Biophysical approaches to protein-induced membrane deformations in trafficking

Membrane traffic requires membrane deformation to generate vesicles and tubules. Strong evidence suggests that assembly of curvature-active proteins can drive such membrane shape changes. Well-documented pathways often involve protein scaffolds, in particular coats (clathrin or COP). However, membrane curvature should, in principle, be influenced by any protein binding asymmetrically on a membrane; large membrane morphological changes could result from their aggregation. In the case of Shiga toxin or viral matrix proteins, tubules and buds appear to result from the cargo-driven formation of protein-lipid nanodomains, showing that collective protein behaviour is crucial in the process. We argue here that a combination of in vitro experiments on giant unilamellar vesicles and theoretical modelling based on statistical physics is ideally suited to tackle these collective effects.     

A new numerical approach to mechanically analyse biological structures

In this work, an advanced discretization meshless technique is used to study the structural response of a human brain due to an impact load. The 2D and 3D brain geometrical models, and surrounding structures, were obtained through the processing of medical images, allowing to achieve a realistic geometry for the virtual model and to define the distribution of the mechanical properties accordingly with the medical images colour scale. Additionally, a set of essential and natural boundary conditions were assumed in order to reproduce a sudden impact force applied to the cranium. Then, a structural numerical analysis was performed using the Natural Neighbour Radial Point Interpolation Method (NNRPIM). The obtained results were compared with the finite element method (FEM) and a solution available in the literature. This work shows that the NNRPIM is a robust and accurate numerical technique, capable to produce results very close to other numerical approaches. In addition, the variable fields obtained with the meshless method are much smoother than the FEM corresponding solution.     

A fuzzy-set-theory-based approach to analyse species membership in DNA barcoding

Reliable assignment of an unknown query sequence to its correct species remains a methodological problem for the growing field of DNA barcoding. While great advances have been achieved recently, species identification from barcodes can still be unreliable if the relevant biodiversity has been insufficiently sampled. We here propose a new notion of species membership for DNA barcoding-fuzzy membership, based on fuzzy set theory-and illustrate its successful application to four real data sets (bats, fishes, butterflies and flies) with more than 5000 random simulations. Two of the data sets comprise especially dense species/population-level samples. In comparison with current DNA barcoding methods, the newly proposed minimum distance (MD) plus fuzzy set approach, and another computationally simple method, 'best close match', outperform two computationally sophisticated Bayesian and BootstrapNJ methods. The new method proposed here has great power in reducing false-positive species identification compared with other methods when conspecifics of the query are absent from the reference database.     

A particle-based computational model to analyse remodelling of the red blood cell cytoskeleton during malaria infections

Red blood cells can withstand the harsh mechanical conditions in the vasculature only because the bending rigidity of their plasma membrane is complemented by the shear elasticity of the underlying spectrin-actin network. During an infection by the malaria parasite Plasmodium falciparum, the parasite mines host actin from the junctional complexes and establishes a system of adhesive knobs, whose main structural component is the knob-associated histidine rich protein (KAHRP) secreted by the parasite. Here we aim at a mechanistic understanding of this dramatic transformation process. We have developed a particle-based computational model for the cytoskeleton of red blood cells and simulated it with Brownian dynamics to predict the mechanical changes resulting from actin mining and KAHRP-clustering. Our simulations include the three-dimensional conformations of the semi-flexible spectrin chains, the capping of the actin protofilaments and several established binding sites for KAHRP. For the healthy red blood cell, we find that incorporation of actin protofilaments leads to two regimes in the shear response. Actin mining decreases the shear modulus, but knob formation increases it. We show that dynamical changes in KAHRP binding affinities can explain the experimentally observed relocalization of KAHRP from ankyrin to actin complexes and demonstrate good qualitative agreement with experiments by measuring pair cross-correlations both in the computer simulations and in super-resolution imaging experiments.     

A mechanostatistical approach to cortical bone remodelling: an equine model

In this study, the development of a mechanostatistical model of three-dimensional cortical bone remodelling informed with in vivo equine data is presented. The equine model was chosen as it is highly translational to the human condition due to similar Haversian systems, availability of in vivo bone strain and biomarker data, and furthermore, equine models are recommended by the US Federal Drugs Administration for comparative joint research. The model was derived from micro-computed tomography imaged specimens taken from the equine third metacarpal bone, and the Frost-based ‘mechanostat’ was informed from both in vivo strain gauges and biomarkers to estimate bone growth rates. The model also described the well-known ‘cutting cone’ phenomena where Haversian canals tunnel and replace bone. In order to make this model useful in practice, a partial least squares regression (PLSR) surrogate model was derived based on training data from finite element simulations with different loads. The PLSR model was able to predict microstructure and homogenised Young’s modulus with errors less than 2.2 % and \(0.6\,\% \), respectively.     

