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Analyses of the status of the membrane spectrin-based skeleton during fludarabine/mitoxantrone/dexamethasone-induced (FND-induced) apoptosis revealed proteolytic degradation of β-spectrin, with the prevalent appearance of a specific fragment with a molecular weight of ~55kDa, containing the actin-binding domain (ABD). Appearance of this fragment was dependent on induction of apoptosis. In silico proteolysis of spectrin identified caspase-8 as a candidate protease responsible for the generation of this ~55kDa ABD-containing fragment. Analyses of spectrin and procaspase-8 localization during early apoptosis indicated temporary (

J. Neurochem. (2012) 122, 681–690. Abstract The interactions of the neurotransmitter dopamine, and its precursor l-dopa, with membrane lipids were investigated through a set of molecular dynamic simulations with all atom resolution. The results obtained indicate that both dopamine and l-dopa have a pronounced association with the lipid head groups, predominantly mediated through H-bonds. As a result the molecules are anchored to the interfacial region of the membrane. The strength of this interaction is dependent on lipid composition – the presence of phosphatidylserine leads to an increase in the strength of this interaction, resulting in an H-bond network with a lifetime much longer than the timescale of our simulations. Also, bilayers that include sphingomieline and cholesterol interact strongly with dopamine and l-dopa. We postulate that the high membrane association that we have observed for both dopamine and l-dopa could have the following effects: 1) when on the plasma membrane exterior, favour the availability of these compounds for cell membrane uptake processes and, 2) when on an internal membrane surface, accentuate the importance of membrane-bound metabolizing enzymes over their soluble counterparts.

Biological membranes are compartmentalized for functional diversity by a variety of specific protein–protein, protein–lipid, and lipid–lipid interactions. A subset of these are the preferential interactions between sterols, sphingolipids, and saturated aliphatic lipid tails responsible for liquid–liquid domain coexistence in eukaryotic membranes, which give rise to dynamic, nanoscopic assemblies whose coalescence is regulated by specific biochemical cues. Microscopic phase separation recently observed in isolated plasma membranes (giant plasma membrane vesicles and plasma membrane spheres) ( i ) confirms the capacity of compositionally complex membranes to phase separate, ( ii ) reflects the nanoscopic organization of live cell membranes, and ( iii ) provides a versatile platform for the investigation of the compositions and properties of the phases. Here, we show that the properties of coexisting phases in giant plasma membrane vesicles are dependent on isolation conditions—namely, the chemicals used to induce membrane blebbing. We observe strong correlations between the relative compositions and orders of the coexisting phases, and their resulting miscibility. Chemically unperturbed plasma membranes reflect these properties and validate the observations in chemically induced vesicles. Most importantly, we observe domains with a continuum of varying stabilities, orders, and compositions induced by relatively small differences in isolation conditions. These results show that, based on the principle of preferential association of raft lipids, domains of various properties can be produced in a membrane environment whose complexity is reflective of biological membranes.

The human epidermal growth factor receptor (EGFR) is a key representative of tyrosine kinase receptors, ubiquitous actors in cell signaling, proliferation, differentiation, and migration. Although the receptor is well-studied, a central issue remains: How does the compositional diversity and functional diversity of the surrounding membrane modulate receptor function? Reconstituting human EGFR into proteoliposomes of well-defined and controlled lipid compositions represents a minimal synthetic approach to systematically address this question. We show that lipid composition has little effect on ligand-binding properties of the EGFR but rather exerts a profound regulatory effect on kinase domain activation. Here, the ganglioside GM3 but not other related lipids strongly inhibited the autophosphorylation of the EGFR kinase domain. This inhibitory action of GM3 was only seen in liposomes compositionally poised to phase separate into coexisting liquid domains. The inhibition by GM3 was released by either removing the neuraminic acid of the GM3 headgroup or by mutating a membrane proximal lysine of EGFR (K642G). Our results demonstrate that GM3 exhibits the potential to regulate the allosteric structural transition from inactive to a signaling EGFR dimer, by preventing the autophosphorylation of the intracellular kinase domain in response to ligand binding.

