Location of dopamine in lipid bilayers and its relevance to neuromodulator function.

Dopamine (DA) is a neurotransmitter that also acts as a neuromodulator, with both functions being essential to brain function. Here, we present the first experimental measurement of DA location in lipid bilayers using x-ray diffuse scattering, solid-state deuterium NMR, and electron paramagnetic resonance. We find that the association of DA with lipid headgroups as seen in electron density profiles leads to an increase of intermembrane repulsion most likely due to electrostatic charging. DA location in the lipid headgroup region also leads to an increase of the cross-sectional area per lipid without affecting the bending rigidity significantly. The order parameters measured by solid-state deuterium NMR decrease in the presence of DA for the acyl chains of PC and PS lipids, consistent with an increase in the area per lipid due to DA. Most importantly, these results support the hypothesis that three-dimensional diffusion of DA to target membranes could be followed by relatively more efficient two-dimensional diffusion to receptors within those membranes.

Visualization of human optic nerve by diffusion tensor mapping and degree of neuropathy.

Diffusion-weighted magnetic resonance imaging of the human optic nerve and tract is technically difficult because of its small size, the inherent strong signal generated by the surrounding fat and the cerebrospinal fluid, and due to eddy current-induced distortions and subject movement artifacts. The effects of the bone canal through which the optic nerve passes, and the proximity of blood vessels, muscles and tendons are generally unknown. Also, the limited technical capabilities of the scanners and the minimization of acquisition times result in poor quality diffusion-weighted images. It is challenging for current tractography methods to accurately track optic pathway fibers that correspond to known anatomy. Despite these technical limitations and low image resolution, here we show how to visualize the optic nerve and tract and quantify nerve atrophy. Our visualization method based on the analysis of the diffusion tensor shows marked differences between a healthy male subject and a male subject with progressive optic nerve neuropathy. These differences coincide with diffusion scalar metrics and are not visible on standard morphological images. A quantification of the degree of optic nerve atrophy in a systematic way is provided and it is tested on 9 subjects from the Human Connectome Project.

Correction: Flexible lipid nanomaterials studied by NMR spectroscopy.

Correction for 'Flexible lipid nanomaterials studied by NMR spectroscopy' by K. J. Mallikarjunaiah et al., Phys. Chem. Chem. Phys., 2019, 21, 18422-18457, DOI: .

Biofabrication of spheroids fusion-based tumor models: computational simulation of glucose effects.

In vitrotumor models consisting of cell spheroids are increasingly used for mechanistic studies and pharmacological testing. However, unless vascularized, the availability of nutrients such as glucose to deeper layers of multicellular aggregates is limited. In addition, recent developments in cells-only biofabrication (e.g. 'scaffold-free bioprinting'), allow the creation of more complex spheroid-based structures, further exposing the cells to nutrient deprivation within these constructs. To explore the impact of glucose availability on such tumor-like structures, we used the CompuCell3D platform for modeling of tumor spheroids. By monitoring the types of cells, fusing pairs geometry and the distance between spheroids centers of mass, we made novel heuristic observations on how binary- and multi-spheroid fusions are impacted by glucose availability. At limiting glucose concentrations mimicking hypoglycemia we noted an abrupt collapse of the tumor spheroids, unexpectedly amplified by the contact with normal cell spheroids. At higher glucose concentrations, we found an increased intermixing of cancerous cells, strong anti-phase oscillations between proliferating and quiescent tumor cells and a structural instability of fusing tumor spheroids, leading to their re-fragmentation. In a model of tumor microenvironment composed of normal cell spheroids fusing around a tumoral one, the competition for glucose lead to either the tumor's disappearance, to a steady state, or to its expansion. Moreover, the invasion of this microenvironment by individual tumor cells was also strongly depended on the available glucose. In conclusion, we demonstrate the value of computational simulations for anticipating the properties of biofabricated tumor models, and in generating testable hypotheses regarding the relationship between cancer, nutrition and diabetes.

Vitamin E Promotes the Inverse Hexagonal Phase via a Novel Mechanism: Implications for Antioxidant Role.

