Membrane Lipid Organization Research Articles

Lipid Hydroperoxidation Effect on the Dynamical Evolution of the Conductance Process in Bilayer Lipid Membranes: A Condition Toward Criticality.

Cell membranes are one of the main targets of oxidative processes mediated by reactive oxygen species (ROS). These chemical species interact with unsaturated fatty acids of membrane lipids, triggering an autocatalytic chain reaction, producing lipid hydroperoxides (LOOHs) as the first relatively stable product of the ROS-mediated lipid peroxidation (LPO) process. Numerous biophysical and computational studies have been carried out to elucidate the LPO impact on the structure and organization of lipid membranes. However, although LOOHs are the major primary product of LPO of polyunsaturated fatty acids (PUFAs), to the best of our knowledge, there is no experimental evidence on the effects of the accumulation of these LPO byproducts on the electrical properties and the underlying dynamics of lipid membranes. In this work, bilayer lipid membranes (BLMs) containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline (POPC) with increasing hydroperoxidized POPC (POPC-OOH) molar proportions (BLMPC/PC-OOH) are used as model membranes to investigate the effect of LOOH-mediated LPO propagation on the electrical behavior of lipid bilayers. Voltage-induced ion current signals are analyzed by applying the fractal method of power spectrum density (PSD) analysis. We experimentally prove that, when certain LOOH concentration and energy threshold are overcome, oxidized membranes evolve toward a critical state characterized by the emergence of non-linear electrical behavior dynamics and the pore-type metastable structures formation. PSD analysis shows that temporal dynamics exhibiting "white" noise (non-time correlations) reflects a linear relationship between the input and output signals, while long-term correlations (β > 0.5) begin to be observed closely to the transition (critical point) from linear (Ohmic) to nonlinear (non-Ohmic) behavior. The generation of lipid pores appears to arise as an optimized energy dissipation mechanism based on the system's ability to self-organize and generate ordered structures capable of dissipating energy gradients more efficiently under stressful oxidative conditions.

Boar spermatozoa proteomic profile varies in sperm collected during the summer and winter

Boar sperm quality is less during the summer as a result of the different photoperiod or ambient temperatures as compared with the winter. The present study was conducted to elucidate possible variations in proteomic profiles of boar spermatozoa collected during the summer and winter. Effects of season on sperm viability, total motility, progressive motility, acrosome status, mitochondrial membrane potential and plasma membrane lipid organization were also analyzed. Only sperm viability and mitochondrial membrane potential were less during the summer (P < 0.05). Spermatozoa were processed and evaluated using the nano LC–MS/MS QTof procedures. A total of 1028 characterized proteins were identified in sperm collected during both seasons of the year (False Discovery Rate < 0.01) and, among the total, 85 proteins differed in sperm collected in the winter and summer, with there being a lesser abundance of these proteins when there were ejaculate collections during the summer (q-value ≤ 0.05). The results from enrichment assessments for these protein networks utilizing UniProtKB procedures for determining reproductive processes indicates there were 23 proteins that were less abundant in the summer than winter. These proteins have essential functions in spermatogenesis, sperm motility, acrosome reaction and fertilization. These results are the first where there was ascertaining of proteomic differences in boar spermatozoa collected in the summer and winter. These results might help to explain the decreased sperm quality and prolificity when semen of boars is used for artificial insemination that is collected during the season of the year when ambient temperatures are relatively greater.

Lipid Organization in Mixed Lipid Membranes Driven by Intrinsic Curvature Difference

