Mridula Dwivedi

Size Influences the Effect of Hydrophobic Nanoparticles on Lung Surfactant Model Systems

The alveolar lung surfactant (LS) is a complex lipid protein mixture that forms an interfacial monolayer reducing the surface tension to near zero values and thus preventing the lungs from collapse. Due to the expanding field of nanotechnology and the corresponding unavoidable exposure of human beings from the air, it is crucial to study the potential effects of nanoparticles (NPs) on the structural organization of the lung surfactant system. In the present study, we investigated both, the domain structure in pure DPPC monolayers as well as in lung surfactant model systems. In the pure lipid system we found that two different sized hydrophobic polymeric nanoparticles with diameter of ~12 nm and ~136 nm have contrasting effect on the functional and structural behavior. The small nanoparticles inserted into fluid domains at the LE-LC phase transition are not visibly disturbing the phase transition but disrupting the domain morphology of the LE phase. The large nanoparticles led to an expanded isotherm and to a significant decrease in the line tension and thus to a drastic disruption of the domain structures at a much lower number of nanoparticles with respect to the lipid. The surface activity of the model LS films again showed drastic variations due to presence of different sized NPs illustrated by the film balance isotherms and the atomic force microscopy. AFM revealed laterally profuse multilayer protrusion formation on compression but only in the presence of 136 nm sized nanoparticles. Moreover we investigated the vesicle insertion process into a preformed monolayer. A severe inhibition was observed only in the presence of ~136 nm NPs compared to minor effects in the presence of ~12 nm NPs. Our study clearly shows that the size of the nanoparticles made of the same material determines the interaction with biological membranes.

Biophysical investigations of the structure and function of the tear fluid lipid layer and the effect of ectoine. Part A: Natural meibomian lipid films

The tear fluid lipid layer is present at the outermost part of the tear film which lines the ocular surface and functions to maintain the corneal surface moist by retarding evaporation. Instability in the structure of the tear fluid lipid layer can cause an increased rate of evaporation and thus dry eye syndrome. Ectoine has been previously shown to fluidize lipid monolayers and alter the phase behavior. In the current study we have investigated the effect of ectoine on the artificial tear fluid lipid layer composed of binary and ternary lipid mixtures of dipalmitoyl phosphatidylcholine (DPPC), cholesteryl esters and tri-acyl-glycerols. The focus of our study was mainly the structural and the biophysical aspects of the artificial tear fluid lipid layer using surface activity studies and topology analysis. The presence of ectoine consistently causes an expansion of the pressure-area isotherm indicating increased intermolecular spacing. The topology studies showed the formation of droplet-like structures due to the addition of ectoine only when tri-acyl-glycerol is present in the mixture of DPPC and chol-palmitate, similar to the natural meibomian lipids. Consequently, the hypothesis of an exclusion of tri/di-acyl-glycerol from the meibomian lipid film in the presence of ectoine in the subphase is confirmed. A model describing the effect of ectoine on meibomian lipid films is further presented which may have an application for the use of ectoines in eye drops as a treatment for the dry eye syndrome.

Influence of isoform-specific Ras lipidation motifs on protein partitioning and dynamics in model membrane systems of various complexity

Abstract The partitioning of the lipidated signaling proteins N-Ras and K-Ras4B into various membrane systems, ranging from single-component fluid bilayers, binary fluid mixtures, heterogeneous raft model membranes up to complex native-like lipid mixtures (GPMVs) in the absence and presence of integral membrane proteins have been explored in the last decade in a combined chemical-biological and biophysical approach. These studies have revealed pronounced isoform-specific differences regarding the lateral distribution in membranes and formation of protein-rich membrane domains. In this context, we will also discuss the effects of lipid head group structure and charge density on the partitioning behavior of the lipoproteins. Moreover, the dynamic properties of N-Ras and K-Ras4B have been studied in different model membrane systems and native-like crowded milieus. Addition of crowding agents such as Ficoll and its monomeric unit, sucrose, gradually favors clustering of Ras proteins in forming small oligomers in the bulk; only at very high crowder concentrations association is disfavored.

Binding of Vinculin to Lipid Membranes in Its Inhibited and Activated States

Phosphoinositols are an important class of phospholipids that are involved in a myriad of cellular processes, from cell signaling to motility and adhesion. Vinculin (Vn) is a major adaptor protein that regulates focal adhesions in conjunction with PIP2 in lipid membranes and other cytoskeletal components. The binding and unbinding transitions of Vn at the membrane interface are an important link to understanding the coordination of cell signaling and motility. Using different biophysical tools, including atomic force microscopy combined with confocal fluorescence microscopy and Fourier transform infrared spectroscopy, we studied the nanoscopic interactions of activated and autoinhibited states of Vn with lipid membranes. We hypothesize that a weak interaction occurs between Vn and lipid membranes, which leads to binding of autoinhibited Vn to supported lipid bilayers, and to unbinding in freestanding lipid vesicles. Likely driving forces may include tethering of the C-terminus to the lipid membrane, as well as hydrophobic helix-membrane interactions. Conversely, activated Vn binds strongly to membranes through specific interactions with clusters of PIP2 embedded in lipid membranes. Activated Vn harbored on PIP2 clusters may form small oligomeric interaction platforms for further interaction partners, which is necessary for the proper function of focal adhesion points.

