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1.
Proc Natl Acad Sci U S A ; 121(36): e2404790121, 2024 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-39186653

RESUMO

Eukaryotic cells are characterized by multiple chemically distinct compartments, one of the most notable being the nucleus. Within these compartments, there is a continuous exchange of information, chemicals, and signaling molecules, essential for coordinating and regulating cellular activities. One of the main goals of bottom-up synthetic biology is to enhance the complexity of synthetic cells by establishing functional compartmentalization. There is a need to mimic autonomous signaling between compartments, which in living cells, is often regulated at the genetic level within the nucleus. This advancement is key to unlocking the potential of synthetic cells as cell models and as microdevices in biotechnology. However, a technological bottleneck exists preventing the creation of synthetic cells with a defined nucleus-like compartment capable of genetically programmed intercompartment signaling events. Here, we present an approach for creating synthetic cells with distinct nucleus-like compartments that can encapsulate different biochemical mixtures in discrete compartments. Our system enables in situ protein expression of membrane proteins, enabling autonomous chemical communication between nuclear and cytoplasmic compartments, leading to downstream activation of enzymatic pathways within the cell.


Assuntos
Células Artificiais , Núcleo Celular , Biologia Sintética , Biologia Sintética/métodos , Núcleo Celular/metabolismo , Núcleo Celular/genética , Células Artificiais/metabolismo , Transdução de Sinais , Citoplasma/metabolismo , Comunicação Celular
2.
Anal Chem ; 95(32): 12006-12014, 2023 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-37526607

RESUMO

Lipid membranes are crucial for cellular integrity and regulation, and tight control of their structural and mechanical properties is vital to ensure that they function properly. Fluorescent probes sensitive to the membrane's microenvironment are useful for investigating lipid membrane properties; however, there is currently a lack of quantitative correlation between the exact parameters of lipid organization and a readout from these dyes. Here, we investigate this relationship for "molecular rotors", or microviscosity sensors, by simultaneously measuring their fluorescence lifetime to determine the membrane viscosity, while using X-ray diffraction to determine the membrane's structural properties. Our results reveal a phase-dependent correlation between the membrane's structural parameters and mechanical properties measured by a BODIPY-based molecular rotor, giving excellent predictive power for the structural descriptors of the lipid bilayer. We also demonstrate that differences in membrane thickness between different lipid phases are not a prerequisite for the formation of lipid microdomains and that this requirement can be disrupted by the presence of line-active molecules. Our results underpin the use of membrane-sensitive dyes as reporters of the structure of lipid membranes.


Assuntos
Corantes Fluorescentes , Bicamadas Lipídicas , Bicamadas Lipídicas/química , Viscosidade , Corantes Fluorescentes/química , Membranas , Fluorescência , Membrana Celular
3.
Phys Chem Chem Phys ; 25(8): 6316-6325, 2023 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-36779289

RESUMO

Understanding the structure-property relationship and nanoscopic behaviour of ionic liquids is of utmost importance for their potential applications. Focusing these studies on sets of homobaric ionic liquids could provide important insight into the effects of specific chemical groups on the overall interaction profile, bringing researchers one step closer to succesfully designing ionic liquids which are tailor-made for specific applications. This work focuses on ionic liquids with 12 total carbons on their side chains, studying both their bulk physical properties (such as densities and viscosities) and their nanostructuring. The results reveal that by keeping the total number of carbons constant, but arranging them differently around the imidazolium ring, either in a linear or in a branched-chain formation, can result in compounds with quite distinct properties. Some of those (such as diffusivity) appear to be more sensitive to symmetry variations, while others (such as density) are not significantly affected. X-ray scattering is used in order to get a clearer understanding of the nanostructuring of the studied compounds and to investigate to what extent the observed macroscopic properties are directly linked to the nanoscale ordering.

4.
Biophys J ; 120(17): 3787-3794, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34273316

RESUMO

Cellular life relies on membranes, which provide a resilient and adaptive cell boundary. Many essential processes depend upon the ease with which the membrane is able to deform and bend, features that can be characterized by the bending rigidity. Quantitative investigations of such mechanical properties of biological membranes have primarily been undertaken in solely lipid bilayers and frequently in the absence of buffers. In contrast, much less is known about the influence of integral membrane proteins on bending rigidity under physiological conditions. We focus on an exemplar member of the ubiquitous major facilitator superfamily of transporters and assess the influence of lactose permease on the bending rigidity of lipid bilayers. Fluctuation analysis of giant unilamellar vesicles (GUVs) is a useful means to measure bending rigidity. We find that using a hydrogel substrate produces GUVs that are well suited to fluctuation analysis. Moreover, the hydrogel method is amenable to both physiological salt concentrations and anionic lipids, which are important to mimic key aspects of the native lactose permease membrane. Varying the fraction of the anionic lipid in the lipid mixture DOPC/DOPE/DOPG allows us to assess the dependence of membrane bending rigidity on the topology and concentration of an integral membrane protein in the lipid bilayer of GUVs. The bending rigidity gradually increases with the incorporation of lactose permease, but there is no further increase with greater amounts of the protein in the membrane.


