RESUMO
One of the deepest branches in the tree of life separates the Archaea from the Bacteria. These prokaryotic groups have distinct cellular systems including fundamentally different phospholipid membrane bilayers. This dichotomy has been termed the lipid divide and possibly bestows different biophysical and biochemical characteristics on each cell type. Classic experiments suggest that bacterial membranes (formed from lipids extracted from Escherichia coli, for example) show permeability to key metabolites comparable to archaeal membranes (formed from lipids extracted from Halobacterium salinarum), yet systematic analyses based on direct measurements of membrane permeability are absent. Here, we develop a new approach for assessing the membrane permeability of approximately 10 µm unilamellar vesicles, consisting of an aqueous medium enclosed by a single lipid bilayer. Comparing the permeability of 18 metabolites demonstrates that diether glycerol-1-phosphate lipids with methyl branches, often the most abundant membrane lipids of sampled archaea, are permeable to a wide range of compounds useful for core metabolic networks, including amino acids, sugars, and nucleobases. Permeability is significantly lower in diester glycerol-3-phosphate lipids without methyl branches, the common building block of bacterial membranes. To identify the membrane characteristics that determine permeability, we use this experimental platform to test a variety of lipid forms bearing a diversity of intermediate characteristics. We found that increased membrane permeability is dependent on both the methyl branches on the lipid tails and the ether bond between the tails and the head group, both of which are present on the archaeal phospholipids. These permeability differences must have had profound effects on the cell physiology and proteome evolution of early prokaryotic forms. To explore this further, we compare the abundance and distribution of transmembrane transporter-encoding protein families present on genomes sampled from across the prokaryotic tree of life. These data demonstrate that archaea tend to have a reduced repertoire of transporter gene families, consistent with increased membrane permeation. These results demonstrate that the lipid divide demarcates a clear difference in permeability function with implications for understanding some of the earliest transitions in cell origins and evolution.
Assuntos
Archaea , Lipossomas Unilamelares , Archaea/genética , Lipossomas Unilamelares/metabolismo , Glicerol/metabolismo , Membrana Celular/metabolismo , Bactérias/metabolismo , Lipídeos de Membrana/metabolismo , Fosfolipídeos/metabolismo , Fosfatos/metabolismo , Bicamadas Lipídicas/análise , Bicamadas Lipídicas/metabolismoRESUMO
Dimethyl sulfoxide (DMSO) is widely used in a number of biological and biotechnological applications, mainly because of its effects on the cell plasma membrane, but the molecular origins of this action are yet to be fully clarified. In this work, we used two- and three-component synthetic membranes (liposomes) and the plasma membrane of human erythrocytes to investigate the effect of DMSO when added to the membrane-solvating environment. Fourier transform infrared spectroscopy and thermal fluctuation spectroscopy revealed significant differences in the response of the two types of liposome systems to DMSO in terms of the bilayer thermotropic behavior, available free volume of the bilayer, its excess surface area, and bending elasticity. DMSO also alters the mechanical properties of the erythrocyte membrane in a concentration-dependent manner and is capable of increasing membrane permeability to ATP at even relatively low concentrations (3% v/v and above). Taken in its entirety, these results show that DMSO is likely to have a differential effect on heterogeneous biological membranes, depending on their local composition and structure, and could affect membrane-hosted biological functions.