UNC119A decreases the membrane-binding of myristoylated c-Src

Plasma membrane localization of myristoylated c‐Src, a proto‐oncogene protein‐tyrosine kinase, is required for its signaling activity. Recent studies proposed that UNC119 protein functions as a solubilizing factor for myristoylated proteins, thereby regulating their subcellular distribution and signaling. The underlying molecular mechanism by which UNC119 regulates the membrane binding of c‐Src has remained elusive. By combining different biophysical techniques, we have found that binding of a myristoylated c‐Src‐derived N‐terminal peptide (Myr‐Src) by UNC119A results in a reduced membrane binding affinity of the peptide, due to the competition of binding to membranes. The dissociation of Myr‐Src from membranes is facilitated in the presence of UNC119A, as a consequence of which the clustering propensity of this peptide on the membrane is partially impaired. By these means, UNC119A is able to regulate c‐Src spatially in the cytoplasm and on cellular membranes, and this has important implications for its cellular signaling.

Interaction of Nanoparticles with Lipid Monolayers and Lung Surfactant Films

It has been shown that the interactions of nanoparticles with lipid and lipid–peptide monolayers mimicking the lung surfactant strongly depend on the physical properties of the nanoparticles, their size, and on the physical properties of the surface film. Hydrophobic nanoparticles have been found inserting into fluid phases of lipid monolayers. They have an adverse effect on the functional properties of the pulmonary surfactant, which strongly depends on the nanoparticle size. But how NPs disturb or inhibit this surfactant function still remains unclear. Experimental evidences gathered under physiologically relevant conditions or from in vivo studies are still lacking. The present review summarizes systematic investigations on simplified model systems of the lung surfactant using high-resolution bioanalytical techniques that have provided valuable hints and indications about the interactions of NPs with the surfactant layer at the molecular level. Further studies are needed in particular for a more detailed understanding of the mechanism by which NPs are capable of crossing the surfactant barrier even though they experience a very different and individual free energy barrier at the interface.

3D Molecular ToF-SIMS Imaging of Artificial Lipid Membranes Using a Discriminant Analysis-Based Algorithm

Artificial lipid membranes play a growing role in technical applications such as biosensors in pharmacological research and as model systems in the investigation of biological lipid films. In the standard procedure for displaying the distribution of membrane components, fluorescence microscopy, the fluorophores used can influence the distribution of the components and usually not all substances can be displayed at the same time. The discriminant analysis-based algorithm used in combination with scanning time-of-flight secondary ion mass spectrometry (ToF-SIMS) enables marker-free, quantitative, simultaneous recording of all membrane components. These data are used for reconstruction of distribution patterns. In the model system used for this survey, a tear fluid lipid layer, the distribution patterns of all lipids correlate well in calculated ToF-SIMS images and epi-fluorescence microscopic images. All epi-fluorescence microscopically viewable structures are visible when using both positive and negative secondary ions and can be reproduced with high lateral resolution in the submicrometer range despite the very low signal intensity and a very low signal-to-noise ratio. In addition, three-dimensional images can be obtained with a subnanometer depth resolution. Furthermore, structures and the distribution of substances that cannot be made visible by epi-fluorescence microscopy can be displayed. This enables new insights that cannot be gained by epi-fluorescence microscopy alone.

The Effect of Ectoins on the Structural Organization of the Tear Fluid Monolayer

We investigate the influence of Ectoin on the structural organization of the natural and artificial tear fluid lipid layers (ATFLL) using surface activity analysis and topographical studies. The natural meibomian lipids exhibit a continuous pressure-area isotherm without any phase transitions. In the presence of ectoin, the isotherm is expanded towards higher area per molecule implying decreased interaction between the lipid molecules. The AFM scans show presence of fiber like structures in the natural meibomian lipid film. In the presence of ectoine, droplet-like structures are observed which are hypothesized to be triacyl glycerols excluded from the lipid film. ATFLL illustrate the fluidizing effect of ectoine on the lipid films where the pressure-area isotherms are expanded in the presence of ectoin. With the addition of a triacyl glycerol to the mixture of DPPC and Chol-Palmitate, we observed the formation of similar drop-like structures in the presence of ectoine as in the case of natural meibomian lipid films. Consequently, the hypothesis explaining the exclusion of tri/di acyl glycerol from the meibomian lipid film in the presence of ectoine in the subphase is confirmed which lead us to a model describing the fluidizing effect of ectoine on meibomian lipid films.