Laurdan fluorescence, novel spectral fitting, and dynamic light scattering were combined to determine lateral lipid organization in mixed lipid membranes of the oxidized lipid, 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC), and each of the three bilayer lipids, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC). Second harmonic spectra were computed to determine the number of elementary emissions present. All mixtures indicated two emissions. Accordingly, spectra were fit to two log-normal distributions. Changes with PGPC mole fraction, XPGPC, of the area of the shorter wavelength line and of dynamic light scattering-derived aggregate sizes show that: DPPC and PGPC form component-separated mixed vesicles for XPGPC ≤ 0.2 and coexisting vesicles and micelles for XPGPC > 0.2 in gel and liquid-ordered phases and for all XPGPC in the liquid-disordered phase; POPC and PGPC form randomly mixed vesicles for XPGPC ≤ 0.2 and component-separated mixed vesicles for XPGPC > 0.2. DOPC and PGPC separate into vesicles and micelles. Component segregation is due to unstable inhomogeneous membrane curvature stemming from lipid-specific intrinsic curvature differences between mixing molecules. PGPC is inverse cone-shaped because its truncated tail with a terminal polar group points into the interface. It is similar to and mixes with POPC, also an inverse cone because of mobility of its unsaturated tail. PGPC is least similar to DOPC because mobilities of both unsaturated tails confer a cone shape to DOPC, and PGPC separates form DOPC. DPPC and PGPC do not mix in the liquid-disordered phase because mobility of both tails in this phase renders DPPC a cone. DPPC is a cylinder in the gel phase and of moderate similarity to PGPC and mixes moderately with PGPC.

Direct Imaging of Nanoscale Lipid Organization in Probe-Free Biomimetic Membranes

The three-dimensional architecture of biological membranes has functional consequences for living cells. In the outer leaflet of the plasma membrane (PM), lipids are thought to organize into ordered yet fluid domains, with diverse evidence supporting participation of these “rafts” in membrane processes including protein sorting and signaling. However, many details of PM domain structure remain elusive due to a lack of methods to directly probe membrane features at the nanoscale. We report direct imaging of coexisting nanoscopic domains in synthetic and bio-derived membranes without extrinsic probes using cryoEM. Analysis of images from vesicles composed of lipids ranging from 14 to 22 carbons in length show that cryoEM can resolve sub-angstrom differences in average bilayer thickness. Features in experimental images were reproduced in simulated images constructed from atomistic molecular dynamics simulations, revealing relationships between bilayer thickness measured with small-angle X-ray scattering and calculated from cryoEM images. We further used simulations to predict two sources of contrast between coexisting phases within the same vesicle, namely 1) differences in membrane thickness, and 2) electron density arising from different lipid packing in the two phases. We observed both sources of contrast in vesicles composed of saturated lipids, unsaturated lipids, and cholesterol. We conclude that cryoEM imaging enables quantitative analysis of membrane parameters such as thickness, domain size, and lipid packing, opening new avenues for direct investigation of nanoscopic membrane organization.

Supported Lipid Membranes at the Au‐Buffer Interface by Solvent Exchange: The Effect of Initial Solvent Concentration

The recently introduced solvent‐assisted lipid bilayer (SALB) formation method has proved a fast, simple, and versatile alternative to vesicle fusion in forming membrane‐mimicking platforms on solid supports such as SiO2, TiO2, graphene, and Au. This confers SALB with a broad applicability for developing biomaterial coatings for implants, biomimetic cell culture platforms, and cancer diagnostics. Although significant understanding on the interplay of the parameters controlling SALB (type of solvent, lipid concentration, and flow rate) has been achieved to date, there still exists several unexplored specific questions to be tackled. Herein, the question of how the initial solvent concentration affects the formation and organization of (zwitterionic) lipid membranes formed by SALB on Au surfaces is addressed. Using quartz crystal microbalance with dissipation monitoring, differences in frequency and dissipation responses are observed upon dissolving the lipid compound dimyristoyl phosphatidylcholine (DMPC) in either pure isopropanol or mixtures of different volume ratios of isopropanol and aqueous buffer. At high and medium initial solvent concentrations, thin and rigid supported lipid bilayers (SLBs) are typically formed at the Au–liquid interface, irrespective of the lipid structures formed in bulk, whereas at low solvent concentrations, irreversible vesicle adsorption takes place hindering the homogeneous SLB formation.

Interaction of Graphene Nanoparticles and Lipid Membranes Displaying Different Liquid Orderings: A Molecular Dynamics Study.

