RESUMO
Polarizable colloids are expected to form crystalline equilibrium phases when exposed to a steady, uniform field. However, when colloids become localized this field-induced phase transition arrests and the suspension persists indefinitely as a kinetically trapped, percolated structure. We anneal such gels formed from magneto-rheological fluids by toggling the field strength at varied frequencies. This processing allows the arrested structure to relax periodically to equilibrium--colloid-rich, cylindrical columns. Two distinct growth regimes are observed: one in which particle domains ripen through diffusive relaxation of the gel, and the other where the system-spanning structure collapses and columnar domains coalesce apparently through field-driven interactions. There is a stark boundary as a function of magnetic field strength and toggle frequency distinguishing the two regimes. These results demonstrate how kinetic barriers to a colloidal phase transition are subverted through measured, periodic variation of driving forces. Such directed assembly may be harnessed to create unique materials from dispersions of colloids.
Assuntos
Coloides/química , Géis/química , Modelos Químicos , Transição de Fase , Cinética , Magnetismo , Reologia , Ausência de PesoRESUMO
Sphingomyelinase (SMase) has been shown to be involved in a variety of cell regulation processes by reorganizing the cell membrane morphology. Here we report that SMase can induce a reaction-induced and a solvent-mediated phase transformation, causing switches of three stationary membrane morphologies and multiple-time-domain ceramide generation in model raft membranes. The reaction-induced phase transformation, triggered by the addition of SMase, transforms a pre-existing morphology to a long-lasting intermediate morphology with coexisting ceramide-enriched (Cer-enriched) and sphingomyelin-enriched (SM-enriched) domains. Solvent-mediated phase transformation ultimately transforms all of the SM-enriched domains of the intermediate morphology into Cer-enriched domains. Labeled SMase experiments suggest that the intermediate morphology results from physical trapping of SM in the SM-enriched domains, which are found to be relatively inaccessible to SMase. The characterization results from confocal fluorescence imaging show that the trigger of the solvent-mediated phase transformation is the formation of a 3-D feature rich in SMase, sphingomyelin, and ceramide. This 3-D feature is hypothesized as a slowly nucleating SMase-enriched phase, where SMase processes sphingomyelin more efficiently. The disparate time-scales of the formation of these SMase-features and the SM-enriched domains allow for the development of a significant duration of the middle intermediate morphology between the two transformations. The results show that SMase can be actively involved in the lipid membrane phase changes. The multistage morphology evolution is not only due to membrane-compositional changes caused by SMase, but also due to the selective binding of SMase, and the SMase's special phase behavior during the solvent-mediated phase transformation.
Assuntos
Ceramidas/biossíntese , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Anticorpos , Bacillus cereus/enzimologia , Ceramidas/metabolismo , Microfluídica , Transição de Fase , Solventes/química , Solventes/metabolismo , Fatores de Tempo , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismoRESUMO
Heterogeneities in cell membranes due to the ordering of lipids and proteins are thought to play an important role in enabling protein and lipid trafficking throughout the secretory pathway and in maintaining cell polarization. Protein-coated vesicles provide a major mechanism for intracellular transport of select cargo, which may be sorted into lipid microdomains; however, the mechanisms and physical constraints for lipid sorting by protein coats are relatively unexplored. We studied the influence of membrane-tethered protein coats on the sorting, morphology, and phase behavior of liquid-ordered lipid domains in a model system of giant unilamellar vesicles composed of dioleoylphosphatidylcholine, sphingomyelin, and cholesterol. We created protein-coated membranes by forming giant unilamellar vesicles containing a small amount of biotinylated lipid, thereby creating binding sites for streptavidin and avidin proteins in solution. We found that individual tethered proteins colocalize with the liquid-disordered phase, whereas ordered protein domains on the membrane surface colocalize with the liquid-ordered phase. These observations may be explained by considering the thermodynamics of this coupled system, which maximizes its entropy by cosegregating ordered protein and lipid domains. In addition, protein ordering inhibits lipid domain rearrangement and modifies the morphology and miscibility transition temperature of the membrane, most dramatically near the critical point in the membrane phase diagram. This observation suggests that liquid-ordered domains are stabilized by contact with ordered protein domains; it also hints at an approach to the stabilization of lipid microdomains by cross-linked protein clusters or ordered protein coats.
