Glass bead probes of local structural and mechanical properties of fluid, supported membranes.
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Chemistry, Physical↗
Biomedical subjects
Atul N Parikh
Publications and source records attributed to Atul N Parikh.
Nonequilibrium patterns of cholesterol-rich chemical heterogenieties within single fluid supported phospholipid bilayer membranes.
We have developed a simple method to introduce cholesterol- and sphingomyelin-rich chemical heterogeneities into controlled densities and concentrations within predetermined regions of another distinct fluid phospholipid bilayer supported on a solid substrate. A contiguous primary phase--a fluid POPC bilayer displaying a well-defined array of lipid-free voids (e.g., 20-100 microm squares)--was first prepared on a clean glass surface by microcontact printing under water using a poly(dimethylsiloxane) stamp. The aqueous-phase primary bilayer pattern was subsequently incubated with secondary-phase small unilamellar vesicles composed of independent chemical compositions. Backfilling by comparable vesicles resulted in gradual mixing between the primary- and secondary-phase lipids, effacing the pattern. When the secondary vesicles consisted of phase-separating mixtures of cholesterol, sphingomyelin, and a phospholipid (2:1:1 POPC/sphingomyelin/cholesterol or 1:1:1 DOPC/sphingomyelin/cholesterol), well-defined spatial patterns of fluorescence, chemical compositions, and fluidities emerged. We conjecture that these patterns form because of the differences in the equilibration rates of the secondary liquid-ordered and liquid-disordered phases with the primary fluid POPC phase. The pattern stability depended strongly on the ambient-phase temperature, cholesterol concentration, and miscibility contrast between the two phases. When cholesterol concentration in the secondary vesicles was below 20 mol %, secondary intercalants gradually diffused within the primary POPC bilayer phase, ultimately dissolving the pattern in several minutes and presumably forming a new quasi-equilibrated lipid mixture. These phase domain micropatterns retain some properties of biological rafts including detergent resistance and phase mixing induced by selective cholesterol extraction. These patterns enable direct comparisons of cholesterol- and sphingomyelin-rich phase domains and fluid phospholipid phases for their functional preferences and may be useful for developing simple, parallelized assays for phase and chemical composition-dependent membrane functionalities.
Cholesterol↗
A class of supported membranes: formation of fluid phospholipid bilayers on photonic band gap colloidal crystals.
We report the formation of a new class of supported membranes consisting of a fluid phospholipid bilayer coupled directly to a broadly tunable colloidal crystal with a well-defined photonic band gap. For nanoscale colloidal crystals exhibiting a band gap at the optical frequencies, substrate-induced vesicle fusion gives rise to a surface bilayer riding onto the crystal surface. The bilayer is two-dimensionally continuous, spanning multiple beads with lateral mobilities which reflect the coupling between the bilayer topography and the curvature of the supporting colloidal surface. In contrast, the spreading of vesicles on micrometer scale colloidal crystals results in the formation of bilayers wrapping individual colloidal beads. We show that simple UV photolithography of colloidal crystals produces binary patterns of crystal wettabilities, photonic stopbands, and corresponding patterns of lipid mono- and bilayer morphologies. We envisage that these approaches will be exploitable for the development of optical transduction assays and microarrays for many membrane-mediated processes, including transport and receptor-ligand interactions.
Colloids↗
Fas signaling induces raft coalescence that is blocked by cholesterol depletion in human RPE cells undergoing apoptosis.