Understanding the effects of graphene-based nanomaterials on lipid membranes is fundamental to determine their environmental impact and the efficiency of their biomedical use. By means of molecular dynamics simulations of simple model lipid bilayers, we analyze in detail the different interaction modes. We have studied bilayers consisting of lipid species (including cholesterol) which display different internal liquid orderings. Nanometric graphene layers can be transiently adsorbed onto the lipid membrane and/or inserted in its hydrophobic region. Once inserted, graphene nanometric flakes display a diffusive dynamics in the membrane plane, they adopt diverse orientations depending on their size and oxidation degree, and they show a particular aversion to be placed close to cholesterol molecules in the membrane. Addition of graphene to phase-segregated ternary membranes is also investigated in the context of the lipid raft model for the lipid organization of biological membranes. Our simulation results show that graphene layers can be inserted indistinctly in the ordered and disordered regions. Once inserted, nanometric flakes migrate to disordered and cholesterol-poor lipid phases.

Effect of boar semen supplementation with recombinant heat shock proteins during summer

Artificial insemination (AI) in pigs is mainly performed with refrigerated boar semen. There is a marked negative seasonal effect on the quality of boar sperm, mainly due to relatively greater ambient temperatures; to counteract this thermal stress, sperm cells possess natural defensive mechanisms such as Heat Shock Proteins (HSPs) that prevent protein denaturation. Thus, the objective of this research was to improve the quality of commercial boar semen collected during the summer when ambient temperatures are greater using recombinant HSPs. For this purpose, different concentrations (0.1, 0.5 and 1 μg/ml) of recombinant heat shock proteins (HSPD1, HSPA8 or HSP86) were added to commercial boar semen and there was cooling for 48 h at 17 °C. After this storage period, sperm quality was assessed by analyzing sperm viability, mitochondrial membrane potential and plasma membrane lipid organization using flow cytometry; additionally, sperm motility was examined using a CASA system. Also, in vitro fertilization (IVF) using HSP-supplemented boar semen was performed and the quality of the embryos produced was evaluated using quantitative real-time polymerase chain reaction (qPCR) analyzing the relative abundance of mRNA transcripts for genes encoding for embryo quality-related proteins (BAX, TFAM, POLG and POG2). Sperm quality variables, blastocyst rates and the abundance of mRNA transcripts for the selected genes were not affected by the presence of recombinant HSPs at any concentration. These results indicate that the supplementation of commercial seminal doses with recombinant HSPs does not improve boar sperm quality or fertility during the summer months when ambient temperatures are greater.

Confocal Microscopy Confirmed that in Phosphatidylcholine Giant Unilamellar Vesicles with very High Cholesterol Content Pure Cholesterol Bilayer Domains Form.

The cholesterol (Chol) content in the fiber cell plasma membranes of the eye lens is extremely high, exceeding the solubility threshold in the lenses of old humans. This high Chol content forms pure Chol bilayer domains (CBDs) and Chol crystals in model membranes and membranes formed from the total lipid extracts from human lenses. CBDs have been detected using electron paramagnetic resonance (EPR) spin-labeling approaches. Here, we confirm the presence of CBDs in giant unilamellar vesicles prepared using the electroformation method from Chol/1-palmitoyl-2-oleoylphosphocholine and Chol/distearoylphosphatidylcholine mixtures. Confocal microscopy experiments using phospholipid (PL) analog (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-5,5'-disulfonic acid) and cholesterol analog fluorescent probes (23-(dipyrrometheneboron difluoride)-24-norcholesterol) were performed, allowing us to make three major conclusions: (1) In all membranes with a Chol/PL mixing ratio (expressed as a molar ratio) >2, pure CBDs were formed within the bulk PL bilayer saturated with Chol. (2) CBDs were present as the pure Chol bilayer and not as separate patches of Chol monolayers in each leaflet of the PL bilayer. (3) CBDs, presented as single large domains, were always located at the top of giant unilamellar vesicles, independent of the change in sample orientation (right-side-up/upside-down). Results obtained with confocal microscopy and fluorescent Chol and PL analogs, combined with those obtained using EPR and spin-labeled Chol and PL analogs, contribute to the understanding of the organization of lipids in the fiber cell plasma membranes of the human eye lens.

Systematic Affinity Purification Coupled to Mass Spectrometry Identified p62 as Part of the Cannabinoid Receptor CB2 Interactome.