Assuntos
Bicamadas Lipídicas , Fosfatidilcolinas , Proteínas de Membrana Transportadoras , Lipossomas Unilamelares
5.
Soft Matter ; 17(23): 5763-5771, 2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34019613

RESUMO

Mixtures of fatty acids and phospholipids can form hexagonal (HII) and inverse bicontinuous cubic phases, the latter of which are implicated in various cellular processes and have wide-ranging biotechnological applications in protein crystallisation and drug delivery systems. Therefore, it is vitally important to understand the formation conditions of inverse bicontinuous cubic phases and how their properties can be tuned. We have used differential scanning calorimetry and synchrotron-based small angle and wide angle X-ray scattering (SAXS/WAXS) to investigate the polymorphic phase behaviour of palmitic acid/partially-methylated phospholipid mixtures, and how headgroup methylation impacts on inverse bicontinuous cubic phase formation. We find that upon partial methylation of the phospholipid headgroup (1 or 2 methyl substituents) inverse bicontinuous cubic phases are formed (of the Im3m spacegroup), which is not the case with 0 or 3 methyl substituents. This shows how important headgroup methylation is for controlling phase behaviour and how a change in headgroup methylation can be used to controllably tune various inverse bicontinuous phase features such as their lattice parameter and the temperature range of their stability.


Assuntos
Ácido Palmítico , Etanolaminas , Metilação , Espalhamento a Baixo Ângulo , Difração de Raios X
6.
Int J Mol Sci ; 22(11)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34200063

RESUMO

The modification of archaeal lipid bilayer properties by the insertion of apolar molecules in the lipid bilayer midplane has been proposed to support cell membrane adaptation to extreme environmental conditions of temperature and hydrostatic pressure. In this work, we characterize the insertion effects of the apolar polyisoprenoid squalane on the permeability and fluidity of archaeal model membrane bilayers, composed of lipid analogues. We have monitored large molecule and proton permeability and Laurdan generalized polarization from lipid vesicles as a function of temperature and hydrostatic pressure. Even at low concentration, squalane (1 mol%) is able to enhance solute permeation by increasing membrane fluidity, but at the same time, to decrease proton permeability of the lipid bilayer. The squalane physicochemical impact on membrane properties are congruent with a possible role of apolar intercalants on the adaptation of Archaea to extreme conditions. In addition, such intercalant might be used to cheaply create or modify chemically resistant liposomes (archeaosomes) for drug delivery.


Assuntos
Archaea/fisiologia , Membrana Celular/fisiologia , Bicamadas Lipídicas/metabolismo , Lipossomos/metabolismo , Fluidez de Membrana , Esqualeno/análogos & derivados , Archaea/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Esqualeno/farmacologia , Temperatura
7.
J Cell Sci ; 131(15)2018 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-29930079

RESUMO

Life is dependent upon the ability of a cell to rapidly respond to changes in the environment. Small perturbations in local environments change the ability of molecules to interact and, hence, communicate. Hydrostatic pressure provides a rapid non-invasive, fully reversible method for modulating affinities between molecules both in vivo and in vitro We have developed a simple fluorescence imaging chamber that allows intracellular protein dynamics and molecular events to be followed at pressures <200 bar in living cells. By using yeast, we investigated the impact of hydrostatic pressure upon cell growth and cell-cycle progression. While 100 bar has no effect upon viability, it induces a delay in chromosome segregation, resulting in the accumulation of long undivided cells that are also bent, consistent with disruption of the cytoskeletons. This delay is independent of stress signalling and induces synchronisation of cell-cycle progression. Equivalent effects were observed in Candida albicans, with pressure inducing a reversible cell-cycle delay and hyphal growth. We present a simple novel non-invasive fluorescence microscopy-based approach to transiently impact molecular dynamics in order to visualise, dissect and study signalling pathways and cellular processes in living cells.