Assuntos
Dimetil Sulfóxido , Lipossomos , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Humanos , Lipossomos/metabolismo , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
The present work explores the potential use of the conjugated cationic polyfluorene {[9,9-bis(6'-N,N,N-trimethylammonium)hexyl]fluorene-phenylene} bromide (HTMA-PFP) as a fluorescent membrane marker. To this end, the interaction of the polyelectrolyte with anionic model membranes has been investigated using different biophysical approaches. High affinity interaction was confirmed through alterations in the fluorescence spectrum of HTMA-PFP and by Förster resonance energy transfer (FRET) analysis. Quenching data indicate that once HTMA-PFP interacts with the membrane, it penetrates in the hydrophobic core embedded in the lipid bilayer where it presents high fluorescence quantum yield and photostability. Leakage experiments and dynamic light scattering (DLS) measurements show that the integrity of the lipid vesicles is maintained after polymer incorporation since no vesicle fusion or decomposition into small fragments is detectable. This conclusion is supported by fluorescence microscopy images, which confirm that polyelectrolyte interacts with the vesicle, labeling the lipid membrane without altering its morphology. Further experiments performed as a function of temperature indicate that the polymer is accommodated in the membrane without inducing significant loss of lipid cooperativity and without altering the packing of lipids within the bilayer. Finally, results show that polyelectrolyte fluorescence is sensitive to the large structural changes taking place in the lipid bilayer at the lipid phase transition. All these results confirm the ability of HTMA-PFP to visualize membrane structures and to monitor membrane processes.
Assuntos
Biomarcadores/química , Fluorenos/química , Polímeros/química , Compostos de Amônio Quaternário/química , Transferência Ressonante de Energia de Fluorescência , Microscopia de FluorescênciaRESUMO
Soft and mechanically responsive actuators hold the promise to revolutionize the design and manufacturing of devices in the areas of microfluidics, soft robotics and biomedical engineering. In many of these applications, the actuators need to operate in a wet environment that can strongly affect their performance. In this paper, we report on the photomechanical response in a biological buffer of azobenzene-containing liquid crystal elastomer (LCE)-based actuators, prepared by four-dimensional (4D) printing. Although the photothermal contribution to the photoresponse is largely cancelled by the heat withdrawing capacity of the employed buffer, a significant photoinduced reversible contraction, in the range of 7% of its initial length, has been achieved under load, taking just a few seconds to reach half of the maximum contraction. Effective photomechanical work performance under physiological conditions has, therefore, been demonstrated in the 4D-printed actuators. Advantageously, the photomechanical response is not sensitive to salts present in the buffer differently to hydrogels with responses highly dependent on the fluid composition. Our work highlights the capabilities of photomechanical actuators, created using 4D printing, when operating under physiological conditions, thus showing their potential for application in the microfluidics and biomedical fields.
Assuntos
Cristais Líquidos , Compostos Azo , Bioengenharia , ElastômerosRESUMO
Environmental and intracellular stresses can perturb protein homeostasis and trigger the formation and accumulation of protein aggregates. It has been recently suggested that the level of protein aggregates accumulated in bacteria correlates with the frequency of persister and viable but nonculturable cells that transiently survive treatment with multiple antibiotics. However, these findings have often been obtained employing fluorescent reporter strains. This enforced heterologous protein expression facilitates the visualization of protein aggregates but could also trigger the formation and accumulation of protein aggregates. Using microfluidics-based single-cell microscopy and a library of green fluorescent protein reporter strains, we show that heterologous protein expression favors the formation of protein aggregates. We found that persister and viable but nonculturable bacteria surviving treatment with antibiotics are more likely to contain protein aggregates and downregulate the expression of heterologous proteins. Our data also suggest that such aggregates are more basic with respect to the rest of the cell. These findings provide evidence for a strong link between heterologous protein expression, protein aggregation, intracellular pH, and phenotypic survival to antibiotics, suggesting that antibiotic treatments against persister and viable but nonculturable cells could be developed by modulating protein aggregation and pH regulation.