Assuntos
Colesterol/química , Bicamadas Lipídicas/metabolismo , Fosfatidilcolinas/química , Esfingomielinas/química , Estreptavidina/metabolismo , Biotina/metabolismo , Bicamadas Lipídicas/química , Microscopia de Fluorescência , Modelos Biológicos , Ligação Proteica , Transporte Proteico , Estreptavidina/química , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismoRESUMO
The cell membrane comprises numerous protein and lipid molecules capable of asymmetric organization between leaflets and liquid-liquid phase separation. We use single supported lipid bilayers (SLBs) to model cell membranes, and study how cholesterol and asymmetrically oriented ganglioside receptor G(M1) affect membrane structure using synchrotron x-ray reflectivity. Using mixtures of cholesterol, sphingomyelin, and 1,2-dioleoyl-sn-glycero-3-phosphocholine, we characterize the structure of liquid-ordered and liquid-disordered SLBs in terms of acyl-chain density, headgroup size, and leaflet thickness. SLBs modeling the liquid-ordered phase are 10 A thicker and have a higher acyl-chain electron density (rho(chain) = 0.33 e(-)/A(3)) compared to SLBs modeling the liquid-disordered phase, or pure phosphatidylcholine SLBs (rho(chain) = 0.28 e(-)/A(3)). Incorporating G(M1) into the distal bilayer leaflet results in membrane asymmetry and thickening of the leaflet of 4-9 A. The structural effect of G(M1) is more complex in SLBs of cholesterol/sphingomyelin/1,2-dioleoyl-sn-glycero-3-phosphocholine, where the distal chains show a high electron density (rho(chain) = 0.33 e(-)/A(3)) and the lipid diffusion constant is reduced by approximately 50%, as measured by fluorescence microscopy. These results give quantitative information about the leaflet asymmetry and electron density changes induced by receptor molecules that penetrate a single lipid bilayer.
Assuntos
Colesterol/química , Bicamadas Lipídicas/química , Fosfolipídeos/química , Receptores de Superfície Celular/química , Conformação Molecular , Síncrotrons , Difração de Raios XRESUMO
We study proteins at the surface of bilayer membranes using streptavidin and avidin bound to biotinylated lipids in a supported lipid bilayer (SLB) at the solid-liquid interface. Using X-ray reflectivity and simultaneous fluorescence microscopy, we characterize the structure and fluidity of protein layers with varied relative surface coverages of crystalline and noncrystalline protein. With continuous bleaching, we measure a 10-15% decrease in the fluidity of the SLB after the full protein layer is formed. We propose that this reduction in lipid mobility is due to a small fraction (0.04) of immobilized lipids bound to the protein layer that create obstacles to membrane diffusion. Our X-ray reflectivity data show a 40 A thick layer of protein, and we resolve an 8 A layer separating the protein layer from the bilayer. We suggest that the separation provided by this water layer allows the underlying lipid bilayer to retain its fluidity and stability.
Assuntos
Bicamadas Lipídicas/química , Proteínas/química , Animais , Avidina/química , Cristalização , Difusão , Corantes Fluorescentes , Técnicas In Vitro , Substâncias Macromoleculares/química , Fluidez de Membrana , Microscopia de Fluorescência , Modelos Moleculares , Marcadores de Spin , Eletricidade Estática , Estreptavidina/química , Água/químicaRESUMO
Cellular membranes can take on a variety of shapes to assist biological processes including endocytosis. Membrane-associated protein domains provide a possible mechanism for determining membrane curvature. We study the effect of tethered streptavidin protein crystals on the curvature of giant unilamellar vesicles (GUVs) using confocal, fluorescence, and differential interference contrast microscopy. Above a critical protein concentration, streptavidin domains align and percolate as they form, deforming GUVs into prolate spheroidal shapes in a size-dependent fashion. We propose a mechanism for this shape transformation based on domain growth and jamming. Osmotic deflation of streptavidin-coated GUVs reveals that the relatively rigid streptavidin protein domains resist membrane bending. Moreover, in contrast to highly curved protein domains that facilitate membrane budding, the relatively flat streptavidin domains prevent membrane budding under high osmotic stress. Thus, crystalline streptavidin domains are shown to have a stabilizing effect on lipid membranes. Our study gives insight into the mechanism for protein-mediated stabilization of cellular membranes.
Assuntos
Biofísica/métodos , Físico-Química/métodos , Estrutura Terciária de Proteína , Proteínas/química , Avidina/química , Biotinilação , Cristalização , Bicamadas Lipídicas , Lipídeos/química , Microscopia de Fluorescência , Osmose , Estreptavidina/químicaRESUMO
We measured the shape and the internal dynamics of starlike dendrimers under good solvent conditions with small-angle neutron scattering and neutron spin-echo (NSE) spectroscopy, respectively. Architectural parameters such as the spacer length and generation were varied in a systematic manner. Structural changes occurring in the dendrimers as a function of these parameters are discussed, i.e., in terms of the fractal dimension and deviations of the radius of gyration from the Gaussian value. A first cumulant evaluation of the NSE spectra for each scattering vector q separately yields the length scale dependent relaxation rates. We observe a local minimum in the normalized relaxation rates Omega(q)q(3) on length scales corresponding to the overall dendrimer dimension. The dynamics is discussed within a Rouse-Zimm approach generalized to the case of starlike dendrimers of arbitrary geometry. The model allows an identification of the modes contributing to the relaxation of the dendrimer in the q and time range of the NSE experiment. The local minimum is due to collective breathing motions of (parts of) the dendrons relative to each other. Shape fluctuations are not observed.