Galectins are unconventionally secreted lectins that participate in the formation of glycoprotein lattices that perform a variety of cell surface functions. Galectins also bind glycosphingolipid headgroups with as yet unclear implications for cellular physiology. We report a specific interaction between galectin-9 and the Forssman glycosphingolipid (FGL) that is important for polarizing Madin–Darby canine kidney epithelial cells. Galectin-9 knockdown leads to a severe loss of epithelial polarity that can be rescued by addition of the recombinant protein. The FGL glycan is identified as the surface receptor that cycles galectin-9 to the Golgi apparatus from which the protein is recycled back to the apical surface. Together our results suggest a model wherein such glycosphingolipid–galectin couples form a circuit between the Golgi apparatus and the cell surface that in an epithelial context facilitates the apical sorting of proteins and lipids.

It was previously shown that ankyrins play a crucial role in the membrane skeleton arrangement. Purifying ankyrinR obtained from erythrocytes is a time-consuming process. Therefore, cloned and bacterially expressed ankyrinR-spectrin-binding domain (AnkSBD) is a demanded tool for studying spectrin-ankyrin interactions. In this communication, we report on the cloning and purification of AnkSBD and describe the results of binding experiments, in which we showed high-affinity interactions between the AnkSBD construct and isolated erythrocyte or non-erythroid spectrins. pEGFP-AnkSBD-transfected cells co-localised with non-erythroid spectrin in HeLa cells. The functional interactions of the AnkSBD construct in vivo and in vitro open many possibilities to study the structure and function of this domain, which has not yet been as extensively studied when compared to the aminoterminal domain of this protein.

The object of this paper is to review briefly the studies on the interactions of erythroid and non-erythroid spectrins with lipids in model and natural membranes. An important progress on the identification of lipid-binding sites has recently been made although many questions remain still unanswered. In particular, our understanding of the physiological role of such interactions is still limited. Another important issue is the occurrence of spectrins in membrane rafts, how they are attached to the raft and what is their function in rafts.

S-Acylation of proteins is a ubiquitous post-translational modification and a common signal for membrane association. The major palmitoylated protein in erythrocytes is MPP1, a member of the MAGUK family and an important component of the ternary complex that attaches the spectrin-based skeleton to the plasma membrane. Here we show that DHHC17 is the only acyltransferase present in red blood cells (RBC). Moreover, we give evidence that protein palmitoylation is essential for membrane organization and is crucial for proper RBC morphology, and that the effect is specific for MPP1. Our observations are based on the clinical cases of two related patients whose RBC had no palmitoylation activity, caused by a lack of DHHC17 in the membrane, which resulted in a strong decrease of the amount of detergent-resistant membrane (DRM) material. We confirmed that this loss of detergent-resistant membrane was due to the lack of palmitoylation by treatment of healthy RBC with 2-bromopalmitic acid (2-BrP, common palmitoylation inhibitor). Concomitantly, fluorescence lifetime imaging microscopy (FLIM) analyses of an order-sensing dye revealed a reduction of membrane order after chemical inhibition of palmitoylation in erythrocytes. These data point to a pathophysiological relationship between the loss of MPP1-directed palmitoylation activity and perturbed lateral membrane organization.

We document a new dimension of surface recognition in which communication is controlled through the collective behavior of lipids. Membrane cholesterol induces a tilt in glycolipid receptor headgroup, resulting in loss of access for ligand binding. This property appears to organize erythrocyte blood group presentation and glycolipid receptor function during the activation of sperm fertility, suggesting that lipid 'allostery' is a means to regulate membrane recognition processes.

The interactions of the neurotransmitter dopamine, and its precursor l-dopa, with membrane lipids were investigated through a set of molecular dynamic simulations with all atom resolution. The results obtained indicate that both dopamine and l-dopa have a pronounced association with the lipid head groups, predominantly mediated through H-bonds. As a result the molecules are anchored to the interfacial region of the membrane. The strength of this interaction is dependent on lipid composition - the presence of phosphatidylserine leads to an increase in the strength of this interaction, resulting in an H-bond network with a lifetime much longer than the timescale of our simulations. Also, bilayers that include sphingomieline and cholesterol interact strongly with dopamine and l-dopa. We postulate that the high membrane association that we have observed for both dopamine and l-dopa could have the following effects: 1) when on the plasma membrane exterior, favour the availability of these compounds for cell membrane uptake processes and, 2) when on an internal membrane surface, accentuate the importance of membrane-bound metabolizing enzymes over their soluble counterparts.

