Labeling Of Lipids Research Articles

Diffusion of Lipid Nanovesicles Bound to a Lipid Membrane Is Associated with the Partial-Slip Boundary Condition.

During diffusion of nanoparticles bound to a cellular membrane by ligand–receptor pairs, the distance to the laterally mobile interface is sufficiently short for their motion to depend not only on the membrane-mediated diffusivity of the tethers but also in a not yet fully understood manner on nanoparticle size and interfacial hydrodynamics. By quantifying diffusivity, velocity, and size of individual membrane-bound liposomes subjected to a hydrodynamic shear flow, we have successfully separated the diffusivity contributions from particle size and number of tethers. The obtained diffusion-size relations for synthetic and extracellular lipid vesicles are not well-described by the conventional no-slip boundary condition, suggesting partial slip as well as a significant diffusivity dependence on the distance to the lipid bilayer. These insights, extending the understanding of diffusion of biological nanoparticles at lipid bilayers, are of relevance for processes such as cellular uptake of viruses and lipid nanoparticles or labeling of cell-membrane-residing molecules.

Translocation of the nonlabeled antimicrobial peptide PGLa across lipid bilayers and its entry into vesicle lumens without pore formation

Fluorescent-probe-labeled peptides are used to study the interactions of peptides with cells and lipid vesicles but labeling peptides with fluorescent probes can significantly change these interactions. We recently developed a new method to detect the entry of nonlabeled peptides into the lumen of single giant unilamellar vesicles (GUVs). Here we applied this method to examine the interaction of the antimicrobial peptide PGLa with single GUVs to elucidate whether PGLa can enter the GUV lumen without pore formation. First, we examined the interaction of nonlabeled PGLa with single GUVs comprising dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) (4/6) whose lumens contain the fluorescent probe AF647 and DOPG/DOPC (8/2)-large unilamellar vesicles encapsulating a high concentration of calcein. After a large lag period from starting the interaction with PGLa, the fluorescence intensity of the GUV lumen due to calcein (Icalcein) increased gradually without leakage of AF647, indicating that PGLa enters the GUV lumen without pore formation in the GUV membrane. The fraction of entry of PGLa increased with increasing PGLa concentration. Simultaneous measurement of the fractional area change of the GUV membrane (δ) and PGLa-induced increase in Icalcein showed that the entry of PGLa occurs only during the second increase in δ, indicating that PGLa enters the lumen during its translocation from the outer leaflet to the inner leaflet. The fraction of entry of PGLa without pore formation increased with increasing membrane tension. Based on these results, we discuss the elementary processes and the mechanism of the entry of PGLa into the GUV lumen.

Confocal Imaging Analysis of Mitochondrial Trafficking of Individual Lipid Species in Live Cells

Lipids exert dynamic biological functions which are determined both by their fatty acyl compositions and precise spatiotemporal distributions inside the cell. However, it remains a daunting task to investigate any of these features in live cells for each of the more than 1000 lipid species in a typical mammalian cell. Here we resolved this issue by developing a de novo lipid labeling method for major lipid species, including glycerolipids, glycerophospholipids, and cholesterol esters by using a single fluorescent probe. The method not only allowed us to probe the precise subcellular distribution and trafficking of individual lipid species in live cells, but also uncovered some unexpected biological functions of previously reported lipid metabolic enzymes that were not possible by conventional biochemical methods. We envision that this method will become an indispensable tool for the functional analysis of individual lipid species and numerous lipid metabolic enzymes and transporters in live cells.

Neutron Spin Echo Spectroscopy as a Unique Probe for Lipid Membrane Dynamics and Membrane-Protein Interactions

This paper describes the protocols for sample preparation, data reduction, and data analysis in neutron spin echo (NSE) studies of lipid membranes. Judicious deuterium labeling of lipids enables access to different membrane dynamics on mesoscopic length and time scales, over which vital biological processes occur.