RESUMO
The membrane binding and model lipid raft interaction of synthetic peptides derived from the caveolin scaffolding domain (CSD) of the protein caveolin-1 have been investigated. CSD peptides bind preferentially to liquid-disordered domains in model lipid bilayers composed of cholesterol and an equimolar ratio of dioleoylphosphatidylcholine (DOPC) and brain sphingomyelin. Three caveolin-1 peptides were studied: the scaffolding domain (residues 83-101), a water-insoluble construct containing residues 89-101, and a water-soluble construct containing residues 89-101. Confocal and fluorescence microscopy investigation shows that the caveolin-1 peptides bind to the more fluid cholesterol-poor phase. The binding of the water-soluble peptide to lipid bilayers was measured using fluorescence correlation spectroscopy (FCS). We measured molar partition coefficients of 10(4) M(-1) between the soluble peptide and phase-separated lipid bilayers and 10(3) M(-1) between the soluble peptide and bilayers with a single liquid phase. Partial phase diagrams for our phase-separating lipid mixture with added caveolin-1 peptides were measured using fluorescence microscopy. The water-soluble peptide did not change the phase morphology or the miscibility transition in giant unilamellar vesicles (GUVs); however, the water-insoluble and full-length CSD peptides lowered the liquid-liquid melting temperature.
Assuntos
Caveolina 1/química , Bicamadas Lipídicas/química , Modelos Biológicos , Peptídeos/química , Adsorção , Sítios de Ligação , Modelos Moleculares , Tamanho da Partícula , Transição de Fase , Propriedades de SuperfícieRESUMO
This study focuses on the design of chemically regulated surfaces that allow for reversible control of the interactions between biological matter (cells and proteins) and planar substrates. As a tunable interlayer, we use a monolayer of a near-monodisperse poly[2-(dimethylamino)ethyl methacrylate-block-methyl methacrylate] (PDMAEMA-PMMA) diblock copolymer. Owing to the relatively large fraction (50%) of the hydrophobic PMMA block, this copolymer forms a stable Langmuir monolayer at the air/water interface. Both in situ and ex situ film balance experiments suggest that the hydrophilic PDMAEMA block adsorbs to the air/water interface in its uncharged state (pH 8.5), but stretches into the subphase in its charged state (pH 5.5). Optimization of the preparation protocols enables us to fabricate stable, homogeneous diblock copolymer films on hydrophobized substrates via Langmuir-Schaefer transfer at well-defined lateral chain densities. Ellipsometry and X-ray reflectivity studies of the transferred films confirm that the film thickness can be systematically regulated by the lateral chain densities. The transferred copolymer films remain stable in water for about a week, suggesting that they are promising materials for the creation of pH-controlled solid substrates for the support of biological matter such as proteins and cells.
Assuntos
Membranas Artificiais , Metilmetacrilatos/química , Termodinâmica , Adsorção , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Soluções , Propriedades de Superfície , Água/químicaRESUMO
A monolayer of the pH-responsive poly[2-(dimethylamino)ethyl methacrylate-block-methyl methacrylate] diblock copolymer [PDMAEMA-PMMA] was transferred from the air/water interface to a silicon substrate for evaluation as a tunable interlayer between biological material and solid substrates. Specular neutron reflectivity experiments revealed that the weak polyelectrolyte PDMAEMA chains at the solid/liquid interface can be reversibly activated by pH modulation. The thickness, scattering length density, and surface roughness of the polymer film can be systematically controlled by pH titration. As a simple model of plasma membranes, a lipid bilayer was deposited onto the polymer film. The membrane-substrate interaction was characterized by neutron reflectivity experiments, demonstrating that the membrane-substrate distance could be reversibly regulated by pH titration. These results confirm the potential of stimuli-responsive polymers for precise control of cell-surface interactions.
Assuntos
Materiais Biocompatíveis/química , Membranas Artificiais , Metilmetacrilatos/química , Concentração de Íons de Hidrogênio , Difração de Nêutrons , Pressão , Propriedades de SuperfícieRESUMO
We present a study of the simultaneous observation of protease reaction and surface diffusion as the enzyme interacts with a model substrate surface. We use micro-fluidic patterning to decorate a bovine serum albumin substrate surface with stripes of adsorbed enzyme in the absence of physical barriers. Spreading of the enzyme from the initial striped region indicates surface diffusion, while removal of the substrate provides a measure of reactivity. Microfluidic patterning provides a means to determine the relative importance of enzyme adsorption, surface diffusion, and reaction on the rate of substrate removal.