The conservation of fluidity is a theme common to all cell membranes. In this study, an analysis of lipid packing was conducted via C-laurdan spectroscopy of cell surface membranes prepared from representative species of Bacteria and Eukarya. We found that despite their radical differences in composition (namely the presence and absence of membrane-rigidifying sterol) the membrane order of all taxa converges on a remarkably similar level. To understand how this similarity is constructed, we reconstituted membranes with either bacterial or eukaryotic components. We found that transmembrane segments of proteins have an important role in buffering lipid-mediated packing. This buffering ensures that sterol-free and sterol-containing membranes exhibit similar barrier properties.

It was shown previously that an ankyrin-sensitive, phosphatidylethanolamine/phosphatidylcholine (PE/PC) binding site maps to the N-terminal part of the ankyrin-binding domain of β-spectrin (ankBDn). Here we have identified the amino acid residues within this domain which are responsible for recognizing monolayers and bilayers composed of PE/PC mixtures. In vitro binding studies revealed that a quadruple mutant with substituted hydrophobic residues W1771, L1775, M1778 and W1779 not only failed to effectively bind PE/PC, but its residual PE/PC-binding activity was insensitive to inhibition with ankyrin. Structure prediction and analysis, supported by in vitro experiments, suggests that “opening” of the coiled-coil structure underlies the mechanism of this interaction. Experiments on red blood cells and HeLa cells supported the conclusions derived from the model and in vitro lipid-protein interaction results, and showed the potential physiological role of this binding. We postulate that direct interactions between spectrin ankBDn and PE-rich domains play an important role in stabilizing the structure of the spectrin-based membrane skeleton.

The physical basis for protein partitioning into lipid rafts remains an outstanding question in membrane biology that has previously been addressed only through indirect techniques involving differential solubilization by nonionic detergents. We have used giant plasma membrane vesicles, a plasma membrane model system that phase separates to include an ordered phase enriching for raft constituents, to measure the partitioning of the transmembrane linker for activation of T cells (LAT). LAT enrichment in the raft phase was dependent on palmitoylation at two juxtamembrane cysteines and could be enhanced by oligomerization. This palmitoylation requirement was also shown to regulate raft phase association for the majority of integral raft proteins. Because cysteine palmitoylation is the only lipid modification that has been shown to be reversibly regulated, our data suggest a role for palmitoylation as a dynamic raft targeting mechanism for transmembrane proteins.

Increasing evidence suggests that biological membranes can be laterally subdivided into domains enriched in specific lipid and protein components and that these domains may be involved in the regulation of a number of vital cellular processes. An example is membrane rafts, which are lipid-mediated domains dependent on preferential association between sterols and sphingolipids and inclusive of a specific subset of membrane proteins. While the lipid and protein composition of rafts has been extensively characterized, the structural details determining protein partitioning to these domains remain unresolved. Here, we review evidence suggesting that post-translation modification by saturated lipids recruits both peripheral and transmembrane proteins to rafts, while short, unsaturated, and/or branched hydrocarbon chains prevent raft association. The most widely studied group of raft-associated proteins are glycophosphatidylinositol- anchored proteins (GPI-AP), and we review a variety of evidence supporting raft-association of these saturated lipid-anchored extracellular peripheral proteins. For transmembrane and intracellular peripheral proteins, S-acylation with saturated fatty acids mediates raft partitioning, and the dynamic nature of this modification presents an exciting possibility of enzymatically regulated raft association. The other common lipid modifications, that is, prenylation and myristoylation, are discussed in light of their likely role in targeting proteins to nonraft membrane regions. Finally, although the association between raft affinity and lipid modification is well-characterized, we discuss several open questions regarding regulation and remodeling of these post-translational modifications as well as their role in transbilayer coupling of membrane domains.