Assuntos
Ciclo Celular/fisiologia , Pressão Hidrostática , Candida albicans/citologia , Candida albicans/metabolismo , Proliferação de Células/fisiologia , Microscopia de Fluorescência , Simulação de Dinâmica Molecular , Schizosaccharomyces/citologia , Schizosaccharomyces/metabolismo
8.
Soft Matter ; 16(25): 5925-5932, 2020 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-32538402

RESUMO

Lipid asymmetry is a crucial property of biological membranes and significantly influences their physical and mechanical properties. It is responsible for maintaining different chemical environments on the external and internal surfaces of cells and organelles and plays a vital role in many biological processes such as cell signalling and budding. In this work we show, using non-equilibrium molecular dynamics (NEMD) simulations, that thermal fields can induce lipid asymmetry in biological membranes. We focus our investigation on cholesterol, an abundant lipid in the plasma membrane, with a rapid flip-flop rate, significantly influencing membrane properties. We demonstrate that thermal fields induce membrane asymmetry with cholesterol showing thermophobic behaviour and therefore accumulating on the cold side of the membrane. This work highlights a possible experimental route to preparing and controlling asymmetry in synthetic membranes.


Assuntos
Colesterol/química , Bicamadas Lipídicas/química , 1,2-Dipalmitoilfosfatidilcolina/química , Simulação de Dinâmica Molecular , Temperatura , Termodinâmica
9.
Proc Natl Acad Sci U S A ; 114(16): 4225-4230, 2017 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-28373555

RESUMO

Invasion of the red blood cell (RBC) by the Plasmodium parasite defines the start of malaria disease pathogenesis. To date, experimental investigations into invasion have focused predominantly on the role of parasite adhesins or signaling pathways and the identity of binding receptors on the red cell surface. A potential role for signaling pathways within the erythrocyte, which might alter red cell biophysical properties to facilitate invasion, has largely been ignored. The parasite erythrocyte-binding antigen 175 (EBA175), a protein required for entry in most parasite strains, plays a key role by binding to glycophorin A (GPA) on the red cell surface, although the function of this binding interaction is unknown. Here, using real-time deformability cytometry and flicker spectroscopy to define biophysical properties of the erythrocyte, we show that EBA175 binding to GPA leads to an increase in the cytoskeletal tension of the red cell and a reduction in the bending modulus of the cell's membrane. We isolate the changes in the cytoskeleton and membrane and show that reduction in the bending modulus is directly correlated with parasite invasion efficiency. These data strongly imply that the malaria parasite primes the erythrocyte surface through its binding antigens, altering the biophysical nature of the target cell and thus reducing a critical energy barrier to invasion. This finding would constitute a major change in our concept of malaria parasite invasion, suggesting it is, in fact, a balance between parasite and host cell physical forces working together to facilitate entry.


Assuntos
Antígenos de Protozoários/metabolismo , Membrana Celular/patologia , Eritrócitos/patologia , Glicoforinas/metabolismo , Malária Falciparum/patologia , Plasmodium falciparum/patogenicidade , Proteínas de Protozoários/metabolismo , Antígenos de Protozoários/genética , Biofísica , Membrana Celular/metabolismo , Membrana Celular/parasitologia , Citoesqueleto , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Glicoforinas/genética , Interações Hospedeiro-Parasita , Humanos , Malária Falciparum/metabolismo , Malária Falciparum/parasitologia , Plasmodium falciparum/isolamento & purificação , Ligação Proteica , Proteínas de Protozoários/genética , Transdução de Sinais
10.
Biophys J ; 116(10): 1984-1993, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-31053255

RESUMO

Gravity-sensitive cellular responses are regularly observed in both specialized and nonspecialized cells. One potential mechanism for this sensitivity is a changing viscosity of the intracellular organelles. Here, we report a novel, to our knowledge, viscosity-sensitive molecular rotor based on mesosubstituted boron-dipyrrin used to investigate the response of viscosity of cellular membranes to hypergravity conditions created at the large diameter centrifuge at the European Space Agency Technology Centre. Mouse osteoblastic (MC3T3-E1) and endothelial (human umbilical vein endothelial cell) cell lines were tested, and an increase in viscosity was found with increasing hypergravity loading. This response is thought to be primarily biologically driven, with the potential for a small, instantaneous physical mechanism also contributing to the observed effect. This work provides the first, to our knowledge, quantitative data for cellular viscosity changes under hypergravity, up to 15 × g.


