Diffusion of Myelin Oligodendrocyte Glycoprotein in Living OLN-93 Cells Investigated by Raster-Scanning Image Correlation Spectroscopy (RICS)

Many membrane proteins and lipids are partially confined in substructures ranging from tens of nanometers to micrometers in size. Evidence for heterogeneities in the membrane of oligodendrocytes, i.e. the myelin-producing cells of the central nervous system, is almost exclusively based on detergent methods. However, as application of detergents can alter the membrane phase behaviour, it is important to investigate membrane heterogeneities in living cells. Here, we report on the first investigations of the diffusion behavior of the myelin-specific protein MOG (myelin oligodendrocyte glycoprotein) in OLN-93 as studied by the recently developed RICS (raster-scanning image correlation spectroscopy) technique. We implemented RICS on a standard confocal laser-scanning microscope with one-photon excitation and analog detection. Measurements on FITC-dextran were used to evaluate the performance of the system and the data analysis procedure. Ellen Gielen and Nick Smisdom contributed equally to this work.     

The impact of the modified Poisson–Boltzmann model on protein bound to a lipid coated silicon nanowire field effect transistor biosensor in an electrolyte environment

The aim of this work was to analyse the electrostatic potential profile, various effects of electrolyte concentrations, and the influences of surface charge on a protein bound to a lipid coated Silicon nanowire field effect transistor (Si-NW FET) biosensor by implementing the modified Poisson–Boltzmann (MPB) model. In this work, we modelled a lipid monolayer-coated Si-NW FET for the sensing of proteins, which consisted of variable amounts of aspartic acid. The electrostatic potential profile, protein charge distributions, the response to various electrolyte concentration, and the impacts of various surface charge were studied by implementing the MPB model with the Si-NW FET biosensor. Additionally, a comparison between the use of the MPB and the Poisson–Boltzmann model in studying the effects of various surface charges was carried out. Taken together, it was found that the MPB model showed a higher resolution in studying the Si-NW FET biosensor model when higher concentrations and surface charges were administered.     

Dynamics of topological defects in the Lβ′ phase of 1,2-dipalmitoylphosphatidycholine bilayers

We use the optical birefringence of 1,2-dipalmitoylphosphatidycholine bilayers (DPPC) in the gel (L_β′) phase to study recombination dynamics of topological defects. The birefringence of anisotropic thin films, such as the L_β′ phase of DPPC bilayers, is related to their molecular polarizability, different on the heads and the acyl chains. When the sample is cooled down into the L_β′ phase, a period of rapid recombination (taking place over a few seconds) is followed by slow dynamics with metastable states existing in excess of several minutes. After this, either another metastable state or a truly stable state remains where no further change is observed, although a spatially non-uniform distribution of the orientation of the birefringence remains. We compare our results with a model for the free energy and the dynamics of the lipid bilayer in the gel state, finding good qualitative agreement.     

Monitoring membrane rafts in colorectal cancer cells by means of correlative fluorescence electron microscopy (CFEM)

Detergent-resistant membrane (DRM) rafts have been shown to play a pivotal role in regulating key cell biological processes, such as signal transduction, cellular transport and cell survival. The fine structure of membrane rafts are studied using various different imaging approaches and the outcomes are largely dependent on the detection methodology applied. All these microscopy techniques which employ light-, laser- and photon-optics, electrons as well as atomic force probing are characterized on their turn by their strengths and limitations for membrane raft identification. This explains in part the diversity of definitions available to describe these peculiar membrane structures. We present herewith an alternative and uncomplicated microscopy tool to study fluorescently labelled DRMs with information at the transmission electron microscopical level of the same cell, enabling us to obtain a snapshot of the morpho-functional relationships between the cell's interior and DRMs. The proposed approach of correlative fluorescence electron microscopy (CFEM) can therefore be considered as an additional alternative imaging approach to unravel DRM structure–function relationships from micro- to nanometre length scales, from the cell to the molecule.     

Ultrastructural analysis of midgut cells from Culex quinquefasciatus (Diptera: Culicidae) larvae resistant to Bacillus sphaericus

The larvicidal action of the entomopathogen Bacillus sphaericus towards Culex quinquefasciatus is due to the binary (Bin) toxin present in crystals, which are produced during bacterial sporulation. The Bin toxin needs to recognize and bind specifically to a single class of receptors, named Cqm1, which are 60-kDa α-glucosidases attached to the apical membrane of midgut cells by a glycosylphosphatidylinositol anchor. C. quinquefasciatus resistance to B. sphaericus has been often associated with the absence of the α-glucosidase Cqm1 in larvae midgut microvilli. In this work, we aimed to investigate, at the ultrastructural level, the midgut cells from C. quinquefasciatus larvae whose resistance relies on the lack of the Cqm1 receptor. The morphological analysis showed that midgut columnar cells from the resistant larvae are characterized by a pronounced production of lipid inclusions, throughout the 4th instar. At the end of this stage, resistant larvae had an increased size and number of these inclusions in the midgut cells, while only a small number were observed in the cells from susceptible larvae. The morphological differences in the midgut cells of resistant larvae found in this work suggested that the lack of the Cqm1 receptor, which also has a physiological role as being an α-glucosidase, can be related to changes in the cell metabolism. The ultrastructural effects of Bin toxin on midgut epithelial cells from susceptible and resistant larvae were also investigated. The cytopathological alterations observed in susceptible larvae treated with a lethal concentration of toxin included breakdown of the endoplasmic reticulum, mitochondrial swelling, microvillar disruption and vacuolization. Some effects were observed in cells from resistant larvae, although those alterations did not lead to larval death, indicating that the receptor Cqm1 is essential to mediate the larvicidal action of the toxin.This is the first ultrastructural study to show differences in the cell morphology of resistant larvae and further investigation is needed to understand the impact of the lack of expression of midgut enzymes on the physiology of resistant insects.     