Assuntos
Enzimas/química , Enzimas/metabolismo , Técnicas Analíticas Microfluídicas/métodos , Animais , Bovinos , Difusão , Recuperação de Fluorescência Após Fotodegradação , Soroalbumina Bovina/metabolismo , Subtilisina/metabolismo , Propriedades de SuperfícieRESUMO
Paramagnetic colloidal particles aggregate into linear chains under an applied external magnetic field. These particles can be chemically linked to create chains that can be magnetically actuated to manipulate microscopic fluid flow. The flexibility of the chain can be adjusted by varying the length of the linker molecule. In this paper, we describe the use of a suspension of linked paramagnetic chains in a rotating magnetic field to perform microscale mixing. The effect of chain rotation and flexibility on the diffusion of molecules is studied by observing the mixing of an acid and base in a microchannel. We show that, as the chain rotation frequency increases, there is marked increase in the effective mixing between fluid streams; however, a maximum frequency exists and above this frequency the chains are no longer effective in mixing. More flexible chains are more effective at mixing over a larger range of frequencies.
Assuntos
Magnetismo , Enzimas/química , Fluoresceína-5-Isotiocianato/química , Espectrometria de FluorescênciaRESUMO
We present a scattering study of a selectively deuterated micelle-forming diblock copolymer. The copolymer comprises a partially deuterated polystyrene (d,h-PS) block and an imidazolium-functionalized PS (IL) block. In toluene solutions, the copolymers assemble into elongated micelles where the IL block forms the micelle core. Through dynamic light scattering (DLS) measurements, we obtain the overall size of the micelles. In our small-angle neutron scattering (SANS) studies, we use contrast matching to characterize the IL core and the PS shell of the micelles independently. The PS block forming the micelle shell exhibits either a starlike or brushlike conformation depending upon the size of the core to which it is tethered. We find the IL block to be in an extended conformation, driving the formation of slightly elongated and relatively stiff micelle cores. The elongated micelle core cross-sectional radius and length depend linearly on the length of the IL block. We find that the micelles can sequester a few water molecules for each IL repeat unit; the addition of water slightly increases the cross section of the elongated micelles.
Assuntos
Imidazóis/química , Micelas , Difração de Nêutrons/métodos , Poliestirenos/química , Deutério/química , Estrutura Molecular , Tamanho da Partícula , Poliestirenos/síntese química , Propriedades de Superfície , Água/químicaRESUMO
Paramagnetic particles have the unique ability to reversibly form magnetic chains. We have taken advantage of this property by permanently linking the chains with three linking chemistries to create flexible chains whose behavior changes with the application of a magnetic field. We study the behavior of these chains in a rotating magnetic field and model them as elastic rods. Rigid chains rotate as a solid body while flexible chains deform under the influence of magnetic, viscous, and elastic stresses. We find that the shapes chains assume in rotating magnetic fields confirm the chain flexibility determined from previous micromechanics measurements.
RESUMO
We synthesize a series of block copolymers comprising a polystyrene (PS) block and an imidazolium-functionalized PS (IL) block and characterize their assembly properties. We use small-angle neutron scattering and dynamic light scattering to determine the micelle size and shape in dilute solutions and to assess the micelle interactions in concentrated solutions. By studying a series of copolymers with fixed PS block length, we find that the length of the IL block governs the micelle dimensions. Our data suggest that these copolymers form elongated micelle structures where the IL block is extended in the micelle core. We find that these micelles can sequester water and that interactions between the micelles lead to structure factor peaks at elevated concentrations.
Assuntos
Imidazolinas/química , Micelas , Nêutrons , Poliestirenos/química , Espalhamento de Radiação , Sítios de Ligação , Polímeros/síntese química , Solubilidade , Soluções/química , Propriedades de Superfície , Água/químicaRESUMO
Magnetorheological particles, permanently linked into chains, provide a magnetically actuated means to manipulate microscopic fluid flow. Paramagnetic colloidal particles form reversible chains by acquiring dipole moments in the presence of an external magnetic field. By chemically connecting paramagnetic colloidal particles, flexible magnetoresponsive chains can be created. We link the paramagnetic microspheres using streptavidin-biotin binding. Streptavidin coated microspheres are placed in a flow cell and a magnetic field is applied, causing the particles to form chains. Then a solution of polymeric linkers of bis-biotin-poly(ethylene glycol) molecules is added in the presence of the field. These linked chains remain responsive to a magnetic field; however, in the absence of an external magnetic field these chains bend and flex due to thermal motion. The chain flexibility is determined by the length of the spacer molecule between particles and is quantified by the flexural rigidity or bending stiffness. To understand the mechanical properties of the chains, we use a variety of optical trapping experiments to measure the flexural rigidity. Increasing the length of the poly(ethylene glycol) chain in the linker increases the flexibility of the chains.