Hyperspectral imaging: a novel approach for microscopic analysis

BACKGROUND: The usefulness of the light microscope has been dramatically enhanced by recent developments in hardware and software. However, current technologies lack the ability to capture and analyze a high-resolution image representing a broad diversity of spectral signatures in a single-pass view. We show that hyperspectral imaging offers such a technology. METHODS AND RESULTS We developed a prototype hyperspectral imaging microscope capable of collecting the complete emission spectrum from a microscope slide. A standard epifluorescence microscope was optically coupled to an imaging spectrograph, with output recorded by a CCD camera. Software was developed for image acquisition and computer display of resultant X--Y images with spectral information. Individual images were captured representing Y-wavelength planes, with the stage successively moved in the X direction, allowing an image cube to be constructed from the compilation of generated scan files. This prototype instrument was tested with samples relevant to cytogenetic, histologic, cell fusion, microarray scanning, and materials science applications. CONCLUSIONS: Hyperspectral imaging microscopy permits the capture and identification of different spectral signatures present in an optical field during a single-pass evaluation, including molecules with overlapping but distinct emission spectra. This instrument can reduce dependence on custom optical filters and, in future imaging applications, should facilitate the use of new fluorophores or the simultaneous use of similar fluorophores.     

A novel biotinylated lipid raft reporter for electron microscopic imaging of plasma membrane microdomains

The submicroscopic spatial organization of cell surface receptors and plasma membrane signaling molecules is readily characterized by electron microscopy (EM) via immunogold labeling of plasma membrane sheets. Although various signaling molecules have been seen to segregate within plasma membrane microdomains, the biochemical identity of these microdomains and the factors affecting their formation are largely unknown. Lipid rafts are envisioned as submicron membrane subdomains of liquid ordered structure with differing lipid and protein constituents that define their specific varieties. To facilitate EM investigation of inner leaflet lipid rafts and the localization of membrane proteins therein, a unique genetically encoded reporter with the dually acylated raft-targeting motif of the Lck kinase was developed. This reporter, designated Lck-BAP-GFP, incorporates green fluorescent protein (GFP) and biotin acceptor peptide (BAP) modules, with the latter allowing its single-step labeling with streptavidin-gold. Lck-BAP-GFP was metabolically biotinylated in mammalian cells, distributed into low-density detergent-resistant membrane fractions, and was readily detected with avidin-based reagents. In EM images of plasma membrane sheets, the streptavidin-gold-labeled reporter was clustered in 20-50 nm microdomains, presumably representative of inner leaflet lipid rafts. The utility of the reporter was demonstrated in an investigation of the potential lipid raft localization of the epidermal growth factor receptor.     

A simple covarion‐based approach to analyse nucleotide substitution rates

Using the ratio of nonsynonymous to synonymous nucleotide substitution rates (K_a/K_s) is a common approach for detecting positive selection. However, calculation of this ratio over a whole gene combines amino acid sites that may be under positive selection with those that are highly conserved. We introduce a new covarion‐based method to sample only the sites potentially under selective pressure. Using ancestral sequence reconstruction over a phylogenetic tree coupled with calculation of K_a/K_s ratios, positive selection is better detected by this simple covarion‐based approach than it is using a whole gene analysis or a windowing analysis. This is demonstrated on a synthetic dataset and is tested on primate leptin, which indicates a previously undetected round of positive selection in the branch leading to Gorilla gorilla.     

A novel approach to prezizaane sesquiterpenes

Prezizaane sesquiterpenes are an olfactorily interesting class of tricyclic natural products, which occur in some precious perfumery raw materials. These compounds are biosynthetically derived from farnesyl pyrophosphate via cyclization, but some questions regarding the stereoselectivity of this process have not yet been answered. We discuss a novel and concise access to the tricyclic framework of these sesquiterpenes, as exemplified by the synthesis of (+/-)-5-epi-sesquithuriferone (5-epi-4).     

A systems biology approach to analyse leaf carbohydrate metabolism in Arabidopsis thaliana

Plant carbohydrate metabolism comprises numerous metabolite interconversions, some of which form cycles of metabolite degradation and re-synthesis and are thus referred to as futile cycles. In this study, we present a systems biology approach to analyse any possible regulatory principle that operates such futile cycles based on experimental data for sucrose (Scr) cycling in photosynthetically active leaves of the model plant Arabidopsis thaliana. Kinetic parameters of enzymatic steps in Scr cycling were identified by fitting model simulations to experimental data. A statistical analysis of the kinetic parameters and calculated flux rates allowed for estimation of the variability and supported the predictability of the model. A principal component analysis of the parameter results revealed the identifiability of the model parameters. We investigated the stability properties of Scr cycling and found that feedback inhibition of enzymes catalysing metabolite interconversions at different steps of the cycle have differential influence on stability. Applying this observation to futile cycling of Scr in leaf cells points to the enzyme hexokinase as an important regulator, while the step of Scr degradation by invertases appears subordinate.     

