RESUMO
Borrowing principles of anhydrobiosis, we have developed a technique for self-assembling proteolipid-supported membranes on demand--simply by adding water. Intact lipid- and proteolipid vesicles dispersed in aqueous solutions of anhydrobiotic trehalose are vitrified on arbitrary substrates, producing glassy coats encapsulating biomolecules. Previous efforts establish that these carbohydrate coats arrest molecular mobilities and preserve native conformations and aggregative states of the embedded biomolecules, thereby enabling long-term storage. Subsequent rehydration, even after an extended period of time (e.g., weeks), devitrifies sugar--releasing the cargo and unmasking the substrate surface--thus triggering substrate-mediated vesicle fusion in real time, producing supported membranes. Using this method, arrays of membranes, including those functionalized with membrane proteins, can be readily produced in situ by spatially addressing vitrification using common patterning tools--useful for multiplexed or stochastic sensing and assaying of target interactions with the fluid and functional membrane surface.
Assuntos
Carboidratos/química , Bicamadas Lipídicas/química , Trealose/química , Vesículas Citoplasmáticas , Proteínas Hemolisinas/química , Água/químicaRESUMO
Substrate-mediated fusion of small polymersomes, derived from mixtures of lipids and amphiphilic block copolymers, produces hybrid, supported planar bilayers at hydrophilic surfaces, monolayers at hydrophobic surfaces, and binary monolayer/bilayer patterns at amphiphilic surfaces, directly responding to local measures of (and variations in) surface free energy. Despite the large thickness mismatch in their hydrophobic cores, the hybrid membranes do not exhibit microscopic phase separation, reflecting irreversible adsorption and limited lateral reorganization of the polymer component. With increasing fluid-phase lipid fraction, these hybrid, supported membranes undergo a fluidity transition, producing a fully percolating fluid lipid phase beyond a critical area fraction, which matches the percolation threshold for the immobile point obstacles. This then suggests that polymer-lipid hybrid membranes might be useful models for studying obstructed diffusion, such as occurs in lipid membranes containing proteins.
Assuntos
Lipídeos/química , Membranas Artificiais , Polímeros/química , Tensoativos/química , Recuperação de Fluorescência Após Fotodegradação , Microscopia de Fluorescência , Propriedades de SuperfícieRESUMO
We describe an ultrafast fiber optical parametric oscillator operating in the 1210 nm to 1340 nm wavelength range. The system consists of a microstructure fiber placed in a Fabry-Perot cavity which is optically pumped with 1030-nm light from an Ytterbium mode-locked fiber laser. The output wavelength is tunable over a 130-nm span by adjusting the position of one cavity mirror. SHG FROG measurements reveal that the output pulse quality varies as a function of pump power and wavelength. Ultrafast sources operating in this range are particularly instrumental for deep-tissue nonlinear biophotonics applications.
Assuntos
Amplificadores Eletrônicos , Tecnologia de Fibra Óptica/instrumentação , Oscilometria/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , MiniaturizaçãoRESUMO
Bilayer vesicles that mimic a real biological cell can be tailored to carry out a specific function by manipulating the molecular composition of the amphiphiles. These bio-inspired and bio-mimetic structures are increasingly being employed for a number of applications from drug delivery to water purification and beyond. Complex hybrid bilayers are the key building blocks for fully synthetic vesicles that can mimic biological cell membranes, which often contain a wide variety of molecular species. While the assembly and morpholgy of pure phospholid bilayer vesicles is well understood, the functionality and structure dramaticlly changes when copolymer and/or carbon nanotube porins (CNTP) are added. The aim of this study is to understand how the collective molecular interactions within hybrid vesicles affect their nanoscale structure and properties. In situ small and wide angle X-ray scattering (SAXS/WAXS) and molecular dynamics simulations (MD) are used to investigate the morphological effect of molecular interactions between polybutadiene polyethylene oxide, lipids and carbon nanotubes (CNT) within the hybrid vesicle bilayer. Within the lipid/copolymer system, the hybrid bilayer morphology transitions from phase separated lipid and compressed copolymer at low copolymer loadings to a mixed bilayer where opposing lipids are mostly separated from the inner region. This transition begins between 60 wt% and 70 wt%, with full homogenization observed by 80 wt% copolymer. The incorporation of CNT into the hybrid vesicles increases the bilayer thickness and enhances the bilayer symmetry. Analysis of the WAXS and MD indicate that the CNT-dioleoyl interactions are much stronger than the CNT-polybutadiene.