PURPOSE: To investigate whether the signaling events occurring in Fas-mediated apoptosis alter raft membrane formation in human RPE cells. METHODS: Formation of lipid rafts in cultured human retinal pigment epithelial cells (ARPE-19) was studied by confocal microscopy, with fluorescein-labeled cholera toxin subunit B binding protein (BODIPY)-labeled ganglioside GM1 lipid after Fas-L induction of apoptosis. Apoptosis was assessed by fluorescein-labeled annexin V detection of phosphatidylserine externalization and quadrant analysis with flow cytometry. Membrane rafts were localized into membrane vesicles by passing BODIPY-labeled GM1 RPE cells through a 2-microm-pore polycarbonate membrane using an extruder device. The labeled fractions, containing vesicles enriched in GM1, were detected by flow cytometry and then analyzed for the presence of Fas protein. RESULTS: Differential punctate staining of membrane rafts was demonstrated in normal and FasL-induced apoptotic human ARPE-19 cells in culture by confocal microscopy, using cholera toxin B and GM1 labeling of extruded vesicles. The lipid raft-associated vesicles were derived by plasma membrane dissociation, via a newly developed whole-cell extrusion technique that produced 2-microm vesicles with both GM1 lipid and Fas protein abundance enriched in a subpopulation of the membrane-derived vesicles. The amount of Fas protein in the vesicles containing raft domains markedly increased in FasL-treated cells. Treatment of human ARPE 19 cells with methyl beta-cyclodextrin after FasL induction of apoptosis resulted in cellular cholesterol depletion and markedly reduced the incidence of Fas-receptor localization in GM1 rafts. CONCLUSIONS: Human ARPE-19 cells in culture contain membrane rafts with apoptotic signaling effectors uniformly distributed in the native state. The cells stimulated to undergo apoptosis appear to use membrane rafts in the death-signaling process by mobilization of rafts to localized regions of the membrane that are now enriched with apoptotic signaling effectors. Fas signaling induces apoptotic raft formation that results in polar condensation, or capping, of the rafts in the late stages of apoptosis. A novel extrusion technique is described that allows localization and enrichment of rafts into membrane vesicles, which can be assayed by flow cytometry. Cholesterol depletion, after Fas ligand activation of apoptosis, reduced raft formation in cells induced to undergo apoptosis. Therapeutic implications for the treatment of retinal disorders are discussed.
Apoptosis↗
Formation of spatially patterned colloidal photonic crystals through the control of capillary forces and template recognition.
We report the formation of microscopic patterns of substrate-supported, 3D planar colloidal crystals using physical confinement in conjunction with surfaces displaying predetermined binary patterns of hydropholicity. The formation process involves a primary self-assembly wherein nano- and microscale colloids order into a photonic fcc lattice via capillary interactions followed by a secondary template-induced crystal cleavage step. Following this method, arbitrary arrays of pattern elements, which preserve structural and orientational properties of the parent crystal, can be easily obtained.
Journal Article↗
Transition from homogeneous Langmuir-Blodgett monolayers to striped bilayers driven by a wetting instability in octadecylsiloxane monolayers.
We show that two dips of an oxidized silicon substrate through a prepolymerized n-octadecylsiloxane monolayer at an air-water interface in a rapid succession produces periodic, linear striped patterns in film morphology extending over macroscopic area of the substrate surface. Langmuir monolayers of n-octadecyltrimethoxysilane were prepared at the surface of an acidic subphase (pH 2) maintained at room temperature (22 +/- 2 degrees C) under relative humidities of 50-70%. The substrate was first withdrawn at a high dipping rate from the quiescent aqueous subphase (upstroke) maintained at several surface pressures corresponding to a condensed monolayer state and lowered soon after at the same rate into the monolayer covered subphase (downstroke). The film structure and morphology were characterized using a combination of optical microscopy, imaging ellipsometry, and Fourier transform infrared spectroscopy. An extended striped pattern, perpendicular to the pushing direction of the second stroke, resulted for all surface pressures when the dipping rate exceeded a threshold value of 40 mm min(-1). Below this threshold value, uniform deposition characterizing formation of a bimolecular film was obtained. Under conditions that favored striped deposition during the downstroke through the monolayer-covered interface, we observed a periodic auto-oscillatory behavior of the meniscus. The stripes appear to be formed by a highly correlated reorganization and/or exchange of the first monolayer, mediated by the Langmuir monolayer at the air-water interface. This mechanism appears distinctly different from nanometer scale stripes observed recently in single transfers of phospholipid monolayers maintained near a phase boundary. The stripes further exhibit wettability patterns useful for spatially selective functionalization, as demonstrated by directed adsorptions of an organic dye (fluorescein) and an oil (hexadecane).
Journal Article↗
Surfactant removal and silica condensation during the photochemical calcination of thin film silica mesophases.