The endocannabinoid system (ECS) consists particularly of cannabinoid receptors 1 and 2 (CB1 and CB2), their endogenous ligands, and enzymes that synthesize and degrade their ligands. It acts in a variety of organs and disease states ranging from cancer progression over neuropathic pain to neurodegeneration. Protein components engaged in the signaling, trafficking, and homeostasis machinery of the G-protein coupled CB2, are however largely unknown. It is therefore important to identify further interaction partners to better understand CB2 receptor functions in physiology and pathophysiology. For this purpose, we used an affinity purification and mass spectrometry-based proteomics approach of Strep-HA-CB2 receptor in HEK293 cells. After subtraction of background interactions and protein frequency library assessment we could identify 83 proteins that were classified by the identification of minimally 2 unique peptides as highly probable interactors. A functional protein association network analysis obtained an interaction network with a significant enrichment of proteins functionally involved in protein metabolic process, in endoplasmic reticulum, response to stress but also in lipid metabolism and membrane organization. The network especially contains proteins involved in biosynthesis and trafficking like calnexin, Sec61A, tubulin chains TUBA1C and TUBB2B, TMED2, and TMED10. Six proteins that were only expressed in stable CB2 expressing cells were DHC24, DHRS7, GGT7, HECD3, KIAA2013, and PLS1. To exemplify the validity of our approach, we chose a candidate having a relatively low number of edges in the network to increase the likelihood of a direct protein interaction with CB2 and focused on the scaffold/phagosomal protein p62/SQSTM1. Indeed, we independently confirmed the interaction by co-immunoprecipitation and immunocytochemical colocalization studies. 3D reconstruction of confocal images furthermore showed CB2 localization in close proximity to p62 positive vesicles at the cell membrane. In summary, we provide a comprehensive repository of the CB2 interactome in HEK293 cells identified by a systematic unbiased approach, which can be used in future experiments to decipher the signaling and trafficking complex of this cannabinoid receptor. Future studies will have to analyze the exact mechanism of the p62-CB2 interaction as well as its putative role in disease pathophysiology.

Switchable Solvatochromic Probes for Live‐Cell Super‐resolution Imaging of Plasma Membrane Organization

AbstractVisualization of the nanoscale organization of cell membranes remains challenging because of the lack of appropriate fluorescent probes. Herein, we introduce a new design concept for super‐resolution microscopy probes that combines specific membrane targeting, on/off switching, and environment sensing functions. A functionalization strategy for solvatochromic dye Nile Red that improves its photostability is presented. The dye is grafted to a newly developed membrane‐targeting moiety composed of a sulfonate group and an alkyl chain of varied lengths. While the long‐chain probe with strong membrane binding, NR12A, is suitable for conventional microscopy, the short‐chain probe NR4A, owing to the reversible binding, enables first nanoscale cartography of the lipid order exclusively at the surface of live cells. The latter probe reveals the presence of nanoscopic protrusions and invaginations of lower lipid order in plasma membranes, suggesting a subtle connection between membrane morphology and lipid organization.

Osh Proteins Control Nanoscale Lipid Organization Necessary for PI(4,5)P2 Synthesis.

SummaryThe plasma membrane (PM) is composed of a complex lipid mixture that forms heterogeneous membrane environments. Yet, how small-scale lipid organization controls physiological events at the PM remains largely unknown. Here, we show that ORP-related Osh lipid exchange proteins are critical for the synthesis of phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2], a key regulator of dynamic events at the PM. In real-time assays, we find that unsaturated phosphatidylserine (PS) and sterols, both Osh protein ligands, synergistically stimulate phosphatidylinositol 4-phosphate 5-kinase (PIP5K) activity. Biophysical FRET analyses suggest an unconventional co-distribution of unsaturated PS and phosphatidylinositol 4-phosphate (PI4P) species in sterol-containing membrane bilayers. Moreover, using in vivo imaging approaches and molecular dynamics simulations, we show that Osh protein-mediated unsaturated PI4P and PS membrane lipid organization is sensed by the PIP5K specificity loop. Thus, ORP family members create a nanoscale membrane lipid environment that drives PIP5K activity and PI(4,5)P2 synthesis that ultimately controls global PM organization and dynamics.