Vitamin E (α-tocopherol) and a range of other biological compounds have long been known to promote the HII (inverted hexagonal) phase in lipids. Now, it has been well established that purely hydrophobic lipids such as dodecane promote the HII phase by relieving extensive packing stress. They do so by residing deep within the hydrocarbon core. However, we argue from X-ray diffraction data obtained with 1-palmitoyl-2-oleoylphosphatidylcholine (POPE) and 1,2-dioleoylphosphatidylcholine (DOPE) that α-tocopherol promotes the HII phase by a different mechanism. The OH group on the chromanol moiety of α-tocopherol anchors it near the aqueous interface. This restriction combined with the relatively short length of α-tocopherol (as compared to DOPE and POPE) means that α-tocopherol promotes the HII phase by relieving compressive packing stress. This observation offers new insight into the nature of packing stress and lipid biophysics. With the deeper understanding of packing stress offered by our results, we also explore the role that molecular structure plays in the primary function of vitamin E, which is to prevent the oxidation of polyunsaturated membrane lipids.

Flexible lipid nanomaterials studied by NMR spectroscopy.

Our review addresses how material properties emerge from atomistic-level interactions in the case of lipid membrane nanostructures. We summarize advances in solid-state nuclear magnetic resonance (NMR) spectroscopy in conjunction with alternative small-angle X-ray and neutron scattering methods for investigating lipid flexibility and dynamics. Solid-state 2H NMR is advantageous in that it provides atomistically resolved information about the order parameters and mobility of phospholipids within liquid-crystalline membranes. Bilayer deformation in response to external perturbations occurs over a range of length scales and allows one to disentangle how the bulk material properties emerge from atomistic forces. Examples include structural parameters such as the area per lipid and volumetric thickness together with the moduli for elastic deformation. Membranes under osmotic stress allow one to further distinguish collective undulations and quasielastic contributions from short-range noncollective effects. Our approach reveals how membrane elasticity involves length scales ranging from the bilayer dimensions on down to the size of the flexible lipid segments. Collective lipid interactions of the order of the bilayer thickness and less occur in the liquid-crystalline state. Emergence of lipid material properties is significant for models of lipid-protein forces acting on the mesoscopic length scale that play key roles in biomembrane functions.

Reorganization of Ternary Lipid Mixtures of Nonphosphorylated Phosphatidylinositol Interacting with Angiomotin.

Phosphatidylinositol (PI) lipids are necessary for many cellular signaling pathways of membrane associated proteins, such as angiomotin (Amot). The Amot family regulates cellular polarity, growth, and migration. Given the low concentration of PI lipids in these membranes, it is likely that such protein-membrane interactions are stabilized by lipid domains or small lipid clusters. By small-angle X-ray scattering, we show that nonphosphorylated PI lipids induce lipid demixing in ternary mixtures of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), likely because of preferential interactions between the head groups of PE and PI. These results were obtained in the presence of buffer containing tris(hydroxymethyl)aminomethane, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, NaCl, ethylenediaminetetraacetic acid, dithiothreitol, and benzamidine at pH 8.0 that in previous work showed an ability to cause PC to phase separate but are necessary to stabilize Amot for in vitro experimentation. Collectively, this provided a framework for determining the effect of Amot on lipid organization. Using fluorescence spectroscopy, we were able to show that the association of Amot with this lipid platform causes significant reorganization of the lipid into a more homogenous structure. This reorganization mechanism could be the basis for Amot membrane association and fusogenic activity previously described in the literature and should be taken into consideration in future protein-membrane interaction studies.

Cation-Selective Channel Regulated by Anions According to Their Hofmeister Ranking.

Specificity of small ions, the Hofmeister ranking, is long-known and has many applications including medicine. Yet it evades consistent theoretical description. Here we study the effect of Hofmeister anions on gramicidin A channels in lipid membranes. Counterintuitively, we find that conductance of this perfectly cation-selective channel increases about two-fold in the H2 PO4-

Direct affinity of dopamine to lipid membranes investigated by Nuclear Magnetic Resonance spectroscopy.

Dopamine, a naturally occurring neurotransmitter, plays an important role in the brain's reward system and acts on sensory receptors in the brain. Neurotransmitters are contained in lipid membraned vesicles and are released by exocytosis. All neurotransmitters interact with transport and receptor proteins in glial cells, on neuronal dendrites, and at the axonal button, and also must interact with membrane lipids. However, the extent of direct interaction between lipid membranes in the absence of receptors and transport proteins has not been extensively investigated. In this report, we use UV and NMR spectroscopy to determine the affinity and the orientation of dopamine interacting with lipid vesicles made of either phosphatidylcholine (PC) or phosphatidylserine (PS) lipids which are primary lipid components of synaptic vesicles. We quantify the interaction of dopamine's aromatic ring with lipid membranes using our newly developed method that involves reference spectra in hydrophobic environments. Our measurements show that dopamine interacts with lipid membranes primarily through the aromatic side opposite to the hydroxyl groups, with this aromatic side penetrating deeper into the hydrophobic region of the membrane. Since dopamine's activity involves its release into extracellular space, we have used our method to also investigate dopamine's release from lipid vesicles. We find that dopamine trapped inside PC and PS vesicles is released into the external solution despite its affinity to membranes. This result suggests that dopamine's interaction with lipid membranes is complex and involves both binding as well as permeation through lipid bilayers, a combination that could be an effective trigger for apoptosis of dopamine-generating cells.