Assuntos
Escherichia coli , Agregados Proteicos , Antibacterianos/farmacologia , Bactérias/genética , Escherichia coli/genética , ProteômicaRESUMO
Remote light exposure of photoresponsive liquid crystalline polymers has drawn great attention over the last years as an attractive strategy to generate mechanical work with high spatial resolution. To tailor these materials into practical engineering devices, it is of key importance to gain control over their morphology and thus precisely program their mechanical response, which must also be fast and relevant in magnitude. In this communication, we report the four-dimensional (4D) printing of azobenzene-containing liquid crystalline elastomers (LCEs) that respond to light. During extrusion of the LCE precursor, mesogen orientation is defined by the needle's moving direction enabling a precise definition of the director, which is later fixed by photopolymerization. Fast mechanical responses have been observed in these 4D printed LCE elements when excited with ultraviolet (UV) light. These 4D printed elements lift objects many times heavier than their own weight, demonstrating a capacity to produce effective work. Photochemical and photothermal contributions to the deformation and force have been identified. Advantageously, the use of blue and UV light excitation enables adjustment of generated forces that can be maintained even in the dark and can be released by light excitation or temperature. The demonstrated ability to generate light-responsive elements quickly delivering sufficient work paves the way for implementing remotely addressed mechanical functions to future soft robotics and engineering.
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In the present work, we have synthesized a novel green-emitter conjugated polyelectrolyte Copoly-{[9,9-bis(6'-N,N,N-trimethylammonium)hexyl]-2,7-(fluorene)-alt-4,7-(2-(phenyl) benzo[d] [1,2,3] triazole)} bromide (HTMA-PFBT) by microwave-assisted Suzuki coupling reaction. Its fluorescent properties have been studied in aqueous media and in presence of model membranes of different composition, in order to explore its ability to be used as a green fluorescent membrane probe. The polyelectrolyte was bound with high affinity to the membrane surface, where it exhibited high fluorescence efficiency and stability. HTMA-PFBT showed lower affinity to zwitterionic membranes as compared to anionic ones, as well as a more external location, near the membrane-aqueous interface. Fluorescence microscopy studies confirmed the interaction of HTMA-PFBT with the model membranes, labelling the lipid bilayer without perturbing its morphology and showing a clear preference towards anionic systems. In addition, the polyelectrolyte was able to label the membrane of bacteria and living mammalian cells, separately. Finally, we explored if the polyelectrolyte can function also as a sensitive probe able of detecting lipid-phase transitions. All these results suggest the potential use of HTMA-PFBT as a green membrane marker for bioimaging and selective recognition of bacteria cell over mammalian ones and as a tool to monitor changes in physical state of lipid membranes.
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This work describes the development of a novel fluorescent biosensor based on the inhibition of alkaline phosphatase (ALP). The biosensor is composed of the enzyme ALP and the conjugated cationic polyfluorene HTMA-PFP. The working principle of the biosensor is based on the fluorescence quenching of this polyelectrolyte by p-nitrophenol (PNP), a product of the hydrolysis reaction of p-nitrophenyl phosphate (PNPP) catalyzed by ALP. Because HTMA-PFP forms unstable aggregates in buffer, with low fluorescence efficiency, previous stabilization of the polyelectrolyte was required before the development of the biosensor. HTMA-PFP was stabilized through its interaction with lipid vesicles to obtain stable blue-emitting nanoparticles (NPs). Fluorescent NPs were characterized, and the ability to be quenched by PNP was evaluated. These nanoparticles were coupled to ALP and entrapped in a sol-gel matrix to produce a biosensor that can serve as a screening platform to identify ALP inhibitors. The components of the biosensor were examined before and after sol-gel entrapment, and the biosensor was optimized to allow the determination of phosphate ion in aqueous medium.