Abstract 784: Eicosapentaenoic Acid, Unlike Other Omega-3 Fatty Acids, Inhibits Membrane Cholesterol Crystalline Domains Under Conditions of Hyperglycemia

Prescription eicosapentaenoic acid (EPA) reduced cardiovascular events in the REDUCE-IT study population, including in patients with diabetes. During hyperglycemia, there is abnormal membrane cholesterol aggregation which has been linked to oxidative stress and plaque instability. EPA at a pharmacologic dose may preserve normal cholesterol distribution as compared to other omega-3 fatty acids (FAs), including docosahexaenoic acid (DHA), eicosatriaenoic acid (ETE) and α-linolenic acid (ALA). Membrane vesicles were prepared from dilinoleoylphosphatidylcholine (DLPC) at a cholesterol-to-phospholipid mole ratio of 0.6:1 and treated with the omega-3 FAs under conditions of hyperglycemia (200 mg/dL). Changes in membrane lipid organization and width were measured using small angle X-ray diffraction approaches. Cholesterol domains were identified by the presence of diffraction peaks corresponding to a unit cell periodicity or width of 34 Å. Results showed that only EPA was able to prevent the formation of cholesterol domains and had a membrane structure characterized by a normal width (54 Å) and cholesterol distribution despite hyperglycemia. But samples containing the other FAs had a biphasic membrane structure containing prominent cholesterol crystalline domains. The relative size of the cholesterol domains was greatest in the order of ALA&gt;DHA&gt;ETE. Thus, EPA preserved normal cholesterol distribution under conditions of hyperglycemia in a manner that was not reproduced with other omega-3 FAs. These data indicate differences between these long chain FAs and support a potential benefit for EPA in reducing atherosclerotic disease.

Effects of γ-zein peptides on lipid membrane organization: Quartz crystal microbalance with dissipation and Langmuir monolayer studies

γ-zein peptides (VHLPPP)n present great potential as efficient molecule carriers across the cell membrane. To further understand the binding behavior of peptides with cell membrane, simplified models consisting of peptides and mono/bi-layer membranes (Langmuir monolayer and liposome) were used to studied the resulting response in the structure of peptide and cell membrane in the present work. The results showed that (VHLPPP)n=1,3 bound to dipalmitoylphosphatidylcholine (DPPC) liposomes and was induced to self-assemble into polyproline II (PP II) structure. The incorporation of (VHLPPP)n=1,3 in the DPPC Langmuir monolayers resulted in an increase in the surface pressure and a decrease in the collapse pressure, suggesting the increase of lipid monolayer fluidity. Fluorescence images further confirmed that the presence of (VHLPPP)n=1,3 promoted the formation of liquid-expanded (LE, dis-ordered) phases in lipid monolayers. Atomic force microscopy images found that (VHLPPP)n=1,3 preferred to penetrate in LE phase domains. In addition, (VHLPPP)3 possesses higher binding affinity with DPPC membrane models compared to (VHLPPP)1.

Differential Aggregation and Phosphorylation of Alpha Synuclein in Membrane Compartments Associated With Parkinson Disease.

The aggregation of α-synuclein (α-syn) is a major factor behind the onset of Parkinson’s disease (PD). Sublocalization of this protein may be relevant for the formation of multimeric α-syn oligomeric configurations, insoluble aggregates that form Lewy bodies in PD brains. Processing of this protein aggregation is regulated by associations with distinct lipid classes. For instance, instability of lipid raft (LR) microdomains, membrane regions with a particular lipid composition, is an early event in the development of PD. However, the relevance of membrane microdomains in the regulation and trafficking of the distinct α-syn configurations associated with PD remains unexplored. In this study, using 6- and 14-month-old healthy and MPTP-treated animals as a model of PD, we have investigated the putative molecular alterations of raft membrane microstructures, and their impact on α-syn dynamics and conformation. A comparison of lipid analyses of LR microstructures and non-raft (NR) fractions showed alterations in gangliosides, cholesterol, polyunsaturated fatty acids (PUFA) and phospholipids in the midbrain and cortex of aged and MPTP-treated mice. In particular, the increase of PUFA and phosphatidylserine (PS) during aging correlated with α-syn multimeric formation in NR. In these aggregates, α-syn was phosphorylated in pSer129, the most abundant post-transductional modification of α-syn promoting toxic aggregation. Interestingly, similar variations in PUFA and PS content correlating with α-syn insoluble accumulation were also detected in membrane microstructures from the human cortex of incidental Parkinson Disease (iPD) and PD, as compared to healthy controls. Furthermore, structural changes in membrane lipid microenvironments may induce rearrangements in raft-interacting proteins involved in other neuropathologies. Therefore, we also investigated the dynamic of other protein markers involved in cognition and memory impairment such as metabotropic glutamate receptor 5 (mGluR5), ionotropic NMDA receptor (NMDAR2B), prion protein (PrPc) and amyloid precursor protein (APP), whose activity depends on membrane lipid organization. We observed a decline of these protein markers in LR fractions with the progression of aging and pathology. Overall, our findings demonstrate that lipid alterations in membranous compartments promoted by brain aging and PD-like injury may have an effect on α-syn aggregation and segregation in abnormal multimeric structures.