Following de novo triglyceride dynamics in ovaries of Aedes aegypti during the previtellogenic stage

Understanding the molecular and biochemical basis of egg development is a central topic in mosquito reproductive biology. Lipids are a major source of energy and building blocks for the developing ovarian follicles. Ultra-High Resolution Mass Spectrometry (UHRMS) combined with in vivo metabolic labeling of follicle lipids with deuterated water (2H2O) can provide unequivocal identification of de novo lipid species during ovarian development. In the present study, we followed de novo triglyceride (TG) dynamics during the ovarian previtellogenic (PVG) stage (2–7 days post-eclosion) of female adult Aedes aegypti. The incorporation of stable isotopes from the diet was evaluated using liquid chromatography (LC) in tandem with the high accuracy (< 0.3 ppm) and high mass resolution (over 1 M) of a 14.5 T Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (14.5 T FT-ICR MS) equipped with hexapolar detection. LC-UHRMS provides effective lipid class separation and chemical formula identification based on the isotopic fine structure. The monitoring of stable isotope incorporation into de novo incorporated TGs suggests that ovarian lipids are consumed or recycled during the PVG stage, with variable time dynamics. These results provide further evidence of the complexity of the molecular mechanism of follicular lipid dynamics during oogenesis in mosquitoes.

Degradation of Photoreceptor Outer Segments by the Retinal Pigment Epithelium Requires Pigment Epithelium-Derived Factor Receptor (PEDF-R).

PurposeTo examine the contribution of pigment epithelium-derived factor receptor (PEDF-R) to the phagocytosis process. Previously, we identified PEDF-R, the protein encoded by the PNPLA2 gene, as a phospholipase A2 in the retinal pigment epithelium (RPE). During phagocytosis, RPE cells ingest abundant phospholipids and protein in the form of photoreceptor outer segment (POS) tips, which are then hydrolyzed. The role of PEDF-R in RPE phagocytosis is not known.MethodsMice in which PNPLA2 was conditionally knocked out (cKO) in the RPE were generated. Mouse RPE/choroid explants were cultured. Human ARPE-19 cells were transfected with siPNPLA2 silencing duplexes. POSs were isolated from bovine retinas. The phospholipase A2 inhibitor bromoenol lactone was used. Transmission electron microscopy, immunofluorescence, lipid labeling, pulse–chase experiments, western blots, and free fatty acid and β-hydroxybutyrate assays were performed.ResultsThe RPE of the cKO mice accumulated lipids, as well as more abundant and larger rhodopsin particles, compared to littermate controls. Upon POS exposure, RPE explants from cKO mice released less β-hydroxybutyrate compared to controls. After POS ingestion during phagocytosis, rhodopsin degradation was stalled both in cells treated with bromoenol lactone and in PNPLA2-knocked-down cells relative to their corresponding controls. Phospholipase A2 inhibition lowered β-hydroxybutyrate release from phagocytic RPE cells. PNPLA2 knockdown also resulted in a decline in fatty acids and β-hydroxybutyrate release from phagocytic RPE cells.ConclusionsPEDF-R downregulation delayed POS digestion during phagocytosis. The findings imply that the efficiency of RPE phagocytosis depends on PEDF-R, thus identifying a novel contribution of this protein to POS degradation in the RPE.

In Vivo Three-Photon Imaging of Lipids using Ultrabright Fluorogens with Aggregation-Induced Emission.

Fluorescent probes capable of in vivo lipids labeling are highly desirable for studying lipid-accumulation-related metabolic diseases, such as nonalcoholic fatty liver disease, type-2 diabetes, and atherosclerosis. However, most of the current lipid-specific fluorophores cannot be used for in vivo labeling due to their strong hydrophobicity. Herein, organic dots from bright luminogens with aggregation-induced emission (AIEgen) are developed for in vivo labeling and three-photon fluorescence imaging of lipid-rich tissues, such as fatty liver, atherosclerotic plaques in brain vasculatures, and carotid arteries. The organic dots show excellent stability in an aqueous medium with high targeting specificity to lipids and strong three-photon fluorescence in the far-red/near-infrared (NIR) region under NIR-II laser excitation, which enables efficient in vivo labeling and imaging of lipids in deep tissues. The study will inspire the development of lipid-targeting fluorophores for in vivo applications.

Local Labeling of Lipids

Local Labeling of Lipids

Liposome Formulations as Adjuvants for Vaccines.

