Molecular changes in the membranes of mouse erythroid cells accompanying differentiation.

The development of the mouse erythroblast to a mature erythrocyte is accompanied by changes in the composition and properties of the plasma membranes of these cells. Using double fluorescence techniques, we have simultaneously determined the distribution of lectin receptors and spectrin on the membranes of these cells. The lateral mobility of the lectin receptors in the membranes decreases as differentiation proceeds, and this is accompanied by an increasing concentration of spectrin associated with the membranes. The most significant concentration of spectrin occurs, however, during the enucleation of the late erythroblast, where we observe a complete segregation of the spectrin to the incipient reticulocyte, as well as a previously observed enrichment of receptors for concanavalin A into the plasma membrane surrounding the extruding nucleus. On the basis of these and other observations, we explore the possible molecular mechanisms involved in erythroblast enucleation and the role of spectrin in the regulation of protein mobility in erythroid cell membranes.     

Dissecting desaturation: plants prove advantageous

Fatty acid desaturases play important roles in controlling the physical properties o f membranes and in the synthesis of signal molecules such as prostaglandins and pheromones. Most desaturases are membrane proteins that have been recalcitrant to characterization by conventional biochemical methods. Only one enzyme o f this class has been characterized from animals or fungi. In this context, plants have proved to be useful sources of experimental materials. Substantial progress has been made in characterizing and manipulating nine classes of desaturases that control the fatty acid composition o f both plant membranes and plant storage lipids, which account for approximately -30% of the calories in the human diet.     

The impact of lipid polyunsaturation on the physical and mechanical properties of lipid membranes

The lipid composition of cellular membranes and the balance between the different lipid components can be impacted by aging, certain pathologies, specific diets and other factors. This is the case in a subgroup of individuals with psychiatric disorders, such as schizophrenia, where cell membranes of patients have been shown to be deprived in polyunsaturated fatty acids (PUFAs), not only in brain areas where the target receptors are expressed but also in peripheral tissues. This PUFA deprivation thus represents a biomarker of such disorders that might impact not only the interaction of antipsychotic medications with these membranes but also the activation and signaling of the targeted receptors embedded in the lipid membrane. Therefore, it is crucial to understand how PUFAs levels alterations modulate the different physical properties of membranes.In this paper, several biophysical approaches were combined (Laurdan fluorescence spectroscopy, atomic force microscopy, differential scanning calorimetry, molecular modeling) to characterize membrane properties such as fluidity, elasticity and thickness in PUFA-enriched cell membranes and lipid model systems reflecting the PUFA imbalance observed in some diseases. The impact of both the number of unsaturations and their position along the chain on the above properties was investigated. Briefly, data revealed that PUFA presence in membranes increases membrane fluidity, elasticity and flexibility and decreases its thickness and order parameter. Both the level of unsaturation and their position affect these membrane properties.     

Modifications of erythrocyte membrane fluidity of in vivo functionally aged human erythrocytes

The process of red blood cell senescence in the blood stream results in many changes in their physical and biochemical properties. In this work we have studied the physico-chemical state of erythrocyte membranes prepared from 5 subpopulations of erythrocytes of different age by using the fluorescence technique. Membrane fluidity has been evaluated by the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and a further study of the fluorescence decay of this probe has been performed by multifrequency phase and modulation fluorometry. DPH fluorescence polarization is significantly increased in the membranes prepared from the youngest fraction of erythrocytes, indicating a decreased fluidity without any significant change in DPH fluorescence decay.     

Morphological and Biochemical Characterization of the Membranous Hepatitis C Virus Replication Compartment

Like all other positive-strand RNA viruses, hepatitis C virus (HCV) induces rearrangements of intracellular membranes that are thought to serve as a scaffold for the assembly of the viral replicase machinery. The most prominent membranous structures present in HCV-infected cells are double-membrane vesicles (DMVs). However, their composition and role in the HCV replication cycle are poorly understood. To gain further insights into the biochemcial properties of HCV-induced membrane alterations, we generated a functional replicon containing a hemagglutinin (HA) affinity tag in nonstructural protein 4B (NS4B), the supposed scaffold protein of the viral replication complex. By using HA-specific affinity purification we isolated NS4B-containing membranes from stable replicon cells. Complementing biochemical and electron microscopy analyses of purified membranes revealed predominantly DMVs, which contained viral proteins NS3 and NS5A as well as enzymatically active viral replicase capable of de novo synthesis of HCV RNA. In addition to viral factors, co-opted cellular proteins, such as vesicle-associated membrane protein-associated protein A (VAP-A) and VAP-B, that are crucial for viral RNA replication, as well as cholesterol, a major structural lipid of detergent-resistant membranes, are highly enriched in DMVs. Here we describe the first isolation and biochemical characterization of HCV-induced DMVs. The results obtained underline their central role in the HCV replication cycle and suggest that DMVs are sites of viral RNA replication. The experimental approach described here is a powerful tool to more precisely define the molecular composition of membranous replication factories induced by other positive-strand RNA viruses, such as picorna-, arteri- and coronaviruses.     