In biological processes, such as fission, fusion and trafficking, it has been shown that lipids of different shapes are sorted into regions with different membrane curvatures. This lipid sorting has been hypothesized to be due to the coupling between the membrane curvature and the lipid's spontaneous curvature, which is related to the lipid's molecular shape. On the other hand, theoretical predictions and simulations suggest that the curvature preference of lipids, due to shape alone, is weaker than that observed in biological processes. To distinguish between these different views, we have directly measured the curvature preferences of several lipids by using a fluorescence-based method. We prepared small unilamellar vesicles of different sizes with a mixture of egg-PC and a small mole fraction of N-nitrobenzoxadiazole (NBD)-labeled phospholipids or lysophospholipids of different chain lengths and saturation, and measured the NBD equilibrium distribution across the bilayer. We observed that the transverse lipid distributions depended linearly on membrane curvature, allowing us to measure the curvature coupling coefficient. Our measurements are in quantitative agreement with predictions based on earlier measurements of the spontaneous curvatures of the corresponding nonfluorescent lipids using X-ray diffraction. We show that, though some lipids have high spontaneous curvatures, they nevertheless showed weak curvature preferences because of the low values of the lipid molecular areas. The weak curvature preference implies that the asymmetric lipid distributions found in biological membranes are not likely to be driven by the spontaneous curvature of the lipids, nor are lipids discriminating sensors of membrane curvature.

Structural basis of wedging the Golgi membrane by FAPP pleckstrin homology domains Overduin and colleagues present the NMR structures of free, micelle and PtdIns(4)P-bound FAPP1-PH domain. The micelle-bound structure reveals how its prominent wedge independently tubulates Golgi membranes by leaflet penetration. A hydrophobic element inserts into and bends membranes, and is conserved in pleckstrin homology domains of CERT and OSBP proteins., The mechanisms underlying Golgi targeting and vesiculation are unknown, although the responsible phosphatidylinositol 4-phosphate (PtdIns(4)P) ligand and four-phosphate-adaptor protein (FAPP) modules have been defined. The micelle-bound structure of the FAPP1 pleckstrin homology domain reveals how its prominent wedge independently tubulates Golgi membranes by leaflet penetration. Mutations compromising the exposed hydrophobicity of full-length FAPP2 abolish lipid monolayer binding and compression. The trafficking process begins with an electrostatic approach, phosphoinositide sampling and perpendicular penetration. Extensive protein contacts with PtdIns(4)P and neighbouring phospholipids reshape the bilayer and initiate tubulation through a conserved wedge with features shared by diverse protein modules.

BACKGROUND: Specific interactions between sphingomyelin (SM) and cholesterol (Ch) are commonly believed to play a key role in the formation of rafts in the biological membranes. A weakness of this model is the implication that these microdomains are confined to the outer bilayer leaflet. The cytoplasmic leaflet, which contains the bulk of phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI), is thought also to harbour half of the membrane cholesterol. Moreover, SLPE (1-stearoyl-2-linoleoyl-sn-glycero-3-phosphatidyl-ethanolamine) has recently been shown to be enriched in isolated detergent-resistant membranes (DRM), and this enrichment was independent of the method of isolation of DRM. METHODOLOGY/PRINCIPAL FINDINGS: Here we present quantitative evidence coming from Langmuir-Blodgett monolayer experiments that SLPE forms complex with Ch similar to that between SM and Ch. The energies of these interactions as calculated form the monolayer studies are highly negative. FRAP analysis showed that NBD-Ch recovery was similar in liposomes composed of DOPC/Ch SM or SLPE but not DPPE, providing further evidence that SLPE may form an l(o) phase in the presence of high Ch concentration. Experiments on the solubility of DOPC liposomes containing DPPE/Ch (1ratio1), SM/Ch (1ratio1) or SLPE/Ch (1ratio1) showed the presence of Triton X-100 insoluble floating fraction (TIFF) in the case of SM/Ch or SLPE/Ch but not in DPPE/Ch containing liposomes. Quantitative determination of particular lipid species in the TIFF fraction confirms the conclusion that SLPE (or similar PE species) could be an important constituent of the inner leaflet raft. CONCLUSION: Such interactions suggest a possible existence of inner-leaflet nanoscale assemblies composed of cholesterol complexes with SLPE or similar unsaturated PE species.

