We prepared highly oriented, multi-lamellar stacks of human red blood cell (RBC) membranes applied on silicon wafers. RBC ghosts were prepared by hemolysis and applied onto functionalized silicon chips and annealed into multi-lamellar RBC membranes. High resolution X-ray diffraction was used to determine the molecular structure of the stacked membranes. We present direct experimental evidence that these RBC membranes consist of nanometer sized domains of integral coiled-coil peptides, as well as liquid ordered (lo) and liquid disordered (ld) lipids. Lamellar spacings, membrane and hydration water layer thicknesses, areas per lipid tail and domain sizes were determined. The common drug aspirin was added to the RBC membranes and found to interact with RBC membranes and preferably partition in the head group region of the lo domain leading to a fluidification of the membranes, i.e., a thinning of the bilayers and an increase in lipid tail spacing. Our results further support current models of RBC membranes as patchy structures and provide unprecedented structural details of the molecular organization in the different domains.

Precise knowledge of the mechanical properties of emerging nanomaterials and nanocomposites is crucial to match their performance with suitable applications. While methods to characterize mechanical properties exist, they are limited by instrument sensitivity and sample requirements. For bio-based nanomaterials this challenge is exacerbated by the extreme dependence of mechanical properties on humidity. This work presents an alternative approach, based on polymer shrinking-induced wrinkling mechanics, to determine the elastic modulus of nanobiocomposite films in a humidity-independent manner. Layer-by-layer (LbL) films containing cellulose nanocrystals (CNCs) and water-soluble polymers were deposited onto pre-stressed polystyrene substrates followed by thermal shrinking, which wrinkled the films to give them characteristic topographies. Three deposition parameters were varied during LbL assembly: (1) polymer type (xyloglucan - XG, or polyethyleneimine - PEI); (2) polymer concentration (0.1 or 1 wt%); and (3) number of deposition cycles, resulting in 10-600 nm thick nanobiocomposite films with tuneable compositions. Fast Fourier transform analysis on electron microscopy images of the wrinkled films was used to calculate humidity-independent moduli of 70 ± 2 GPa for CNC-XG, 72 ± 2 GPa for CNC-PEI, and 32.2 ± 0.8 GPa for CNC-PEI films. This structuring method is straightforward and amenable to a wide range of supported thin films.

RNA is a linear polymer of nucleotides linked by a ribose-phosphate backbone. Polymerization of nucleotides occurs in a condensation reaction in which phosphodiester bonds are formed. However, in the absence of enzymes and metabolism there has been no obvious way for RNA-like molecules to be produced and then encapsulated in cellular compartments. We investigated 5′-adenosine monophosphate (AMP) and 5′-uridine monophosphate (UMP) molecules confined in multi-lamellar phospholipid bilayers, nanoscopic films, ammonium chloride salt crystals and Montmorillonite clay, previously proposed to promote polymerization. X-ray diffraction was used to determine whether such conditions imposed a degree of order on the nucleotides. Two nucleotide signals were observed in all matrices, one corresponding to a nearest neighbour distance of 4.6 Å attributed to nucleotides that form a disordered, glassy structure. A second, smaller distance of 3.4 Å agrees well with the distance between stacked base pairs in the RNA backbone, and was assigned to the formation of pre-polymers, i.e., the organization of nucleotides into stacks of about 10 monomers. Such ordering can provide conditions that promote the nonenzymatic polymerization of RNA strands under prebiotic conditions. Experiments were modeled by Monte-Carlo simulations, which provide details of the molecular structure of these pre-polymers.

We combine confocal imaging, microfluidics and image analysis to record 3D-images of cells in flow. This enables us to recover the full 3D representation of several hundred living cells per minute. Whereas 3D confocal imaging has thus far been limited to steady specimen, we overcome this restriction and present a method to access the 3D shape of moving objects. The key of our principle is a tilted arrangement of the micro-channel with respect to the focal plane of the microscope. This forces cells to traverse the focal plane in an inclined manner. As a consequence, individual layers of passing cells are recorded which can then be assembled to obtain the volumetric representation. The full 3D information allows for a detailed comparisons with theoretical and numerical predictions unfeasible with e.g.\ 2D imaging. Our technique is exemplified by studying flowing red blood cells in a micro-channel reflecting the conditions prevailing in the microvasculature. We observe two very different types of shapes: `croissants' and `slippers'. Additionally, we perform 3D numerical simulations of our experiment to confirm the observations. Since 3D confocal imaging of cells in flow has not yet been realized, we see high potential in the field of flow cytometry where cell classification thus far mostly relies on 1D scattering and fluorescence signals

Lateral organization of lipids in the cell membrane appears to be an ancient feature of the cell, given the existence of lipid rafts in both eukaryotic and prokaryotic cells. Currently seen as platforms for protein partitioning, we posit that lipid rafts are capable of playing another role: stabilizing membrane physical properties over varying temperatures and other environmental conditions. Membrane composition defines the mechanical and viscous properties of the bilayer. The composition also varies strongly with temperature, with systematic changes in the partitioning of high and low melting temperature membrane components. In this way, rafts function as buffers of membrane physical properties, progressively counteracting environmental changes via compositional changes; i.e., more high melting lipids partition to the fluid phase with increasing temperature, increasing the bending modulus and viscosity, as thermal effects decrease these same properties. To provide an example of this phenomenon, we have performed neutron scattering experiments and atomistic molecular dynamics simulations on a phase separated model membrane. The results demonstrate a buffering effect in both the lateral diffusion coefficient and the bending modulus of the fluid phase upon changing temperature. This demonstration highlights the potentially advantageous stabilizing effect of complex lipid compositions in response to temperature and potentially other membrane destabilizing environmental conditions.