Development of liposome-based formulations as vaccine adjuvants has been intimately associated with, and dependent on, and informed by, a fundamental understanding of biochemical and biophysical properties of liposomes themselves. The Walter Reed Army Institute of Research (WRAIR) has a fifty-year history of experience of basic research on liposomes; and development of liposomes as drug carriers; and development of liposomes as adjuvant formulations for vaccines. Uptake of liposomes by phagocytic cells in vitro has served as an excellent model for studying the intracellular trafficking patterns of liposomal antigen. Differential fluorescent labeling of proteins and liposomal lipids, together with the use of inhibitors, has enabled the visualization of physical locations of antigens, peptides, and lipids to elucidate mechanisms underlying the MHC class I and class II pathways in phagocytic APCs. Army Liposome Formulation (ALF) family of vaccine adjuvants, which have been developed and improved since 1986, and which range from nanosize to microsize, are currently being employed in phase 1 studies with different types of candidate vaccines.

Metabolic labeling of glycerophospholipids via clickable analogs derivatized at the lipid headgroup

Metabolic labeling, in which substrate analogs containing diminutive tags can infiltrate biosynthetic pathways and generate labeled products in cells, has led to dramatic advancements in the means by which complex biomolecules can be detected and biological processes can be elucidated. Within this realm, metabolic labeling of lipid products, particularly in a manner that is headgroup-specific, brings about a number of technical challenges including the complexity of lipid metabolic pathways as well as the simplicity of biosynthetic precursors to headgroup functionality. As such, only a handful of strategies for metabolic labeling of lipids have thus far been reported. However, these approaches provide enticing examples of how strategic modifications to substrate structures, particularly by introducing clickable moieties, can enable the hijacking of lipid biosynthesis. Furthermore, early work in this field has led to an explosion in diverse applications by which these techniques have been exploited to answer key biological questions or detect and track various lipid-containing biological entities. In this article, we review these efforts and emphasize recent advancements in the development and application of lipid metabolic labeling strategies.

Biguanides Induce Acute de novo Lipogenesis in Human Primary Sebocytes.

IntroductionAcne arises during puberty, in part, due to elevated hormones and growth factors which stimulate de novo lipogenesis (DNL) in primary sebocytes to significantly increase sebum production. Oral isotretinoin is an effective acne therapy, reducing sebum production through inducing apoptosis in sebocytes. However, isotretinoin is teratogenic and has additional unwanted side effects, including an initial acne flare-up, which limits its utility. The biguanide, metformin has been found to alleviate severe acne in women with polycystic ovary syndrome (PCOS) through normalization of their insulin and androgen hormone levels. Metformin’s broader effectiveness to improve acne in non-PCOS populations lacks significant clinical support. In an effort to determine whether biguanides directly affect sebogenesis, we investigated their ability to alter DNL in cell-based assays in vitro.MethodsDe novo lipogenesis was measured in human primary sebocytes using [14C]-acetate labeling. Lipid species analysis was performed by extracting newly synthesized lipids and subjecting them to thin layer chromatography. Gene expression changes in sebocytes were identified through qPCR analysis of isolated RNA. Metabolic parameters including oxygen consumption rate, lactate production and activation of adenosine monophosphate-dependent protein kinase (AMPK) were assessed in human primary sebocytes.ResultsUsing human primary sebocytes, we found that biguanides, isotretinoin and azithromycin induced an acute dose and time-dependent increase in [14C]-acetate labeling of neutral lipids, while AICAR, an AMPK activator, inhibited this DNL response. Biguanides did not activate AMPK in sebocytes, however, they significantly reduced oxygen consumption rate and increased lactate production. Treatment with biguanides, but not isotretinoin, significantly upregulated ACSS2 gene expression in primary sebocytes and showed synergism with lipogenic activators to induce DNL genes.DiscussionThese changes are consistent with an acute increase in sebocyte lipogenesis and support the potential of biguanides to cause an initial flare-up in patients suffering from severe acne.