New insight into phase behavior and permeability of skin lipid models based on sphingosine and phytosphingosine ceramides

The intercellular lipid matrix of the stratum corneum (SC), which consist mainly of ceramides (CERs), free fatty acids and cholesterol, is fundamental to the skin barrier function. These lipids assemble into two lamellar phases, known as the long and short periodicity phases (LPP and SPP respectively). The LPP is unique in the SC and is considered important for the skin barrier function. Alterations in CER composition, as well as impaired skin barrier function, are commonly observed in diseased skin, yet the understanding of this relationship remains insufficient. In this study, we have investigated the influence of non-hydroxy and α-hydroxy sphingosine-based CERs and their phytosphingosine counterparts on the permeability and lipid organization of model membranes, which were adjusted in composition to enhance formation of the LPP. The permeability was compared by diffusion studies using ethyl-p-aminobenzoate as a model drug, and the lipid organization was characterized by X-ray diffraction and infrared spectroscopy. Both the sphingosine- and phytosphingosine-based CER models formed the LPP, while the latter exhibited a longer LPP repeat distance. The ethyl-p-aminobenzoate flux across the sphingosine-based CER models was higher when compared to the phytosphingosine counterparts, contrary to the fact that the α-hydroxy phytosphingosine-based CER model had the lowest chain packing density. The unanticipated low permeability of the α-hydroxy phytosphingosine-based model is probably associated with a stronger headgroup hydrogen bonding network. Our findings indicate that the increased level of sphingosine-based CERs at the expense of phytosphingosine-based CERs, as observed in the diseased skin, may contribute to the barrier function impairment.

The influence of 2-hydroxyoleic acid – an anticancer drug – on model membranes of different fluidity modulated by the cholesterol content

2-Hydroxyoleic acid (2OHOA, Minerval) is a Membrane Lipid Therapy drug with anti-cancer activity. Therapeutic effect of this substance is associated with its incorporation into cell membrane. However, despite its effectiveness in cancer treatment and the lack of the side effects, the molecular mechanism of Minerval action is not fully clarified. In this study the influence of 2OHOA on fluidity, permeability and molecular organization of artificial lipid membranes in the context of its membrane-related anticancer properties was investigated. The experiments were performed for raft-like model, composed of the equimolar mixture of sphingomyelin, palmitoyloleoylphosphatidylcholine and cholesterol, as well as for the systems with lower cholesterol content. As model membranes Langmuir films and liposomes were applied. The obtained findings reveal that the incorporation of 2OHOA into artificial lipid membranes leads to the alterations in the properties of the systems. It was found that 2OHOA provokes the chain-disordering, fluidization and the increase in permeability of the studied systems. Both the results of monolayers and bilayers studies show that the strongest 2OHOA effect is observed for raft like mixture. The latter may indicate that the molecular mechanism of action of this compound is closely related to its interactions with lipid rafts – domains that are responsible for the regulation of various cellular functions.