The safe storage of blood is of fundamental importance to health care systems all over the world. Currently, plastic bags are used for the collection and storage of donated blood and are typically made of poly­(vinyl chloride) (PVC) plasticized with di-2-ethylhexyl phthalate (DEHP). DEHP is known to migrate into packed red blood cells (RBC) and has been found to extend their shelf life. It has been speculated that DEHP incorporates itself into the RBC membrane and alters membrane properties, thereby reducing susceptibility to hemolysis and morphological deterioration. Here, we used high-resolution X-ray diffraction and molecular dynamics (MD) simulations to study the interaction between DEHP and model POPC lipid membranes at high (9 mol %) and low (1 mol %) concentrations of DEHP. At both concentrations, DEHP was found to spontaneously partition into the bilayer. At high concentrations, DEHP molecules were found to aggregate in the aqueous phase before inserting as clusters into the membrane. The presence of DEHP in the bilayers resulted in subtle, yet statistically significant, alterations in several membrane properties in both the X-ray diffraction experiments and MD simulations. DEHP led to (1) an increase of membrane width and (2) an increase in the area per lipid. It was also found to (3) increase the deuterium order parameter, however, (4) decrease membrane orientation, indicating the formation of thicker, stiffer membranes with increased local curvature. The observed effects of DEHP on lipid bilayers may help to better understand its effect on RBC membranes in increasing the longevity of stored blood by improving membrane stability.

Blood banks around the world store blood components for several weeks ensuring its availability for transfusion medicine. Red blood cells (RBCs) are known to undergo compositional changes during storage, which may impact the cells’ function and eventually the recipients’ health. We extracted the RBC’s cytoplasmic membrane (RBCcm) to study the effect of storage on the membranes’ molecular structure and bending rigidity by a combination of X-ray diffraction (XRD), X-ray diffuse scattering (XDS) and coarse grained Molecular Dynamics (MD) simulations. Blood was stored in commercial blood bags for 2 and 5 weeks, respectively and compared to freshly drawn blood. Using mass spectrometry, we measured an increase of fatty acids together with a slight shift towards shorter tail lengths. We observe an increased fraction (6%) of liquid ordered (lo) domains in the RBCcms with storage time, and an increased lipid packing in these domains, leading to an increased membrane thickness and membrane order. The size of both, lo and liquid disordered (ld) lipid domains was found to decrease with increased storage time by up to 25%. XDS experiments reveal a storage dependent increase in the RBCcm’s bending modulus κ by a factor of 2.8, from 1.9 kBT to 5.3 kBT. MD simulations were conducted in the absence of proteins. The results show that the membrane composition has a small contribution to the increased bending rigidity and suggests additional protein-driven mechanisms.

The formation of amyloid-β plaques is one of the hallmarks of Alzheimer’s disease. The presence of an amphiphatic cell membrane can accelerate the formation of amyloid-β aggregates, making it a potential druggable target to delay the progression of Alzheimer’s disease. We have prepared unsaturated anionic membranes made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS) and added the trans-membrane segment Aβ25–35. Peptide plaques spontaneously form in these membranes at high peptide concentrations of 20 mol%, which show the characteristic cross-β motif (concentrations are relative to the number of membrane lipids and indicate the peptide-to-lipid ratio). We used atomic force microscopy, fluorescence microscopy, x-ray microscopy, x-ray diffraction, UV-vis spectroscopy and Molecular Dynamics (MD) simulations to study three membrane-active molecules which have been speculated to have an effect in Alzheimer’s disease: melatonin, acetylsalicyclic acid (ASA) and curcumin at concentrations of 5 mol% (drug-to-peptide ratio). Melatonin did not change the structural parameters of the membranes and did not impact the size or extent of peptide clusters. While ASA led to a membrane thickening and stiffening, curcumin made membranes softer and thinner. As a result, ASA was found to lead to the formation of larger peptide aggregates, whereas curcumin reduced the volume fraction of cross-β sheets by ~70%. We speculate that the interface between membrane and peptide cluster becomes less favorable in thick and stiff membranes, which favors the formation of larger aggregates, while the corresponding energy mismatch is reduced in soft and thin membranes. Our results present evidence that cross-β sheets of Aβ25–35 in anionic unsaturated lipid membranes can be re-dissolved by changing membrane properties to reduce domain mismatch.