Assuntos
Gravitação , Espaço Intracelular/metabolismo , Células 3T3 , Animais , Fenômenos Biomecânicos , Compostos de Boro/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Camundongos , Viscosidade
11.
Langmuir ; 35(50): 16521-16527, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31702159

RESUMO

Dispersions of nonlamellar lipid membrane assemblies are gaining increasing interest for drug delivery and protein therapeutic application. A key bottleneck has been the lack of rational design rules for these systems linking different lipid species and conditions to defined lattice parameters and structures. We have developed robust methods to form cubosomes (nanoparticles with porous internal structures) with water channel diameters of up to 171 Å, which are over 4 times larger than archetypal cubosome structures. The water channel diameter can be tuned via the incorporation of cholesterol and the charged lipid DOPA, DOPG, or DOPS. We have found that large molecules can be incorporated into the porous cubosome structure and that these molecules can interact with the internal cubosome membrane. This offers huge potential for accessible encapsulation and protection of biomolecules and development of confined interfacial reaction environments.


Assuntos
Colesterol/química , Engenharia , Glicerofosfolipídeos/química
12.
Phys Chem Chem Phys ; 21(11): 5999-6010, 2019 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-30809621

RESUMO

Ionic liquid (IL) mixtures enable the design of fluids with finely tuned structural and physicochemical properties for myriad applications. In order to rationally develop and design IL mixtures with the desired properties, a thorough understanding of the structural origins of their physicochemical properties and the thermodynamics of mixing needs to be developed. To elucidate the structural origins of the excess molar volume within IL mixtures containing ions with different alkyl chain lengths, 3 IL mixtures containing 1-alkyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide ILs have been explored in a joint small angle X-ray scattering (SAXS) and 129Xe NMR study. The apolar domains of the IL mixtures were shown to possess similar dimensions to the largest alkyl chain of the mixture with the size evolution determined by whether the shorter alkyl chain was able to interact with the apolar domain. 129Xe NMR results illustrated that the origin of excess molar volume in these mixtures was due to fluctuations within these apolar domains arising from alkyl chain mismatch, with the formation of a greater number of smaller voids within the IL structure. These results indicate that free volume effects for these types of mixtures can be predicted from simple considerations of IL structure and that the structural basis for the formation of excess molar volume in these mixtures is substantially different to IL mixtures formed of different types of ions.

13.
Biochemistry ; 57(43): 6228-6233, 2018 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-30265526

RESUMO

The ability of the amyloid-ß peptide to bind to redox active metals and act as a source of radical damage in Alzheimer's disease has been largely accepted as contributing to the disease's pathogenesis. However, a kinetic understanding of the molecular mechanism, which underpins this radical generation, has yet to be reported. Here we use a sensitive fluorescence approach, which reports on the oxidation state of the metal bound to the amyloid-ß peptide and can therefore shed light on the redox kinetics. We confirm that the redox goes via a low populated, reactive intermediate and that the reaction proceeds via the Component I coordination environment rather than Component II. We also show that while the reduction step readily occurs (on the 10 ms time scale) it is the oxidation step that is rate-limiting for redox cycling.


Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Cobre/química , Cobre/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Cinética , Oxirredução , Estresse Oxidativo
14.
J Am Chem Soc ; 140(45): 15384-15392, 2018 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-30351920

RESUMO

The reliable preparation of functional, ordered, nanostructured frameworks would be a game changer for many emerging technologies, from energy storage to nanomedicine. Underpinned by the excellent molecular recognition of nucleic acids, along with their facile synthesis and breadth of available functionalizations, DNA nanotechnology is widely acknowledged as a prime route for the rational design of nanostructured materials. Yet, the preparation of crystalline DNA frameworks with programmable structure and functionality remains a challenge. Here we demonstrate the potential of simple amphiphilic DNA motifs, dubbed "C-stars", as a versatile platform for the design of programmable DNA crystals. In contrast to all-DNA materials, in which structure depends on the precise molecular details of individual building blocks, the self-assembly of C-stars is controlled uniquely by their topology and symmetry. Exploiting this robust self-assembly principle, we design a range of topologically identical, but structurally and chemically distinct C-stars that following a one-pot reaction self-assemble into highly porous, functional, crystalline frameworks. Simple design variations allow us to fine-tune the lattice parameter and thus control the partitioning of macromolecules within the frameworks, embed responsive motifs that can induce isothermal disassembly, and include chemical moieties to capture target proteins specifically and reversibly.