Inferring Models of Bacterial Dynamics toward Point Sources.

Experiments have shown that bacteria can be sensitive to small variations in chemoattractant (CA) concentrations. Motivated by these findings, our focus here is on a regime rarely studied in experiments: bacteria tracking point CA sources (such as food patches or even prey). In tracking point sources, the CA detected by bacteria may show very large spatiotemporal fluctuations which vary with distance from the source. We present a general statistical model to describe how bacteria locate point sources of food on the basis of stochastic event detection, rather than CA gradient information. We show how all model parameters can be directly inferred from single cell tracking data even in the limit of high detection noise. Once parameterized, our model recapitulates bacterial behavior around point sources such as the "volcano effect". In addition, while the search by bacteria for point sources such as prey may appear random, our model identifies key statistical signatures of a targeted search for a point source given any arbitrary source configuration.

Equivalent Isopropanol Concentrations of Aromatic Amino Acids Interactions with Lipid Vesicles.

We show that the interaction of aromatic amino acids with lipid bilayers can be characterized by conventional 1D [Formula: see text]H NMR spectroscopy using reference spectra obtained in isopropanol-d8/D[Formula: see text]O solutions. We demonstrate the utility of this method with three different peptides containing tyrosine, tryptophan, or phenylalanine amino acids in the presence of 1,2-dioleoyl-sn-glycero-3-phosphocholine or 1,2-dioleoyl-sn-glycero-3-phosphoserine lipid membranes. In each case, we determine an equivalent isopropanol concentration (EIC) for each hydrogen site of aromatic groups, in essence constructing a map of the chemical environment. These EIC maps provide information on relative affinities of aromatic side chains for either PC or PS bilayers and also inform on amino acid orientation preference when bound to membranes.

Elastic deformation and area per lipid of membranes: atomistic view from solid-state deuterium NMR spectroscopy.

This article reviews the application of solid-state ²H nuclear magnetic resonance (NMR) spectroscopy for investigating the deformation of lipid bilayers at the atomistic level. For liquid-crystalline membranes, the average structure is manifested by the segmental order parameters (SCD) of the lipids. Solid-state ²H NMR yields observables directly related to the stress field of the lipid bilayer. The extent to which lipid bilayers are deformed by osmotic pressure is integral to how lipid-protein interactions affect membrane functions. Calculations of the average area per lipid and related structural properties are pertinent to bilayer remodeling and molecular dynamics (MD) simulations of membranes. To establish structural quantities, such as area per lipid and volumetric bilayer thickness, a mean-torque analysis of ²H NMR order parameters is applied. Osmotic stress is introduced by adding polymer solutions or by gravimetric dehydration, which are thermodynamically equivalent. Solid-state NMR studies of lipids under osmotic stress probe membrane interactions involving collective bilayer undulations, order-director fluctuations, and lipid molecular protrusions. Removal of water yields a reduction of the mean area per lipid, with a corresponding increase in volumetric bilayer thickness, by up to 20% in the liquid-crystalline state. Hydrophobic mismatch can shift protein states involving mechanosensation, transport, and molecular recognition by G-protein-coupled receptors. Measurements of the order parameters versus osmotic pressure yield the elastic area compressibility modulus and the corresponding bilayer thickness at an atomistic level. Solid-state ²H NMR thus reveals how membrane deformation can affect protein conformational changes within the stress field of the lipid bilayer.

Phase coexistence in single-lipid membranes induced by buffering agents.

Recent literature has shown that buffers affect the interaction between lipid bilayers through a mechanism that involves van der Waals forces, electrostatics, hydration forces and membrane bending rigidity. This letter shows an additional peculiar effect of buffers on the mixed chain 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayers, namely phase coexistence similar to what was reported by Rappolt et al. for alkali chlorides. The data presented suggest that one phase appears to dehydrate below the value in pure water, while the other phase swells as the concentration of buffer is increased. However, since the two phases must be in osmotic equilibrium with one another, this behavior challenges theoretical models of lipid interactions.

Effects of lipid interactions on model vesicle engulfment by alveolar macrophages.