Dependence of chlorophyll fluorescence quenching on the lipid-to-protein ratio in reconstituted light-harvesting complex II membranes containing lipid labels

The quenching of chlorophyll-a fluorescence was investigated in plant light-harvesting complex II (LHCII) embedded in reconstituted membranes containing thylakoid lipids and a lipid label. The proteoliposomes were further separated by density and the protein and lipid contents of the fractions were quantified spectrophotometrically, allowing tighter control over the L/P ratios in a wide range of values. Using time-resolved fluorescence, we found a strong correlation between the fluorescence quenching and the L/P ratio, in line with other studies reporting progressive quenching at low L/P ratios, presumably triggered by self-clustering in the membrane. The average fluorescence lifetimes decreased to 0.3 ns at L/P ratios below 50:1; these values are comparable to the quenching observed in plants under excess-light conditions and are accompanied by a similar far-red fluorescence signature. It is hypothesized that plants can exploit the intrinsic quenching propensity of LHCII-only membrane domains to safely store extra antenna units.

The Devil Is in the Details: What Do We Really Track in Single-Particle Tracking Experiments of Diffusion in Biological Membranes?

Single-particle tracking (SPT) is an experimental technique that allows one to follow the dynamics of individual molecules in biological membranes with unprecedented precision. Given the importance of lipid and membrane protein diffusion in the formation of nanoscale functional complexes, it is critical to understand what exactly is measured in SPT experiments. To clarify this issue, we employed nanoscale computer simulations designed to match SPT experiments that exploit streptavidin-functionalized Au nanoparticles (AuNPs). The results show that lipid labeling interferes critically with the diffusion process; thus, the diffusion measured in SPT is a far more complex process than what has been assumed. It turns out that the influence of AuNP-based labels on the dynamics of probe lipids includes not only the AuNP-induced viscous drag that is the more significant the larger the NP but, more importantly, also the effects related to the interactions of the streptavidin linker with membrane lipids. Due to these effects, the probe lipid moves in a concerted manner as a complex with the linker protein and numerous unlabeled lipids, which can slow down the motion of the probe by almost an order of magnitude. Furthermore, our simulations show that nonlinker streptavidin tetramers on the AuNP surface are able to interact with the membrane lipids, which could potentially lead to multivalent labeling of the NPs by the probe lipids. Our results further demonstrate that in the submicrosecond time domain the motion of the probe lipid is uncorrelated with the motion of the AuNP, showing that there is a 1 μs limit for the temporal resolution of the SPT technique. However, this limit for the temporal resolution depends on the nanoparticle size and increases rapidly with growing AuNPs. Overall, the results provide a molecular-scale framework to accurately interpret SPT data and to design protocols that minimize label-induced artifacts.

A Spotlight on Viruses-Application of Click Chemistry to Visualize Virus-Cell Interactions.

The replication of a virus within its host cell involves numerous interactions between viral and cellular factors, which have to be tightly controlled in space and time. The intricate interplay between viral exploitation of cellular pathways and the intrinsic host defense mechanisms is difficult to unravel by traditional bulk approaches. In recent years, novel fluorescence microscopy techniques and single virus tracking have transformed the investigation of dynamic virus-host interactions. A prerequisite for the application of these imaging-based methods is the attachment of a fluorescent label to the structure of interest. However, their small size, limited coding capacity and multifunctional proteins render viruses particularly challenging targets for fluorescent labeling approaches. Click chemistry in conjunction with genetic code expansion provides virologists with a novel toolbox for site-specific, minimally invasive labeling of virion components, whose potential has just recently begun to be exploited. Here, we summarize recent achievements, current developments and future challenges for the labeling of viral nucleic acids, proteins, glycoproteins or lipids using click chemistry in order to study dynamic processes in virus-cell interactions.

In Vitro Measurement of Sphingolipid Intermembrane Transport Illustrated by GLTP Superfamily Members.