Complex Coacervate Formation on a Heterogeneous Membrane

Mixtures of oppositely charged polymers in salt solutions can undergo liquid-liquid phase separation through a process called complex coacervation. Polyelectrolyte coacervates are liquid droplets enriched in both polymers and stabilized via electrostatic interactions that occur in three dimensions. Multicomponent lipid membranes can also undergo a liquid-liquid phase separation within the two-dimensional plane of the membrane. Phase separated membrane domains are enriched in particular lipid species and are stabilized via interactions between lipids that are highly temperature dependent. Here we investigate the coupling of these two distinct phase transitions by tethering polyelectrolytes directly to a lipid bilayer. We demonstrate that constraining one polymer to a uniform supported membrane supports polymer coacervation at much lower polymer concentrations than are required to form droplets in three-dimensions, consistent with previous reports. We are also investigating coacervate formation on multicomponent membranes capable of forming liquid-ordered and liquid-disordered phases, and can selectively conjugate polyelectrolytes to lipids that partition strongly into one phase. Our overall goal of this work is to investigate regimes where polymer interactions impact the organization of membrane lipids, or where heterogeneity of membrane lipids contributes to the stabilization of coacervates. Analogous coupling of phase transitions is hypothesized to occur in a variety of biological contexts, including at sites of immunoreceptor activation and neuronal synapses.

Osh Proteins Control Nanoscale Lipid Organization Necessary for PI(4,5)P &lt;sub&gt;2&lt;/sub&gt; Synthesis

The plasma membrane (PM) is composed of a complex lipid mixture that forms heterogeneous membrane environments. Yet how small-scale lipid organization controls physiological events at the PM remains largely unknown. Here, we show that ORP/Osh lipid exchange proteins are critical for the synthesis of phosphatidylinositol (4,5)-bisphosphate, PI(4,5)P2, a key regulator of dynamic events at the PM. In real-time assays, we find that unsaturated phosphatidylserine (PS) and sterols, both ORP/Osh protein ligands, synergistically stimulate phosphatidylinositol 4-phosphate 5-kinase (PIP5K) activity. Our biophysical and simulation analyses further reveal an unconventional co-segregation of unsaturated PS and phosphatidylinositol 4-phosphate (PI4P) species in sterol-containing membrane bilayers. Moreover, we show that Osh protein-mediated unsaturated PI4P/PS membrane lipid organization is sensed by the PIP5K specificity loop. Thus, ORP/Osh family members create an unsaturated phospholipid nanoscale environment that drives PIP5K activity and PI(4,5)P2 synthesis that ultimately controls global PM organization and dynamics.

Exploring membrane organization at varying spatiotemporal resolutions utilizing fluorescence-based approaches: implications in membrane biology.

Biological membranes are characterized by lateral inhomogeneities, termed as membrane domains, which are regions enriched with specific types of lipids and proteins. While the functional consequences of membrane domains are well understood, the physicochemical study of domains has proved to be elusive, mainly due to varying spatiotemporal scales associated with them. In this perspective, we provide an overview of representative experimental approaches based on dynamic fluorescence microscopy to analyze organization and dynamics of membrane lipids and proteins. We further elucidate variation of dynamics as a function of area of observation, a unique feature of biological membranes, and its modulation with membrane components such as cholesterol and the actin cytoskeleton. In terms of spatial resolution, we provide examples from super resolution techniques that overcome the diffraction limit encountered in conventional optical microscopes. We conclude that judicious use of a combination of approaches of varying spatiotemporal resolutions, commensurate with spatiotemporal scales of a given membrane process, would provide a comprehensive dynamic model of the biological membrane in terms of membrane organization, dynamics and function.

Membrane Lipid Nanodomains.

Lipid membranes can spontaneously organize their components into domains of different sizes and properties. The organization of membrane lipids into nanodomains might potentially play a role in vital functions of cells and organisms. Model membranes represent attractive systems to study lipid nanodomains, which cannot be directly addressed in living cells with the currently available methods. This review summarizes the knowledge on lipid nanodomains in model membranes and exposes how their specific character contrasts with large-scale phase separation. The overview on lipid nanodomains in membranes composed of diverse lipids (e.g., zwitterionic and anionic glycerophospholipids, ceramides, glycosphingolipids) and cholesterol aims to evidence the impact of chemical, electrostatic, and geometric properties of lipids on nanodomain formation. Furthermore, the effects of curvature, asymmetry, and ions on membrane nanodomains are shown to be highly relevant aspects that may also modulate lipid nanodomains in cellular membranes. Potential mechanisms responsible for the formation and dynamics of nanodomains are discussed with support from available theories and computational studies. A brief description of current fluorescence techniques and analytical tools that enabled progress in lipid nanodomain studies is also included. Further directions are proposed to successfully extend this research to cells.