Assuntos
Técnicas Biossensoriais , Fosfatase Alcalina , Fluorenos , Lipossomos , Nanopartículas , Nitrofenóis , FosfatosRESUMO
The design and development of fluorescent conjugated polyelectrolytes (CPEs) emitting in the red region of the visible spectrum is at present of great interest for bioimaging studies. However, despite the wide variety of CPEs available, stable bright red-emitters remain scarce due to their low solubility and instability in aqueous media, consequently limiting their applications. In this work, we have synthesized and characterized a new red-emitting cationic conjugated polyelectrolyte copoly-{[9,9-bis(6'-N,N,N-trimethylammonium)hexyl]-2,7-(fluorene)-alt-1,4-(naphtho[2,3c]-1,2,5-thiadiazole)} bromide (HTMA-PFNT), based on the incorporation of naphtha[2,3c][1,2,5] thiadiazole on fluorene backbone to increase the bathochromic emission, extending the conjugation length in the polymer backbone. Water stabilization was achieved by binding the polyelectrolyte to two different biological systems which are currently used as nanocarriers: human serum albumin (HSA) and lipid vesicles. Using both systems, stable nanostructures of different composition were obtained and their properties were characterized. The properties of the protein-based nanoparticles are consistent with polyelectrolyte aggregates covered with HSA molecules, while the liposome system is composed of lipid vesicles coated with polyelectrolyte chains partially inserted in the bilayer. Both protein and vesicle structural integrity were not affected after their interaction with HTMA-PFNT, as well as the carrier properties, allowing for the binding and transport of ligands. In addition, the nanoparticles displayed the ability of labeling the cell membrane of living cells. All these results extend the potential applications of these novel multifunctional nanoparticles as therapeutic carriers and bioimaging probes.
Assuntos
Portadores de Fármacos/química , Eletrólitos/química , Luz , Imagem Molecular/métodos , Sondas Moleculares/química , Soluções Tampão , Linhagem Celular , Dicroísmo Circular , Eletrólitos/síntese química , Humanos , Hidrodinâmica , Lipossomos/química , Modelos Teóricos , Nanopartículas/química , Nanopartículas/ultraestrutura , Tamanho da Partícula , Albumina Sérica/química , Espectrometria de Fluorescência , TemperaturaRESUMO
This paper explores the interaction mechanism between the conjugated polyelectrolyte {[9,9-bis(6'-N,N,N-trimethylammonium)hexyl]fluorene-phenylene}bromide (HTMA-PFP) and model lipid membranes. The study was carried out using different biophysical techniques, mainly fluorescence spectroscopy and microscopy. Results show that despite the preferential interaction of HTMA-PFP with anionic lipids, HTMA-PFP shows affinity for zwitterionic lipids; although the interaction mechanism is different as well as HTMA-PFP's final membrane location. Whilst the polyelectrolyte is embedded within the lipid bilayer in the anionic membrane, it remains close to the surface, forming aggregates that are sensitive to the physical state of the lipid bilayer in the zwitterionic system. The different interaction mechanism is reflected in the polyelectrolyte fluorescence spectrum, since the maximum shifts to longer wavelengths in the zwitterionic system. The intrinsic fluorescence of HTMA-PFP was used to visualize the interaction between polymer and vesicles via fluorescence microscopy, thanks to its high quantum yield and photostability. This technique allows the selectivity of the polyelectrolyte and higher affinity for anionic membranes to be observed. The results confirmed the appropriateness of using HTMA-PFP as a membrane fluorescent marker and suggest that, given its different behaviour towards anionic and zwitterionic membranes, HTMA-PFP could be used for selective recognition and imaging of bacteria over mammalian cells.
RESUMO
Interaction between poly[9,9-bis(6'-bromohexyl)-2,7-fluorene-co-alt-1,4-phenylene] (PFPBr2), a neutral conjugated polyfluorene which is completely insoluble in water, and zwitterionic phospholipids has been investigated in order to generate new fluorescent structures which are stable in aqueous media as a means of extending the biological applications of these kinds of polymers. Two types of differently shaped and composed fluorescent structures were identified and then isolated and characterized separately using different biophysical techniques. The first structure type, corresponding to liposomal complexes, showed a fluorescence band centered around 405 nm and maximum absorption at 345 nm, while the second, corresponding to polymer-phospholipid aggregates of variable sizes with lower lipid content, absorbed at longer wavelengths and displayed a well resolved fluorescence spectrum with a maximum centered at 424 nm. Both structures were stable in a large range of pH, and their fluorescence intensity remained practically unaltered for 10 days; it then began to decrease, which was probably because of aggregation. Encapsulation of these structures within the pores of a sol-gel matrix did not affect their fluorescent properties but increased their stability, avoiding further aggregation and subsequent precipitation.