Herein, we describe methodological approaches for measuring in vitro transfer of sphingolipids (SLs) between membranes. The approaches rely on direct tracking of the lipid. Typically, direct tracking involves lipid labeling via attachment of fluorophores or introduction of radioactivity. Members of the GlycoLipid Transfer Protein (GLTP) superfamily are used to illustrate two broadly applicable methods for direct lipid tracking. One method relies on Förster resonance energy transfer (FRET) that enables continuous assessment of fluorophore-labeled SL transfer in real time between lipid donor and acceptor vesicles. The second method relies on tracking of radiolabeled SL transfer by separation of lipid donor and acceptor vesicles at discrete time points. The assays are readily adjustable for assessing lipid transfer (1) between various model membrane assemblies (vesicles, micelles, bicelles, nanodiscs), (2) involving other lipid types by other lipid transfer proteins, (3) with protein preparations that are either crudely or highly purified, and (4) that is spontaneous and occurs in the absence of protein.

Towards measuring growth rates of pathogens during infections by D2 O-labeling lipidomics.

Microbial growth rate is an important physiological parameter that is challenging to measure in situ, partly because microbes grow slowly in many environments. Recently, it has been demonstrated that generation times of S. aureus in cystic fibrosis (CF) infections can be determined by D2 O-labeling of actively synthesized fatty acids. To improve species specificity and allow growth rate monitoring for a greater range of pathogens during the treatment of infections, it is desirable to accurately quantify trace incorporation of deuterium into phospholipids. Lipid extracts of D2 O-treated E. coli cultures were measured on liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) instruments equipped with time-of-flight (TOF) and orbitrap mass analyzers, and used for comparison with the analysis of fatty acids by isotope-ratio gas chromatography (GC)/MS. We then developed an approach to enable tracking of lipid labeling, by following the transition from stationary into exponential growth in pure cultures. Lastly, we applied D2 O-labeling lipidomics to clinical samples from CF patients with chronic lung infections. Lipidomics facilitates deuterium quantification in lipids at levels that are useful for many labeling applications (>0.03 at% D). In the E. coli cultures, labeling dynamics of phospholipids depend largely on their acyl chains and between phospholipids we notice differences that are not obvious from absolute concentrations alone. For example, cyclopropyl-containing lipids reflect the regulation of cyclopropane fatty acid synthase, which is predominantly expressed at the beginning of stationary phase. The deuterium incorporation into a lipid that is specific for S. aureus in CF sputum indicates an average generation time of the pathogen on the order of one cell doubling per day. This study demonstrates how trace level measurement of stable isotopes in intact lipids can be used to quantify lipid metabolism in pure cultures and provides guidelines that enable growth rate measurements in microbiome samples after incubation with a low percentage of D2 O.

Labeling of Phosphatidylinositol Lipid Products in Cells through Metabolic Engineering by Using a Clickable myo-Inositol Probe.

Phosphatidylinositol (PI) lipids control critical biological processes, so aberrant biosynthesis often leads to disease. As a result, the capability to track the production and localization of these compounds in cells is vital for elucidating their complex roles. Herein, we report the design, synthesis, and application of clickable myo-inositol probe 1 a for bioorthogonal labeling of PI products. To validate this platform, we initially conducted PI synthase assays to show that 1 a inhibits PI production in vitro. Fluorescence microscopy experiments next showed probe-dependent imaging in T-24 human bladder cancer and Candida albicans cells. Growth studies in the latter showed that replacement of myo-inositol with probe 1 a led to an enhancement in cell growth. Finally, fluorescence-based TLC analysis and mass spectrometry experiments support the labeling of PI lipids. This approach provides a promising means for tracking the complex biosynthesis and trafficking of these lipids in cells.

Vesicular Delivery of the Antifungal Antibiotics of Lysobacter enzymogenes C3.