Interactions of human islet amyloid polypeptide with lipid structure of different curvatures

Curvature is one of the most important features of lipid membranes in living cells, which significantly influences the structure of lipid membranes and their interaction with proteins. Taken the human islet amyloid polypeptide (hIAPP), an important protein related to the pathogenesis of type II diabetes, as an example, we performed molecular dynamics (MD) simulations to study the interaction between the protein and the lipid structures with varied curvatures. We found that the lipids in the high curvature membrane pack loosely with high mobility. The hIAPP initially forms H-bonds with the membrane surface that anchored the protein, and then inserts into the membrane through the hydrophobic interactions between the residues and the hydrophobic tails of the lipids. hIAPP can insert into the membrane more deeply with a larger curvature and with a stronger binding strength. Our result provided important insights into the mechanism of the membrane curvature-dependent property of proteins with molecular details.     

Lipid membranes in external electric fields: Kinetics of large pore formation causing rupture

About 40 years ago, Helfrich introduced an elastic model to explain shapes and shape transitions of cells (Z Naturforsch C, 1973; 28:693). This seminal article stimulated numerous theoretical as well as experimental investigations and created new research fields. In particular, the predictive power of his approach was demonstrated in a large variety of lipid model system. Here in this review, we focus on the development with respect to planar lipid membranes in external electric fields. Stimulated by the early work of Helfrich on electric field forces acting on liposomes, we extended his early approach to understand the kinetics of lipid membrane rupture. First, we revisit the main forces determining the kinetics of membrane rupture followed by an overview on various experiments. Knowledge on the kinetics of defect formation may help to design stable membranes or serve for novel mechanism for controlled release.     

Dynamic interaction between oppositely charged vesicles: aggregation, lipid mixing, and disaggregation

We investigated dynamic interactions between oppositely charged small unilamellar vesicles using positively charged vesicles containing 1,2-dioleoyl-3-trimethylammonium-propane or 3beta-[N-(N('),N(')-dimethylaminoethane)-carbamoyl] cholesterol and negatively charged vesicles containing L-alpha-phosphatidyl-DL-glycerol. Aggregation, lipid bilayer mixing, contents mixing and contents leakage were systematically examined using optical density measurements, fluorescence resonance energy transfer assays, fluorescence quenching assays, light-scattering analyses, and freeze-fracture transmission electron microscopy. The oppositely charged vesicles aggregated immediately. Lipid mixing was observed, but there was no mixing of the contents. The vesicle aggregates disaggregated spontaneously after several minutes. The surface potential of the disaggregated vesicles was neutralized. From these results, we infer that the lipids in the external monolayers were exchanged between the oppositely charged vesicles while the internal monolayers remained intact. The two types of cationic lipids used exhibited different speeds of disaggregation.     

Review of structural design guiding the development of lipid nanoparticles for nucleic acid delivery

Lipid nanoparticles (LNPs) are the most versatile and successful gene delivery systems, notably highlighted by their use in vaccines against COVID-19. LNPs have a well-defined core–shell structure, each region with its own distinctive compositions, suited for a wide range of in vivo delivery applications. Here, we discuss how a detailed knowledge of LNP structure can guide LNP formulation to improve the efficiency of delivery of their nucleic acid payload. Perspectives are detailed on how LNP structural design can guide more efficient nucleic acid transfection. Views on key physical characterization techniques needed for such developments are outlined including opinions on biophysical approaches both correlating structure with functionality in biological fluids and improving their ability to escape the endosome and deliver they payload.     

Activatable NIR-II Photothermal Lipid Nanoparticles for Improved Messenger RNA Delivery

Endosomal escape remains a central issue limiting the high protein expression of mRNA therapeutics. Here, we present second near-infrared (NIR-II) lipid nanoparticles (LNPs) containing pH activatable NIR-II dye conjugated lipid (Cy-lipid) for potentiating mRNA delivery efficiency via a s timulus-responsive p hotothermal-promoted e ndosomal e scape d elivery (SPEED) strategy. In acidic endosomal microenvironment, Cy-lipid is protonated and turns on NIR-II absorption for light-to-heat transduction mediated by 1064 nm laser irradiation. Then, the heat-promoted LNPs morphology change triggers rapid escape of NIR-II LNPs from the endosome, allowing about 3-fold enhancement of enhanced green fluorescent protein (eGFP) encoding mRNA translation capacity compared to the NIR-II light free group. In addition, the bioluminescence intensity induced by delivered luciferase encoding mRNA in the mouse liver region shows positive correlation with incremental radiation dose, indicating the validity of the SPEED strategy.