid as confirmed by fluorescence recovery after photobleaching. We could introduce multiple bilayer patches having different lipid compositions into the neighboring corrals. The present results demonstrate that the combination of a patterned polymeric bilayer and inkjet printing technology enables efficient, reliable, and scalable generation of the model membrane microarrays having varied compositions.

Surface functionalization of a polymeric lipid bilayer for coupling a model biological membrane with molecules, cells, and microstructures.

We describe a stable and functional model biological membrane based on a polymerized lipid bilayer with a chemically modified surface. A polymerized lipid bilayer was formed from a mixture of two diacetylene-containing phospholipids, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DiynePC) and 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphoethanolamine (DiynePE). DiynePC formed a stable bilayer structure, whereas the ethanolamine headgroup of DiynePE enabled functional molecules to be grafted onto the membrane surface. Copolymerization of DiynePC and DiynePE resulted in a robust bilayer. Functionalization of the polymeric bilayer provided a route to a robust and biomimetic surface that can be linked with biomolecules, cells, and three-dimensional (3D) microstructures. Biotin and peptides were grafted onto the polymeric bilayer for attaching streptavidin and cultured mammalian cells by molecular recognition, respectively. Nonspecific adsorption of proteins and cells on polymeric bilayers was minimum. DiynePE was also used to attach a microstructure made of an elastomer (polydimethylsiloxan: PDMS) onto the membrane, forming a confined aqueous solution between the two surfaces. The microcompartment enabled us to assay the activity of a membrane-bound enzyme (cyochrome P450). Natural (fluid) lipid bilayers were incorporated together with membrane-bound proteins by lithographically polymerizing DiynePC/DiynePE bilayers. The hybrid membrane of functionalized polymeric bilayers and fluid bilayers offers a novel platform for a wide range of biomedical applications including biosensor, bioassay, cell culture, and cell-based assay.

Effects of membrane interaction and aggregation of amyloid ß-peptide on lipid mobility and membrane domain structure.

Alzheimer's disease (AD) is the most prevalent age-dependent form of dementia, characterized by extracellular amyloid deposits comprising amyloid ß-peptide (Aß) in the cerebral cortex. Increasing evidence has indicated that ganglioside GM1 (GM1) in lipid rafts plays a pivotal role in amyloid deposition of Aß and the related cytotoxicity in AD. Despite recent efforts to characterize Aß-lipid interactions, the effect of Aß aggregation on dynamic properties and organization of lipid membranes is poorly understood. In this study, we examined the aggregation of Aß on supported lipid bilayers containing raft components (i.e., cholesterol, sphingomyelin, and GM1) and its effects on the membrane properties. We showed that the lateral fluidity of membranes was significantly affected by membrane binding and subsequent aggregation of Aß. Microscopic observations of the membrane surfaces demonstrated an enhancement in phase separation of lipids as a result of interactions between Aß and GM1 during induced aggregation of Aß. The uptake of GM1 into Aß aggregates and the attendant membrane damage were also observed under a microscope when the membrane-anchored aggregates were formed. On the basis of these observations, we propose that Aß aggregates formed in the presence of lipid membranes have a latent ability to trigger the uptake of raft components accompanied by phase separation of lipids.

Retarded Diffusion and Confinement of Membrane-Bound Molecules in a Patterned Hybrid Membrane of Phospholipid Bilayers and Monolayers.

The biological membrane is a complex two-dimensional fluid, in which various molecular interactions regulate the lateral diffusion of membrane-associated molecules. Pinning of membrane proteins or lipids by extra-membrane proteins impedes the diffusion. In addition, coupling between two monolayer leaflets within a phospholipid bilayer via interdigitation plays important roles, though this effect remains elusive. Here, we fabricate a substrate-supported model membrane with patterned bilayer/monolayer regions to explore the influences of interleaflet coupling. A patterned monolayer of polymerized diacetylene phospholipid, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DiynePC), was lithographically generated and used to form patterned lipid bilayers and monolayers. A phospholipid monolayer was formed on top of the polymerized monolayer. The bilayer/monolayer hybrid membrane was continuous and fluid, but lateral diffusion in the monolayer region was significantly retarded, suggesting the influences of interleaflet coupling. We reconstituted photoreceptor rhodopsin (Rh) and G-protein transducin (Gt) as model transmembrane and peripheral proteins. Rh diffused laterally only in the bilayer region, whereas Gt diffused in both bilayer and monolayer regions. The patterned hybrid bilayer/monolayer membrane reproduces the retarded diffusion and confinement of membrane-bound molecules in a controlled manner and provides insight into the physicochemical and functional roles of semipermeable corrals in the cell membrane.