The object of the chapter is to review the studies on the interactions of erythroid and nonerythroid spectrins and other membrane-skeletal proteins with lipids in model membranes. An important progress on the identification of lipid-binding sites has recently been made although many questions remain still unanswered. In particular, our understanding of the physiological role of such interactions is still limited. Important issue is the mechanism(s) involved in these interactions.

Understanding drug-membrane and drug-membrane protein interactions would be a crucial step towards understanding the action and biological properties of anthracyclines, as the cell membrane with its integral and peripheral proteins is the first barrier encountered by these drugs. In this paper, we briefly describe mitoxantrone-monolayer and mitoxantrone-bilayer interactions, focusing on the effect of mitoxantrone on the interactions between erythroid or nonerythroid spectrin with phosphatidylethanolamine-enriched mono- and bilayers. We found that mitoxantrone markedly modifies the interaction of erythroid and nonerythroid spectrins with phosphatidylethanolamine/phosphatidylcholine (PE/PC) monolayers. The change in delta pi induced by spectrins is several-fold larger in the presence of 72 nM mitoxantrone than in its absence: spectrin/mitoxantrone complexes induced a strong compression of the monolayer. Spin-labelling experiments showed that spectrin/mitoxantrone complexes caused significant changes in the order parameter measured using a 5'-doxyl stearate probe in the bilayer, but they practically did not affect the mobility of 16'-doxyl stearate. These results indicate close-to-surface interactions/penetrations without significant effect on the mid-region of the hydrophobic core of the bilayer. The obtained apparent equilibrium dissociation constants indicated relatively similar mitoxantrone-phospholipid and mitoxantrone-spectrin (erythroid and nonerythroid) binding affinities. These results might in part, explain the effect of mitoxantrone on spectrin distribution in the living cells.

Gene therapy is a new approach in the treatment of numerous diseases caused by damage to or lack of certain genes. Its application seems to be very effective, especially in the case of cancer diseases, where genetic drugs can be designed to selectively eliminate damaged cells. Due to the chemical properties of nucleic acids, their introduction into the cell is very difficult. DNA is a highly polar and negatively charged molecule unable to cross the plasma membrane. Moreover, "naked DNA" is exposed to enzymatic degradation by nucleases present within cells and biological fluids. Thus, for efficient and selective DNA delivery into the cells, it is necessary to use a suitable carrier system that can protect it from nucleases and enable it to cross the plasma membrane. Cationic lipid- and synthetic polymer-based carriers are very promising tools for gene delivery into different cell types, but there are still many obstacles to overcome before efficient gene therapy using synthetic vectors will be truly viable. Studies on the nature of interaction between genetic drugs and their lipid carriers, as well as on the mechanism of gene delivery into the cell allowed improvements to be made to currently existing synthetic vectors, and newer and more effective systems to be created. It is crucial that the lipid carriers and genetic drugs have a low degree of toxicity and high degree of specificity for effective gene delivery to occur. That is why the efforts of many laboratories are focused on designing and producing new forms of genetic drugs and derivatives of lipids in order to facilitate the creation of suitable gene delivery systems.

The object of this paper is to review briefly the studies on the interactions of erythroid and non-erythroid spectrins with lipids in model and natural membranes. An important progress on the identification of lipid-binding sites has recently been made although many questions remain still unanswered. In particular, our understanding of the physiological role of such interactions is still limited. Another important issue is the occurrence of spectrins in membrane rafts, how they are attached to the raft and what is their function in rafts.

Liposomes containing the polyethylene glycol (PEG) derivative of phosphatidyl ethanolamine (PE) have recently been found to be promising drug carriers, as they facilitate controlled and target-oriented release of therapeutics. They also reduce the side effects of many drugs. Here, we present the results of a study on antiliposomal properties of rabbit sera obtained after weekly injections of small liposomes containing 20% PEG-PE. The effect was analysed as the level of induced carboxyfluorescein release from these liposomes in vitro. The incubation of liposomes with rabbit serum taken after the injections induced the release of carboxyfluorescein at a higher level than was seen for incubation with untreated animal's serum. The strongest effect was observed for serum obtained after the second injection, i.e. during the second week of the study. The effect was much smaller after the serum samples were preheated at 56 degrees C. The binding of serum proteins by PEGylated liposomes was analysed via gel filtration and via the immunoblot technique using goat anti-rabbit IgG; this revealed that the serum protein which bound to the liposomes in vitro had a molecular weight of 55 kD and reacted with the anti-IgG antibody. Competition with PEG or lipids indicate that this IgG has an anti-PEG activity. We therefore assume that these antibodies are responsible for the activation of complement and leakage induction of PEG-liposomes. Such antibodies could be responsible for increased phagocytosis by RES macrophages (in particular liver macrophages) and decreased circulation time.