The evolution of photochemical surfactant removal and silica condensation from organically templated thin film silica nanocomposites with mesoscopic ordering has been probed using a combined application of Fourier transform infrared (FT-IR) spectroscopy and single wavelength ellipsometry. Thin films of silica nanocomposites were prepared by a previously reported evaporation-induced self-assembly process. Specifically, oxidized silicon and gold substrates were withdrawn at 25 mm/min from a subcritical micelle concentration solution containing an ethylene oxide surfactant as a structure-directing agent and tetraethyl orthosilicate as a silica precursor. Real-time grazing incidence difference FT-IR spectra of the nanocomposite films on gold taken during exposure to short-wavelength ultraviolet light (184-257 nm) show that surfactant removal and silica condensation occur gradually and concomitantly. Surfactant removal and silica reconstructions were found to be nearly complete after 90 min of exposure. Further, a transient feature was observed in the FT-IR spectra around 1713 cm(-1) during the UV exposure process and was assigned to a carbonyl (C=O) stretching mode absorption, reflecting the transient formation of a partially oxidized surfactant intermediate. From these data we propose a stepwise model for surfactant removal from the nanocomposite films. Ellipsometrically determined index of refraction values collected as a function of UV exposure are also shown to support such a stepwise mechanism of surfactant removal from the ordered nanocomposite silica thin film mesophases studied here.
Carbon↗
Phospholipid morphologies on photochemically patterned silane monolayers.
We have studied the spreading of phospholipid vesicles on photochemically patterned n-octadecylsiloxane monolayers using epifluorescence and imaging ellipsometry measurements. Self-assembled monolayers of n-octadecylsiloxanes were patterned using short-wavelength ultraviolet radiation and a photomask to produce periodic arrays of patterned hydrophilic domains separated from hydrophobic surroundings. Exposing these patterned surfaces to a solution of small unilamellar vesicles of phospholipids and their mixtures resulted in a complex lipid layer morphology epitaxially reflecting the underlying pattern of hydrophilicity. The hydrophilic square regions of the photopatterned OTS monolayer reflected lipid bilayer formation, and the hydrophobic OTS residues supported lipid monolayers. We further observed the existence of a boundary region composed of a nonfluid lipid phase and a lipid-free moat at the interface between the lipid monolayer and bilayer morphologies spontaneously corralling the fluid bilayers. The outer-edge of the boundary region was found to be accessible for subsequent adsorption by proteins (e.g., streptavidin and BSA), but the inner-edge closer to the bilayer remained resistant to adsorption by protein or vesicles. Mechanistic implications of our results in terms of the effects of substrate topochemical character are discussed. Furthermore, our results provide a basis for the construction of complex biomembrane models, which exhibit fluidity barriers and differentiate membrane properties based on correspondence between lipid leaflets. We also envisage the use of this construct where two-dimensionally fluid, low-defect lipid layers serve as sacrificial resists for the deposition of protein and other material patterns.
Adsorption↗
Neutron reflectivity study of lipid membranes assembled on ordered nanocomposite and nanoporous silica thin films.
Single bilayer membranes of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were formed on ordered nanocomposite and nanoporous silica thin films by fusion of small unilamellar vesicles. The structure of these membranes was investigated using neutron reflectivity. The underlying thin films were formed by evaporation induced self-assembly to obtain periodic arrangements of silica and surfactant molecules in the nanocomposite thin films, followed by photocalcination to oxidatively remove the organics and render the films nanoporous. We show that this platform affords homogeneous and continuous bilayer membranes that have promising applications as model membranes and sensors.
Lipid Bilayers↗
Direct photochemical patterning and refunctionalization of supported phospholipid bilayers.
A wet photolithographic route for micropatterning fluid phospholipid bilayers is demonstrated in which spatially directed illumination by short-wavelength ultraviolet radiation results in highly localized photochemical degradation of the exposed lipids. Using this method, we can directly engineer patterns of hydrophilic voids within a fluid membrane as well as isolated membrane corrals over large substrate areas. We show that the lipid-free regions can be refilled by the same or other lipids and lipid mixtures which establish contiguity with the existing membrane, thereby providing a synthetic means for manipulating membrane compositions, engineering metastable membrane microdomains, probing 2D lipid-lipid mixing, and designing membrane-embedded arrays of soluble proteins. Following this route, new constructs can be envisaged for high-throughput membrane proteomic, biosensor array, and spatially directed, aqueous-phase material synthesis.
Dimyristoylphosphatidylcholine↗