Effects of Quenching Mechanism and Type of Quencher Association on Stern-Volmer Plots in Compartmentalized Systems

Fluorescence quenching techniques have been used extensively in recent years to examine reaction rates and the compartmentalization of components in lipid micelles and membranes. Steady-state fluorescence methods are frequently employed in such studies but the interpretation of the resulting Stern-Volmer plots is often hampered by uncertainties regarding the mode of association of the quencher with the lipid structure and the nature of the quenching mechanism. This paper presents a method for simulating steady-state Stern-Volmer plots in two phase systems, and shows how the forms of such plots are influenced by the type of association of the quencher with the membrane or micelle (partition and/or binding) and by the type of quenching mechanism (dynamic and/or static). Comparisons of simulated plots with experimental data must take into account the possible combinations of quencher association(s) and quenching mechanism(s). The methods presented are applicable to synthetic and natural membranes and provide a basis for comparing the quenching of fluorescent molecules in biological membranes of differing composition.     

O2-dependent lipid peroxidation does not affect the molecular order in hepatoma microsomes

Microsomal membranes from rat liver and from the fast-growing Morris hepatoma 3942A have been peroxidized to different extents and the order parameter of the membranes measured by fluorescence depolarization of the probe 1,6-diphenyl-1,3,5-hexatriene. The data have been analysed by applying a mathematical approach that takes into account simultaneously static and dynamic fluorescence parameters. It appears that tumour membranes are more ordered than the control and their order parameter does not increase with greater exposure to the action of O2 radicals in contrast to liver membranes. The fatty acid composition of the membrane lipids has been studied under different experimental conditions and correlated to the behaviour of the physical parameter.     

Liposome destabilization induced by synthetic lipopeptides corresponding to envelope and non-structural domains of GBV-C/HGV virus. Conformational requirements for leakage

Liposomes have been used primarily as a model system for studying biological membranes. Numerous chemical, biochemical and biophysical methods have been used to elucidate the various aspects of the interaction between proteins or peptides and phospholipids. Having in mind the potential use of synthetic lipopeptides as antiviral therapies and aiming for a better understanding of the molecular interaction of the GBV-C/HGV with liposomes as model membranes, epitopes of GBV-C/HGV located at the E2 (99-118) and NS3(440-460) regions were selected. Peptides were modified at the N-terminus with acyl chains of different length (C(14) and C(16)) yielding the corresponding myristoil and palmytoil lipopeptides. The main aim of the present study was to get insight into the membrane-interacting properties of the above-described synthetic lipopeptides and to study their inhibition of the capacity of perturbing model membranes of fusion peptide of HIV-1 using fluorescence spectroscopy. In an attempt to establish a relationship between peptide membrane activity and structure, we use Circular Dichroism (CD) and Fourier-Transform Infrared Spectroscopy (FTIR).     

Molecular Simulations of Gram-Negative Bacterial Membranes: A Vignette of Some Recent Successes

In the following review we use recent examples from the literature to discuss progress in the area of atomistic and coarse-grained molecular dynamics simulations of selected bacterial membranes and proteins, with a particular focus on Gram-negative bacteria. As structural biology continues to provide increasingly high-resolution data on the proteins that reside within these membranes, simulations have an important role to play in linking these data with the dynamical behavior and function of these proteins. In particular, in the last few years there has been significant progress in addressing the issue of biochemical complexity of bacterial membranes such that the heterogeneity of the lipid and protein components of these membranes are now being incorporated into molecular-level models. Thus, in future we can look forward to complementary data from structural biology and molecular simulations combining to provide key details of structure-dynamics-function relationships in bacterial membranes.     