Self-Quenching Behavior of a Fluorescent Probe Incorporated within Lipid Membranes Explored Using Electrophoresis and Fluorescence Lifetime Imaging Microscopy.

Fluorescent probes are useful in biophysics research to assess the spatial distribution, mobility, and interactions of biomolecules. However, fluorophores can undergo "self-quenching" of their fluorescence intensity at high concentrations. A greater understanding of concentration-quenching effects is important for avoiding artifacts in fluorescence images and relevant to energy transfer processes in photosynthesis. Here, we show that an electrophoresis technique can be used to control the migration of charged fluorophores associated with supported lipid bilayers (SLBs) and that quenching effects can be quantified with fluorescence lifetime imaging microscopy (FLIM). Confined SLBs containing controlled quantities of lipid-linked Texas Red (TR) fluorophores were generated within 100 × 100 µm corral regions on glass substrates. Application of an electric field in-plane with the lipid bilayer induced the migration of negatively charged TR-lipid molecules toward the positive electrode and created a lateral concentration gradient across each corral. The self-quenching of TR was directly observed in FLIM images as a correlation of high concentrations of fluorophores to reductions in their fluorescence lifetime. By varying the initial concentration of TR fluorophores incorporated into the SLBs from 0.3% to 0.8% (mol/mol), the maximum concentration of fluorophores reached during electrophoresis could be modulated from 2% up to 7% (mol/mol), leading to the reduction of fluorescence lifetime down to 30% and quenching of the fluorescence intensity down to 10% of their original levels. As part of this work, we demonstrated a method for converting fluorescence intensity profiles into molecular concentration profiles by correcting for quenching effects. The calculated concentration profiles have a good fit to an exponential growth function, suggesting that TR-lipids can diffuse freely even at high concentrations. Overall, these findings prove that electrophoresis is effective at producing microscale concentration gradients of a molecule-of-interest and that FLIM is an excellent approach to interrogate dynamic changes to molecular interactions via their photophysical state.

Binding of islet amyloid polypeptide to supported lipid bilayers and amyloid aggregation at the membranes.

Amyloid deposition of human islet amyloid polypeptide (hIAPP) in the islets of Langerhans is closely associated with the pathogenesis of type II diabetes mellitus. Despite substantial evidence linking amyloidogenic hIAPP to loss of ß-cell mass and decreased pancreatic function, the molecular mechanism of hIAPP cytotoxicity is poorly understood. We here investigated the binding of hIAPP and nonamyloidogenic rat IAPP to substrate-supported planar bilayers and examined the membrane-mediated amyloid aggregation. The membrane binding of IAPP in soluble and fibrillar states was characterized using quartz crystal microbalance with dissipation monitoring, revealing significant differences in the binding abilities among different species and conformational states of IAPP. Patterned model membranes composed of polymerized and fluid lipid bilayer domains were used to microscopically observe the amyloid aggregation of hIAPP in its membrane-bound state. The results have important implications for lipid-mediated aggregation following the penetration of hIAPP into fluid membranes. Using the fluorescence recovery after photobleaching method, we show that the processes of membrane binding and subsequent amyloid aggregation are accompanied by substantial changes in membrane fluidity and morphology. Additionally, we show that the fibrillar hIAPP has a potential ability to perturb the membrane structure in experiments of the fibril-mediated aggregation of lipid vesicles. The results obtained in this study using model membranes reveal that membrane-bound hIAPP species display a pronounced membrane perturbation ability and suggest the potential involvement of the oligomeic forms of hAPP in membrane dysfunction.

Photoregulation of cytochrome P450 activity by using caged compound.

Cytochrome P450 (P450) species play an important role in the metabolism of xenobiotics, and assaying the activities of P450 is important for evaluating the toxicity of chemicals in drugs and food. However, the lag time caused by the introduction and mixing of sample solutions can become sources of error as the throughput is heightened by increasing the sample number and decreasing the sample volume. To amend this technological obstacle, we developed a methodology to photoregulate the activity of P450 by using photoprotected (caged) compounds. We synthesized caged molecules of nicotinamide adenine dinucleotide phosphate (NADP(+)) and glucose 6-phosphate (G6P), which are involved in the generation of NADPH (cofactor of P450). The use of caged-G6P completely blocked the P450 catalysis before the UV illumination, whereas caged-NADP(+) resulted in a little background reaction. Upon UV illumination, more than 90% of the enzymatic activity could be restored. The use of caged-G6P enabled assays in isolated microchambers (width, 50 µm; height, 50 µm) by encapsulating necessary ingredients in advance and initiating the reaction by UV illumination. The initiation of enzymatic reaction could be observed in a single microchamber. Minimizing uncertainties caused by the introduction and mixing of solutions led to significantly reduced errors of obtained kinetic constants.