Carbocyanine dye orientation in red cell membrane studied by microscopic fluorescence polarization.

The orientation of an amphipathic, long acyl chain fluorescent carbocyanine dye [diI-C18-(3)] in a biological membrane is examined by steady-state fluorescence polarization microscopy on portions of single erythrocyte ghosts. The thermodynamically plausible orientation model most consistent with the experimental data is one in which the diI-C18-(3) conjugated bridge chromophore is parallel to the surface of the cell and the acyl chains are imbedded in the bilayer parallel to the phospholipid acyl chains. Comparison of the predictions of this model with the experimental data yields information on the intramolecular orientations of the dye's transition dipoles and on the dye's rate of rotation in the membrane around an axis normal to the membrane. To interpret the experimental data, formulae are derived to account for the effect of high aperture observation on fluorescence polarization ratios. These formulae are generally applicable to any high aperture polarization studied on microscopic samples, such as portions of single cells.     

Pyrene as a membrane depth gauge: wavelength selective fluorescence approach to monitor pyrene localizations in the membrane

We take the advantage of pyrene's unique spectral properties as a reliable polarity indicator to monitor pyrene localizations in the membrane depth by using wavelength selective fluorescence approach. We show that fine structure of pyrene fluorescence emission spectra and excimerization rate in model and native phospholipid membranes depend on the excitation wavelength. This phenomenon is not observed in neat solvents. In membranes, the dependence on the excitation wavelength reflects selective excitation of pyrene molecules located close to the membrane-water polar interface, or deep in the hydrophobic core of the membrane, verified with the aid of pyrene derivatives of fatty acids of various lengths.     

A novel approach to identify bovine sperm membrane proteins that interact with receptors on the vitelline membrane of bovine oocytes

At fertilization, the sperm triggers intracellular calcium oscillations, which are pivotal to oocyte activation and development. A working hypothesis for the interaction between the sperm and the oocyte is that disintegrin ligands on the inner acrosomal membrane of the sperm bind to integrin receptors on the oocyte vitelline membrane. The aim of these experiments was to find and identify the sperm protein ligands involved in bovine sperm-oocyte interactions. In situ fluorescent labeling of proteins and 2-D gel electrophoresis were used to identify specific sperm membrane proteins that interact with proteins in the oocyte vitelline membrane. Sperm were labeled with a fluorescent dye and used to fertilize zona-free oocytes. Sperm-oocyte complexes were either lysed immediately, or following covalent cross-linking of proteins with dibromobimane. The cross-linking reagent serves the critical function of covalently linking proteins together so that they will remain as a unit through lysis of the cells and 2-D gel analysis, and which can be subsequently identified by mass spectrometry. Lysates were electrophoretically run on the same 2-D gel. The comparison of uncross-linked and cross-linked protein spots revealed that some proteins shifted position based on binding. These spots were picked and proteins identified by mass spectrometry. These results provide a list of specific sperm proteins that interact with oocyte membrane proteins and establish a group of candidate ligands, one or more of which may be responsible for induction of outside-in signaling resulting in oocyte activation and fusion of the gametes.     

Exploring membrane organization and dynamics by the wavelength-selective fluorescence approach

Wavelength-selective fluorescence comprises a set of approaches based on the red edge effect in fluorescence spectroscopy which can be used to directly monitor the environment and dynamics around a fluorophore in a complex biological system. A shift in the wavelength of maximum fluorescence emission toward higher wavelengths, caused by a shift in the excitation wavelength toward the red edge of absorption band, is termed red edge excitation shift (REES). This effect is mostly observed with polar fluorophores in motionally restricted media such as very viscous solutions or condensed phases where the dipolar relaxation time for the solvent shell around a fluorophore is comparable to or longer than its fluorescence lifetime. REES arises from slow rates of solvent relaxation (reorientation) around an excited state fluorophore which is a function of the motional restriction imposed on the solvent molecules in the immediate vicinity of the fluorophore. Utilizing this approach, it becomes possible to probe the mobility parameters of the environment itself (which is represented by the relaxing solvent molecules) using the fluorophore merely as a reporter group. Further, since the ubiquitous solvent for biological systems is water, the information obtained in such cases will come from the otherwise 'optically silent' water molecules. This makes REES and related techniques extremely useful since hydration plays a crucial modulatory role in a large number of important cellular events, including lipid-protein interactions and ion transport. The interfacial region in membranes, characterized by unique motional and dielectric characteristics, represents an appropriate environment for displaying wavelength-selective fluorescence effects. The application of REES and related techniques (wavelength-selective fluorescence approach) as a powerful tool to monitor the organization and dynamics of probes and peptides bound to membranes, micelles, and reverse micelles is discussed.