Assuntos
DNA/química , Nanoestruturas/química , Tensoativos/química , Proteínas de Fluorescência Verde/química , Nanotecnologia , Conformação de Ácido Nucleico , Concentração Osmolar , Tamanho da Partícula
15.
Anal Chem ; 90(23): 13915-13921, 2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30395442

RESUMO

Microfluidics has become recognized as a powerful platform technology associated with a constantly increasing array of applications across the life sciences. This surge of interest over recent years has led to an increased demand for microfluidic chips, resulting in more time being spent in the cleanroom fabricating devices using soft lithography-a slow and expensive process that requires extensive materials, training and significant engineering resources. This bottleneck limits platform complexity as a byproduct of lengthy delays between device iterations and affects the time spent developing the final application. To address this problem, we report a new, rapid, and economical approach to microfluidic device fabrication using dry resist films to laminate laser cut sheets of acrylic. We term our method laser lithography and show that our technique can be used to engineer 200 µm width channels for assembling droplet generators capable of generating monodisperse water droplets in oil and micromixers designed to sustain chemical reactions. Our devices offer high transparency, negligible device to device variation, and low X-ray background scattering, demonstrating their suitability for real-time X-ray-based characterization applications. Our approach also requires minimal materials and apparatus, is cleanroom free, and at a cost of around $1.00 per chip could significantly democratize device fabrication, thereby increasing the interdisciplinary accessibility of microfluidics.

16.
Nano Lett ; 17(5): 3276-3281, 2017 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-28417635

RESUMO

Many emerging technologies require materials with well-defined three-dimensional nanoscale architectures. Production of these structures is currently underpinned by self-assembling amphiphilic macromolecules or engineered all-DNA building blocks. Both of these approaches produce restricted ranges of crystal geometries due to synthetic amphiphiles' simple shape and limited specificity, or the technical difficulties in designing space-filling DNA motifs with targeted shapes. We have overcome these limitations with amphiphilic DNA nanostructures, or "C-Stars", that combine the design freedom and facile functionalization of DNA-based materials with robust hydrophobic interactions. C-Stars self-assemble into single crystals exceeding 40 µm in size with lattice parameters exceeding 20 nm.


Assuntos
DNA/química , Nanoestruturas/química , Cristalização , Interações Hidrofóbicas e Hidrofílicas , Substâncias Macromoleculares/química , Conformação de Ácido Nucleico , Tamanho da Partícula
17.
Biophys J ; 113(6): 1200-1211, 2017 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-28801104

RESUMO

A persistent challenge in membrane biophysics has been to quantitatively predict how membrane physical properties change upon addition of new amphiphiles (e.g., lipids, alcohols, peptides, or proteins) in order to assess whether the changes are large enough to plausibly result in biological ramifications. Because of their roles as general anesthetics, n-alcohols are perhaps the best-studied amphiphiles of this class. When n-alcohols are added to model and cell membranes, changes in membrane parameters tend to be modest. One striking exception is found in the large decrease in liquid-liquid miscibility transition temperatures (Tmix) observed when short-chain n-alcohols are incorporated into giant plasma membrane vesicles (GPMVs). Coexisting liquid-ordered and liquid-disordered phases are observed at temperatures below Tmix in GPMVs as well as in giant unilamellar vesicles (GUVs) composed of ternary mixtures of a lipid with a low melting temperature, a lipid with a high melting temperature, and cholesterol. Here, we find that when GUVs of canonical ternary mixtures are formed in aqueous solutions of short-chain n-alcohols (n ≤ 10), Tmix increases relative to GUVs in water. This shift is in the opposite direction from that reported for cell-derived GPMVs. The increase in Tmix is robust across GUVs of several types of lipids, ratios of lipids, types of short-chain n-alcohols, and concentrations of n-alcohols. However, as chain lengths of n-alcohols increase, nonmonotonic shifts in Tmix are observed. Alcohols with chain lengths of 10-14 carbons decrease Tmix in ternary GUVs of dioleoyl-PC/dipalmitoyl-PC/cholesterol, whereas 16 carbons increase Tmix again. Gray et al. observed a similar influence of the length of n-alcohols on the direction of the shift in Tmix. These results are consistent with a scenario in which the relative partitioning of n-alcohols between liquid-ordered and liquid-disordered phases evolves as the chain length of the n-alcohol increases.