The engulfment function of macrophages relies on complex molecular interactions involving both lipids and proteins. In particular, the clearance of apoptotic bodies (efferocytosis) is enabled by externalization on the cell target of phosphatidylserine lipids, which activate receptors on macrophages, suggesting that (local) specific lipid-protein interactions are required at least for the initiation of efferocytosis. However, in addition to apoptotic cells, macrophages can engulf foreign bodies that vary substantially in size from a few nanometers to microns, suggesting that nonspecific interactions over a wide range of length scales could be relevant. Here, we use model lipid membranes (made of phosphatidylcholine, phosphatidylserine, and ceramide) and rat alveolar macrophages to show how lipid bilayer properties probed by small-angle x-ray scattering and solid-state (2)H NMR correlate with engulfment rates measured by flow cytometry. We find that engulfment of protein-free model lipid vesicles is promoted by the presence of phosphatidylserine lipids but inhibited by ceramide, in accord with a previous study of apoptotic cells. We conclude that the roles of phosphatidylserine and ceramide in phagocytosis is based, at least in part, on lipid-mediated modification of membrane physical properties, including interactions at large length scales as well as local lipid ordering and possible domain formation.

Elastic properties of polyunsaturated phosphatidylethanolamines influence rhodopsin function.

Membranes with a high content of polyunsaturated phosphatidylethanolamines (PE) facilitate formation of metarhodopsin-II (M(II)), the photointermediate of bovine rhodopsin that activates the G protein transducin. We determined whether M(II)-formation is quantitatively linked to the elastic properties of PEs. Curvature elasticity of monolayers of the polyunsaturated lipids 18 : 0-22 : 6(n - 3)PE, 18 : 0-22 : 5(n)- 6PE and the model lipid 18 : 1(n - 9)-18 : 1,(n- 9)PE were investigated in the inverse hexagonal phase. All three lipids form lipid monolayers with rather low spontaneous radii of curvature of 26-28 angstroms. In membranes, all three PEs generate high negative curvature elastic stress that shifts the equilibrium of MI(I)/M(II) photointermediates of rhodopsin towards M(II) formation.

Functional effects of nanoparticle exposure on Calu-3 airway epithelial cells.

High concentrations of manufactured carbon nanoparticles (CNP) are known to cause oxidative stress, inflammatory responses and granuloma formation in respiratory epithelia. To examine the effects of lower, more physiologically relevant concentrations, the human airway epithelial cell line, Calu-3, was used to evaluate potential alterations in transepithelial permeability and cellular function of airway epithelia after exposure to environmentally realistic concentrations of carbon nanoparticles. Three common carbon nanoparticles, fullerenes, single- and multi-wall carbon nanotubes (SWCNT, MWCNT) were used in these experiments. Electrophysiological measurements were performed to assay transepithelial electrical resistance (TEER) and epinephrine-stimulated chloride (Cl(-)) ion secretion of epithelial cell monolayers that had been exposed to nanoparticles for three different times (1 h, 24 h and 48 h) and over a 7 log unit range of concentrations. Fullerenes did not have any effect on the TEER or stimulated ion transport. However, the carbon nanotubes (CNT) significantly decreased TEER and inhibited epinephrine-stimulated Cl(-) secretion. The changes were time dependent and at more chronic exposures caused functional effects which were evident at concentrations substantially lower than have been previously examined. The functional changes manifested in response to physiologically relevant exposures would inhibit mucociliary clearance mechanisms and compromise the barrier function of airway epithelia.

Effect of cigarette smoke exposure and structural modifications on the α-1 Antitrypsin interaction with caspases.