Assuntos
Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Nanotubos de Carbono/química , Porinas/química , Difração de Raios XRESUMO
We report on the generation of 70-fs pulses at a center wavelength of 880 nm using a microstructure-fiber-based optical parametric oscillator pumped by a fiber laser operating at 1032 nm. We present optical spectra and autocorrelation measurements that illustrate the generation of ultrashort pulses and the onset of saturation at sufficiently high pump powers. Generation of ultrafast pulses with nanojoule energies provides new opportunities for extending the functionality of mode-locked fiber lasers.
RESUMO
Biological membranes provide a fascinating example of a separation system that is multifunctional, tunable, precise, and efficient. Biomimetic membranes, which mimic the architecture of cellular membranes, have the potential to deliver significant improvements in specificity and permeability. Here, a fully synthetic biomimetic membrane is reported that incorporates ultra-efficient 1.5 nm diameter carbon nanotube porin (CNTPs) channels in a block-copolymer matrix. It is demonstrated that CNTPs maintain high proton and water permeability in these membranes. CNTPs can also mimic the behavior of biological gap junctions by forming bridges between vesicular compartments that allow transport of small molecules.
Assuntos
Interações Hidrofóbicas e Hidrofílicas , Membranas Artificiais , Nanotubos de Carbono/química , Polímeros/química , Porinas/químicaRESUMO
A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ''protein-centric" view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a ''receptor independent" transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. Finally, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death.
Assuntos
Membrana Celular/metabolismo , Lactalbumina/metabolismo , Ácidos Oleicos/metabolismo , Transdução de Sinais , Apoptose , Morte Celular , Permeabilidade da Membrana Celular , Humanos , Ligantes , Bicamadas Lipídicas/metabolismo , Lipídeos de Membrana/metabolismo , Ligação Proteica , Receptores de Superfície Celular/metabolismo , Proteínas ras/metabolismoRESUMO
We report the experimental observation of osmotically induced transient pearling instabilities in vesicular membranes. Giant phospholipid vesicles subjected to negative osmotic gradient, which drives the influx of water in to the vesicular interior, produces transient cylindrical protrusions. These protrusions exhibit a remarkable pearling intermediate, which facilitates their subsequent retraction. The pearling front propagates from the distal free end of the protrusion toward the vesicular source and accompanies gradual shortening of the protrusion via pearl-pearl coalescence. Real-time introduction of a positive osmotic gradient, on the other hand, drives vigorous shape fluctuations, which in turn produce cylindrical, prolate- and pear-shaped intermediates presumably due to an increased vesicular area relative to the encapsulated volume. These intermediates transiently produce a pearled state prior to their fission. In both cases, the transient pearling state gives rise to an array of stable spherical daughter vesicles, which may be connected to one another by fine tethers not resolved in our experiments. These results may have implications for self-reproduction in primitive, protein-free, cells.
Assuntos
Membrana Celular/química , Fosfolipídeos/química , Microscopia de Fluorescência , Osmose , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Sacarose/química , Difração de Raios XRESUMO
We report observations of large-scale, in-plane and out-of-plane membrane deformations in giant uni- and multilamellar vesicles composed of binary and ternary lipid mixtures in the presence of net transvesicular osmotic gradients. The lipid mixtures we examined consisted of binary mixtures of DOPC and DPPC lipids and ternary mixtures comprising POPC, sphingomyelin and cholesterol over a range of compositions - both of which produce co-existing phases for selected ranges of compositions at room temperature under thermodynamic equilibrium. In the presence of net osmotic gradients, we find that the in-plane phase separation potential of these mixtures is non-trivially altered and a variety of out-of-plane morphological remodeling events occur. The repertoire of membrane deformations we observe display striking resemblance to their biological counterparts in live cells encompassing vesiculation, membrane fission and fusion, tubulation and pearling, as well as expulsion of entrapped vesicles from multicompartmental giant unilamellar vesicles through large, self-healing transient pores. These observations suggest that the forces introduced by simple osmotic gradients across membrane boundaries could act as a trigger for shape-dependent membrane and vesicle trafficking activities. We speculate that such coupling of osmotic gradients with membrane properties might have provided lipid-mediated mechanisms to compensate for osmotic stress during the early evolution of membrane compartmentalization in the absence of osmoregulatory protein machinery.