Assuntos
Álcoois/química , Membrana Celular/química , Temperatura de Transição , Lipossomas Unilamelares/química , Álcoois/farmacologia , Animais , Fenômenos Biomecânicos , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Colesterol/química , Colesterol/metabolismo , Microscopia , Fosfatidilcolinas/química , Ratos , Soluções , Água/química
18.
Biophys J ; 111(7): 1528-1540, 2016 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-27705775

RESUMO

The viscosity is a highly important parameter within the cell membrane, affecting the diffusion of small molecules and, hence, controlling the rates of intracellular reactions. There is significant interest in the direct, quantitative assessment of membrane viscosity. Here we report the use of fluorescence lifetime imaging microscopy of the molecular rotor BODIPY C10 in the membranes of live Escherichia coli bacteria to permit direct quantification of the viscosity. Using this approach, we investigated the viscosity in live E. coli cells, spheroplasts, and liposomes made from E. coli membrane extracts. For live cells and spheroplasts, the viscosity was measured at both room temperature (23°C) and the E. coli growth temperature (37°C), while the membrane extract liposomes were studied over a range of measurement temperatures (5-40°C). At 37°C, we recorded a membrane viscosity in live E. coli cells of 950 cP, which is considerably higher than that previously observed in other live cell membranes (e.g., eukaryotic cells, membranes of Bacillus vegetative cells). Interestingly, this indicates that E. coli cells exhibit a high degree of lipid ordering within their liquid-phase plasma membranes.


Assuntos
Membrana Celular/química , Microscopia de Fluorescência/métodos , Viscosidade , Algoritmos , Compostos de Boro , Membrana Celular/metabolismo , Difusão , Escherichia coli , Corantes Fluorescentes , Lipossomos/química , Lipossomos/metabolismo , Microscopia Confocal/métodos , Modelos Biológicos , Esferoplastos/química , Esferoplastos/metabolismo , Temperatura
19.
Biophys J ; 111(3): 537-545, 2016 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-27508437

RESUMO

Diverse molecules induce general anesthesia with potency strongly correlated with both their hydrophobicity and their effects on certain ion channels. We recently observed that several n-alcohol anesthetics inhibit heterogeneity in plasma-membrane-derived vesicles by lowering the critical temperature (Tc) for phase separation. Here, we exploit conditions that stabilize membrane heterogeneity to further test the correlation between the anesthetic potency of n-alcohols and effects on Tc. First, we show that hexadecanol acts oppositely to n-alcohol anesthetics on membrane mixing and antagonizes ethanol-induced anesthesia in a tadpole behavioral assay. Second, we show that two previously described "intoxication reversers" raise Tc and counter ethanol's effects in vesicles, mimicking the findings of previous electrophysiological and behavioral measurements. Third, we find that elevated hydrostatic pressure, long known to reverse anesthesia, also raises Tc in vesicles with a magnitude that counters the effect of butanol at relevant concentrations and pressures. Taken together, these results demonstrate that ΔTc predicts anesthetic potency for n-alcohols better than hydrophobicity in a range of contexts, supporting a mechanistic role for membrane heterogeneity in general anesthesia.


Assuntos
Álcoois/farmacologia , Anestesia , Microdomínios da Membrana/efeitos dos fármacos , Álcoois/química , Animais , Comportamento Animal/efeitos dos fármacos , Linhagem Celular Tumoral , Interações Hidrofóbicas e Hidrofílicas , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Ratos , Temperatura , Xenopus laevis
20.
Soft Matter ; 12(7): 2108-17, 2016 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-26758699

RESUMO

We investigate the structural organization of cholesterol (CHOL) analogues in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers using coarse-grained molecular dynamics simulations and the MARTINI forcefield. Different sterol molecules are modelled by increasing (CHOLL) or decreasing (CHOLS) the diameter of the sterol beads employed in the MARTINI model of CHOL. At high sterol concentrations, (xsterol = 0.5), typical of liquid ordered phases, we find that the sterol arrangement and sterol-DPPC interactions strongly depend on the sterol size. Smaller sterols (CHOLS and CHOL) form linear clusters, while the larger sterols (CHOLL) arrange themselves into disc shaped clusters. By combining structural and dynamical properties we also investigate the So→ Ld transition for the CHOLL and CHOLS sterols. We show that small changes in the sterol size significantly affect the stability of the gel phase with the gel phase stabilized by the small sterols, but destabilized by large sterols. The general dependence of the phase behaviour of the membrane with sterol content is reminiscent of the one observed in naturally occurring membranes. The relevance of our results to understand current cholesterol-bilayer structural models is discussed.


Assuntos
1,2-Dipalmitoilfosfatidilcolina/análogos & derivados , Colesterol/análogos & derivados , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , 1,2-Dipalmitoilfosfatidilcolina/química , Géis , Transição de Fase , Termodinâmica
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