α-1 Antitrypsin (A1AT) is a serpin with a major protective effect against cigarette smoke-induced emphysema development, and patients with mutations of the A1AT gene display a markedly increased risk for developing emphysema. We reported that A1AT protects lung endothelial cells from apoptosis and inhibits caspase-3 activity. It is not clear if cigarette smoking or A1AT mutations alter the caspase-3 inhibitory activity of A1AT and if this serpin alters the function of other caspases. We tested the hypothesis that the caspase-3 inhibitory activity of A1AT is impaired by cigarette smoking and that the A1AT RCL, the key antiprotease domain of the serpin, is required for its interaction with the caspase. We examined the caspase-3 inhibitory activity of human A1AT purified from plasma of actively smoking and nonsmoking individuals, either affected or unaffected with chronic obstructive pulmonary disease. We also tested the caspase inhibitory activity of two mutant forms of A1AT, the recombinant human piZZ and the RCL-deleted (RCL-null) A1AT forms. A1AT purified from the blood of active smokers exhibited marked attenuation in its caspase-3 inhibitory activity, independent of disease status. In vitro exposure of the normal (MM) form of A1AT to cigarette smoke extract reduced its ability to interact with caspase-3, measured by isothermal titration calorimetry, as did the deletion of the RCL, but not the ZZ point mutation. In cell-free assays A1AT was capable of inhibiting all executioner caspases, -3, -7 and especially -6, but not the initiator or inflammatory caspases. The inhibitory effect of A1AT against caspase-6 was tested in vivo, where overexpression of both human MM and ZZ-A1AT via adeno-associated virus transduction significantly protected against apoptosis and against airspace damage induced by intratracheal instillation of caspase-6 in mice. These data indicate a specific inhibitory effect of A1AT on executioner caspases, which is profoundly attenuated by active exposure to cigarette smoking and is dependent on the protein RCL, but is not affected by the PiZZ mutation.

Electrodynamics of lipid membrane interactions in the presence of zwitterionic buffers.

Due to thermal motion and molecular polarizability, electrical interactions in biological systems have a dynamic character. Zwitterions are dipolar molecules that typically are highly polarizable and exhibit both a positive and a negative charge depending on the pH of the solution. We use multilamellar structures of common lipids to identify and quantify the effects of zwitterionic buffers that go beyond the control of pH. We use the fact that the repeat spacing of multilamellar lipid bilayers is a sensitive and accurate indicator of the force balance between membranes. We show that common buffers can in fact charge up neutral membranes. However, this electrostatic effect is not immediately recognized because of the concomitant modification of dispersion (van der Waals) forces. We show that although surface charging can be weak, electrostatic forces are significant even at large distances because of reduced ionic screening and reduced van der Waals attraction. The zwitterionic interactions that we identify are expected to be relevant for interfacial biological processes involving lipid bilayers, and for a wide range of biomaterials, including amino acids, detergents, and pharmaceutical drugs. An appreciation of zwitterionic electrodynamic character can lead to a better understanding of molecular interactions in biological systems and in soft materials in general.

Composite polarizability and the construction of an invariant function of refraction and mass density for solutions.

Re-examination of dynamical ionic polarizabilities in water solutions leads to the formulation of a solution function r(c), which combines the indices of refraction and mass densities of solutions. We show that this function should be independent of ionic concentration if the composite polarizabilities of hydrated solute clusters are constant. Using existing experimental data for a number of aqueous salt and organic solutions, we find that the r(c) function is either constant or varies linearly with concentration, in most cases with negligible slope. We use this function to compare ionic polarizabilities of crystals and aqueous solutions and to highlight how solute polarizabilities at infinite dilution scale with the electronic valence shell of cations and anions. The proposed r(c) function can be used generally to verify the consistency of experimental measurements and of simulation results, and it provides a test of assumptions in current theories of ionic polarizabilities.

Solid-state ²H NMR shows equivalence of dehydration and osmotic pressures in lipid membrane deformation.

Lipid bilayers represent a fascinating class of biomaterials whose properties are altered by changes in pressure or temperature. Functions of cellular membranes can be affected by nonspecific lipid-protein interactions that depend on bilayer material properties. Here we address the changes in lipid bilayer structure induced by external pressure. Solid-state ²H NMR spectroscopy of phospholipid bilayers under osmotic stress allows structural fluctuations and deformation of membranes to be investigated. We highlight the results from NMR experiments utilizing pressure-based force techniques that control membrane structure and tension. Our ²H NMR results using both dehydration pressure (low water activity) and osmotic pressure (poly(ethylene glycol) as osmolyte) show that the segmental order parameters (S(CD)) of DMPC approach very large values of ≈ 0.35 in the liquid-crystalline state. The two stresses are thermodynamically equivalent, because the change in chemical potential when transferring water from the interlamellar space to the bulk water phase corresponds to the induced pressure. This theoretical equivalence is experimentally revealed by considering the solid-state ²H NMR spectrometer as a virtual osmometer. Moreover, we extend this approach to include the correspondence between osmotic pressure and hydrostatic pressure. Our results establish the magnitude of the pressures that lead to significant bilayer deformation including changes in area per lipid and volumetric bilayer thickness. We find that appreciable bilayer structural changes occur with osmotic pressures in the range of 10-100 atm or lower. This research demonstrates the applicability of solid-state ²H NMR spectroscopy together with bilayer stress techniques for investigating the mechanism of pressure sensitivity of membrane proteins.