Atomistic molecular dynamics simulations were used to investigate the interactions between the neurotransmitter dopamine and its precursor L-dopa with membrane lipids. The results indicate that these molecules strongly interact with the lipid head groups e.g. via hydrogen bonds. These interactions anchor the dopamine and L-dopa to the membrane interfacial region. The strength of this bonding is dependent on lipids present in membrane. Presence of phosphatidylserine resulted in increased level of bonding strength with a lifetime longer than the timescale of our simulations.The high membrane association of dopamine and L-dopa both, extracellularly, might favour the availability of these compounds for cell membrane uptake processes and, intracellularly, can accentuate the importance of membrane-bound metabolizing enzymes over their soluble counterparts.We can also hypothesize that excessive association of dopamine and its precursor L-dopa with the membranes in the situation, when the concentration of phosphatidylserine is increased, may eventually limit the free use of dopamine as a synaptic transmitter what could possibly be a molecular level mechanism responsible for some of neurodegenerative disorders.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Spectrin and ankyrin are the key components of the erythrocyte cytoskeleton. The recently published crystal structure of the spectrin–ankyrin complex has indicated that their binding involves complementary charge interactions as well as hydrophobic interactions. However, only the former is supported by biochemical evidence. We now show that nonpolar interactions are important for high affinity complex formation, excluding the possibility that the binding is exclusively mediated by association of distinctly charged surfaces. Along these lines we report that substitution of a single hydrophobic residue, F917S in ankyrin, disrupts the structure of the binding site and leads to complete loss of spectrin affinity. Finally, we present data showing that minimal ankyrin binding site in spectrin is formed by helix 14C together with the loop between helices 15 B/C.

article Keywords: Lipid Partitioning GUV GPMV Raft STED-FCS Several simplified membrane models featuring coexisting liquid disordered (Ld) and ordered (Lo) lipid phases have been developed to mimic the heterogeneous organization of cellular membranes, and thus, aid our understanding of the nature and functional role of ordered lipid-protein nanodomains, termed "rafts". In spite of their greatly reduced complexity, quantitative characterization of local lipid environments using model membranes is not trivial, and the parallels that can be drawn to cellular membranes are not al- ways evident. Similarly, various fluorescently labeled lipid analogs have been used to study membrane orga- nization and function in vitro, although the biological activity of these probes in relation to their native counterparts often remains uncharacterized. This is particularly true for raft-preferring lipids ("raft lipids", e.g. sphingolipids and sterols), whose domain preference is a strict function of their molecular architecture, and is thus susceptible to disruption by fluorescence labeling. Here, we analyze the phase partitioning of a multitude of fluorescent raft lipid analogs in synthetic Giant Unilamellar Vesicles (GUVs) and cell-derived Giant Plasma Membrane Vesicles (GPMVs). We observe complex partitioning behavior dependent on label size, polarity, charge and position, lipid headgroup, and membrane composition. Several of the raft lipid an- alogs partitioned into the ordered phase in GPMVs, in contrast to fully synthetic GUVs, in which most raft lipid analogs mis-partitioned to the disordered phase. This behavior correlates with the greatly enhanced order difference between coexisting phases in the synthetic system. In addition, not only partitioning, but also ligand binding of the lipids is perturbed upon labeling: while cholera toxin B binds unlabeled GM1 in the Lo phase, it binds fluorescently labeled GM1 exclusively in the Ld phase. Fluorescence correlation spec- troscopy (FCS) by stimulated emission depletion (STED) nanoscopy on intact cellular plasma membranes consistently reveals a constant level of confined diffusion for raft lipid analogs that vary greatly in their par- titioning behavior, suggesting different physicochemical bases for these phenomena.