Phagocytosis of IgG-coated polystyrene beads by macrophages induces and requires high membrane order

The biochemical composition and biophysical properties of cell membranes are hypothesized to affect cellular processes such as phagocytosis. Here, we examined the plasma membranes of murine macrophage cell lines during the early stages of uptake of immunoglobulin G (IgG)-coated polystyrene particles. We found that the plasma membrane undergoes rapid actin-independent condensation to form highly ordered phagosomal membranes, the biophysical hallmark of lipid rafts. Surprisingly, these membranes are depleted of cholesterol and enriched in sphingomyelin and ceramide. Inhibition of sphingomyelinase activity impairs membrane condensation, F-actin accumulation at phagocytic cups and particle uptake. Switching phagosomal membranes to a cholesterol-rich environment had no effect on membrane condensation and the rate of phagocytosis. In contrast, preventing membrane condensation with the oxysterol 7-ketocholesterol, even in the presence of ceramide, blocked F-actin dissociation from nascent phagosomes and particle uptake. In conclusion, our results suggest that ordered membranes function to co-ordinate F-actin remodelling and that the biophysical properties of phagosomal membranes are essential for phagocytosis.     

Diffusion-based determination of protein homodimerization on reconstituted membrane surfaces

The transient interactions between cellular components, particularly on membrane surfaces, are critical in the proper function of many biochemical reactions. For example, many signaling pathways involve dimerization, oligomerization, or other types of clustering of signaling proteins as a key step in the signaling cascade. However, it is often experimentally challenging to directly observe and characterize the molecular mechanisms such interactions—the greatest difficulty lies in the fact that living cells have an unknown number of background processes that may or may not participate in the molecular process of interest, and as a consequence, it is usually impossible to definitively correlate an observation to a well-defined cellular mechanism. One of the experimental methods that can quantitatively capture these interactions is through membrane reconstitution, whereby a lipid bilayer is fabricated to mimic the membrane environment, and the biological components of interest are systematically introduced, without unknown background processes. This configuration allows the extensive use of fluorescence techniques, particularly fluorescence fluctuation spectroscopy and single-molecule fluorescence microscopy. In this review, we describe how the equilibrium diffusion of two proteins, K-Ras4B and the PH domain of Bruton’s tyrosine kinase (Btk), on fluid lipid membranes can be used to determine the kinetics of homodimerization reactions.     

Fluorescence approaches for determining protein conformations, interactions and mechanisms at membranes

Processes that occur at membranes are essential for the viability of every cell, but such processes are the least well understood at the molecular level. The complex nature and physical properties of the molecular components involved, as well as the requirement for two separated aqueous compartments, restrict the experimental approaches that can be successfully applied to examine the structure, conformational changes and interactions of the membrane-bound proteins that accomplish these processes. In particular, to accurately elucidate the molecular mechanisms that effect and regulate such processes, one must use experimental approaches that do not disrupt the structural integrity or functionality of the protein-membrane complexes being examined. To best accomplish this goal, especially when large multicomponent complexes and native membranes are involved, the optimal experimental approach to use is most often fluorescence spectroscopy. Using multiple independent fluorescence techniques, one can determine structural information in real time and in intact membranes under native conditions that cannot be obtained by crystallography, electron microscopy and NMR techniques, among others. Furthermore, fluorescence techniques provide a comprehensive range of information, from kinetic to thermodynamic, about the assembly, structure, function and regulation of membrane-bound proteins and complexes. This article describes the use of various fluorescence techniques to characterize different aspects of proteins bound to or embedded in membranes.     

Structure and dynamics of supported intermembrane junctions

Supported intermembrane junctions, formed by rupture of giant unilamellar vesicles onto conventional supported lipid membranes, have recently emerged as model systems for the study of biochemical processes at membrane interfaces. Using intermembrane fluorescence resonance energy transfer and optical standing wave fluorescence interferometry, we characterize the nanometer-scale topography of supported intermembrane junctions and find two distinct association states. In one state, the two membranes adhere in close apposition, with intermembrane separations of a few nanometers. In the second state, large intermembrane spacings of approximately 50 nm are maintained by a balance between Helfrich (entropic) repulsion and occasional sites of tight adhesion that pin the two membranes together. Reversible transitions between these two states can be triggered with temperature changes. We further examine the physical properties of membranes in each state using a membrane mixture near its miscibility phase transition temperature. Thermodynamic characteristics of the phase transition and diffusive mobility of individual lipids are comparable. However, collective Brownian motion of phase-separated domains and compositional fluctuations are substantially modulated by intermembrane spacing. The scaling properties of diffusion coefficient with particle size are determined from detailed analysis of domain motion in the different junction types. The results provide experimental verification of a theoretical model for two-dimensional mobility in membranes, which includes frictional coupling across an interstitial water layer.     

Two-dimensional electrophoretic analysis of secretory-granule,granule-membrane,and plasma-membrane proteins of rat parotid cells

Secretory granules and plasma membranes were isolated from rat parotid cells and characterized enzymatically and by electron microscopy. The proteins of the secretory granule membranes, the secretory granules and the plasma membranes were characterized by two-dimensional polyacrylamide gel electrophoresis and visualized by silver staining. The granule membrane contains 166 polypeptides of which only 26 are also present in the granule contents. The membrane proteins have isoelectric points between 4.75 and 6.45 and apparent molecular weights of 17 000 to 190 000 Daltons. The granule content proteins are surprisingly complex and contain 122 polypeptides with molecular weights of 11 000 to 138 000 and isoelectric points of 4.8 to 6.55. Thirteen of these peptides are present as major species. The plasma membrane contains 172 polypeptide species with molecular weights from 17 000 to 200 000 Daltons and isoelectric points of 5.0 to 6.8. Thirty-five of the plasma membrane proteins are also present in the secretory granule membranes indicating that the two membranes have some enzymatic or structural properties in common. Thus, secretory granule membranes and plasma membranes from parotid cells have a more complex polypeptide composition than has previously been shown for membranes of this type. The systems developed are suitable for the analysis of regulatory events such as protein phosphorylation, proteolytic processing, and other types of post-translational modifications that may be important to the secretory mechanism.     

Combining mechanical and optical approaches to dissect cellular mechanobiology

Mechanical force modulates a wide array of cell physiological processes. Cells sense and respond to mechanical stimuli using a hierarchy of structural complexes spanning multiple length scales, including force-sensitive molecules and cytoskeletal networks. Understanding mechanotransduction, i.e., the process by which cells convert mechanical inputs into biochemical signals, has required the development of novel biophysical tools that allow for probing of cellular and subcellular components at requisite time, length, and force scales and technologies that track the spatio-temporal dynamics of relevant biomolecules. In this review, we begin by discussing the underlying principles and recent applications of atomic force microscopy, magnetic twisting cytometry, and traction force microscopy, three tools that have been widely used for measuring the mechanical properties of cells and for probing the molecular basis of cellular mechanotransduction. We then discuss how such tools can be combined with advanced fluorescence methods for imaging biochemical processes in living cells in the context of three specific problem spaces. We first focus on fluorescence resonance energy transfer, which has enabled imaging of intra- and inter-molecular interactions and enzymatic activity in real time based on conformational changes in sensor molecules. Next, we examine the use of fluorescence methods to probe force-dependent dynamics of focal adhesion proteins. Finally, we discuss the use of calcium ratiometric signaling to track fast mechanotransductive signaling dynamics. Together, these studies demonstrate how single-cell biomechanical tools can be effectively combined with molecular imaging technologies for elucidating mechanotransduction processes and identifying mechanosensitive proteins.     

The significance of lipid composition for membrane activity: new concepts and ways of assessing function

In the last decade or so, it has been realised that membranes do not just have a lipid-bilayer structure in which proteins are embedded or with which they associate. Structures are dynamic and contain areas of heterogeneity which are vital for their formation. In this review, we discuss some of the ways in which these dynamic and heterogeneous structures have implications during stress and in relation to certain human diseases. A particular stress is that of temperature which may instigate adaptation in poikilotherms or appropriate defensive responses during fever in mammals. Recent data emphasise the role of membranes in sensing temperature changes and in controlling a regulatory loop with chaperone proteins. This loop seems to need the existence of specific membrane microdomains and also includes association of chaperone (heat stress) proteins with the membrane. The role of microdomains is then discussed further in relation to various human pathologies such as cardiovascular disease, cancer and neurodegenerative diseases. The concept of modifying membrane lipids (lipid therapy) as a means for treating such pathologies is then introduced. Examples are given when such methods have been shown to have benefit. In order to study membrane microheterogeneity in detail and to elucidate possible molecular mechanisms that account for alteration in membrane function, new methods are needed. In the second part of the review, we discuss ultra-sensitive and ultra-resolution imaging techniques. These include atomic force microscopy, single particle tracking, single particle tracing and various modern fluorescence methods. Finally, we deal with computing simulation of membrane systems. Such methods include coarse-grain techniques and Monte Carlo which offer further advances into molecular dynamics. As computational methods advance they will have more application by revealing the very subtle interactions that take place between the lipid and protein components of membranes - and which are so essential to their function.     

Prediction of cell penetrating peptides by support vector machines

Cell penetrating peptides (CPPs) are those peptides that can transverse cell membranes to enter cells. Once inside the cell, different CPPs can localize to different cellular components and perform different roles. Some generate pore-forming complexes resulting in the destruction of cells while others localize to various organelles. Use of machine learning methods to predict potential new CPPs will enable more rapid screening for applications such as drug delivery. We have investigated the influence of the composition of training datasets on the ability to classify peptides as cell penetrating using support vector machines (SVMs). We identified 111 known CPPs and 34 known non-penetrating peptides from the literature and commercial vendors and used several approaches to build training data sets for the classifiers. Features were calculated from the datasets using a set of basic biochemical properties combined with features from the literature determined to be relevant in the prediction of CPPs. Our results using different training datasets confirm the importance of a balanced training set with approximately equal number of positive and negative examples. The SVM based classifiers have greater classification accuracy than previously reported methods for the prediction of CPPs, and because they use primary biochemical properties of the peptides as features, these classifiers provide insight into the properties needed for cell-penetration. To confirm our SVM classifications, a subset of peptides classified as either penetrating or non-penetrating was selected for synthesis and experimental validation. Of the synthesized peptides predicted to be CPPs, 100% of these peptides were shown to be penetrating.     

The force-velocity relationship for the actin-based motility of Listeria monocytogenes

The intracellular movement of the bacterial pathogen Listeria monocytogenes has helped identify key molecular constituents of actin-based motility (recent reviews ). However, biophysical as well as biochemical data are required to understand how these molecules generate the forces that extrude eukaryotic membranes. For molecular motors and for muscle, force-velocity curves have provided key biophysical data to distinguish between mechanistic theories. Here we manipulate and measure the viscoelastic properties of tissue extracts to provide the first force-velocity curve for Listeria monocytogenes. We find that the force-velocity relationship is highly curved, almost biphasic, suggesting a high cooperativity between biochemical catalysis and force generation. Using high-resolution motion tracking in low-noise extracts, we find long trajectories composed exclusively of molecular-sized steps. Robust statistics from these trajectories show a correlation between the duration of steps and macroscopic Listeria speed, but not between average step size and speed. Collectively, our data indicate how the molecular properties of the Listeria polymerization engine regulate speed, and that regulation occurs during molecular-scale pauses.     

Luminescent metal-ligand complexes as probes of macromolecular interactions and biopolymer dynamics

The knowledge of microsecond dynamics is important for an understanding of the mechanism and function of biological systems. Fluorescent techniques are well established in biophysical studies, but their applicability to probe microsecond timescale processes is limited. Luminescent metal-ligand complexes (MLCs) have created interest mainly due to their unique luminescent properties, such as the exceptionally long decay times and large fundamental anisotropy values, allowing examination of microsecond dynamics by fluorescence methods. MLC properties also greatly simplify instrumentation requirements and enable the use of light emitting diode excitation for time-resolved measurements. Recent literature illustrates how MLC labels take full advantage of well developed fluorescence techniques and how those methods can be extended to timescales not easily accessible with nanosecond probes. MLCs are now commercially available as reactive labels which give researchers access to methods that previously required more complex approaches. The present paper gives an overview of the applications of MLC probes to studies of molecular dynamics and interactions of proteins, membranes and nucleic acids.     

Quantitative Imaging of Molecular Order in Lipid Membranes Using Two-Photon Fluorescence Polarimetry

We present a polarimetric two-photon microscopy technique to quantitatively image the local static molecular orientational behavior in lipid and cell membranes. This approach, based on a tunable excitation polarization state complemented by a polarized readout, is easily implementable and does not require hypotheses on the molecular angular distribution such as its mean orientation, which is a main limitation in traditional fluorescence anisotropy measurements. The method is applied to the investigation of the molecular angular distribution in giant unilamellar vesicles formed by liquid-ordered and liquid-disordered micro-domains, and in COS-7 cell membranes. The highest order contrast between ordered and disordered domains is obtained for dyes locating within the membrane acyl chains.