Lysobacter enzymogenes C3 is a predatory strain of Gram-negative gliding bacteria that produces antifungal antibiotics by the polyketide synthetic pathway. Outer membrane vesicles (OMV) are formed as a stress response and can deliver virulence factors to host cells. The production of OMV by C3 and their role in antifungal activity are reported here. Vesicles in the range of 130 to 150 nm in diameter were discovered in the cell-free supernatants of C3 cultures. These OMV contain molecules characteristic of bacterial outer membranes, such as lipopolysaccharide and phospholipids. In addition, they contain chitinase activity and essentially all of the heat-stable antifungal activity in cell supernatants. We show here that C3 OMV can directly inhibit growth of the yeast Saccharomyces cerevisiae as well as that of the filamentous fungus Fusarium subglutinans The activity is dependent on physical contact between OMV and the cells. Furthermore, fluorescent lipid labeling of C3 OMV demonstrated transfer of the membrane-associated probe to yeast cells, suggesting the existence of a mechanism of delivery for membrane-associated molecules. Mass spectrometric analysis of C3 OMV extracts indicates the presence of molecules with molecular weights identical to some of the previously identified antifungal products of C3. These data together suggest that OMV act as an important remote mobile component of predation by LysobacterIMPORTANCE The data presented here suggest a newly discovered function of outer membrane vesicles (OMV) that are produced from the outer membrane of the bacterial species Lysobacter enzymogenes strain C3. We show that these OMV can be released from the surface of the cells to deliver antibiotics to target fungal organisms as a mechanism of killing or growth inhibition. Understanding the role of OMV in antibiotic delivery can generally lead to improved strategies for dealing with antibiotic-resistant organisms. These results also add to the evidence that some bacterially produced antibiotics can be discovered and purified using methods designed for isolation of nanoscale vesicles. Information on these systems can lead to better identification of active molecules or design of delivery vehicles for these molecules.

Changes in the IGF-1 and TNF-α synthesis pathways before and after three-month reduction diet with low glicemic index in women with PCOS.

An increase in IGF-I and TNF-α may be a cardioprotective effect. To examine the relationships between IGF-I and TNF-α and test the anthropometric and biochemical parameters before and after a low-glycemic index reduction diet using a correlation matrix. Twenty-two women diagnosed with PCOS according to Rotterdam's criteria were eligible for this study, which analysed the results before and after a three months dietary intervention. Body composition measurements were determined by bioimpedance and performed twice, along with the labelling of lipid, carbohydrate and hormonal profiles. IGF-I and TNF-α were also determined in the serum. Before dietary intervention, a significant correlation was observed. A correlation was also noted between the increase in TNF-α and DHEA-SO4, FSH, glucose level and total cholesterol. The increase in IGF-I was not related to anth-ropometric measurements: however, its concentration was observed to be related to the level of SHBG and HDL. After dietary intervention, the correlation between TNF-α and muscle mass percentage was confirmed, as was the correlation between WHR and fasting blood glucose levels. A significant negative correlation was observed between extracellular water, provided in litres, and SHBG level. One important role of IGF-I in PCOS pathogenesis is the stimulation of increased synthesis of SHBG and HDL. The increased level of IGF-I after the reduction diet had a cardioprotective effect. TNF-α inhibits FSH synthesis, preventing the growth of numerous follicles. Its synthesis is also related to DHEA-SO₄. After three-month reduction diet does not significantly reduce TNF-α.

Lipid Domains at the Plasma Membrane of Red Blood Cells: Organization and Involvement in Deformation

Erythrocytes are highly deformable cells that go through capillaries eight times-narrower than their diameter to deliver oxygen throughout the body. This deformability is linked to erythrocytes specific features such as their biconcave shape, their highly regulated hemoglobin concentration and the strong anchorage of their membrane to the underlying cytoskeleton. Based on our recent evidence for submicrometric lipid domains upon labeling of cholesterol and polar lipids (sphingomyelin, phosphatidylcholine, ganglioside GM1) in erythrocyte outer plasma membrane leaflet, we here explore the mapping of lipid domains in erythrocytes at resting state and under deformation. Thanks to differential properties between cholesterol- and polar lipid-enriched domains (curvature association, lipid order, temperature dependence, cholesterol content dependence) and partial spatial dissociation, we suggest the coexistence of at least two types of lipid domains at the erythrocyte surface at resting state: those enriched in cholesterol and preferentially associated with high-curvature membranes and those mostly enriched in phosphatidylcholine and ganglioside GM1 and associated with low-curvature membranes. Biophysical properties of these domains are currently explored by atomic force microscopy and force correlation spectroscopy. Lipid domains abundance, composition and biophysical properties are differentially modulated by (i) impairment of erythrocyte deformability (energy depletion, modulation of anchorage to the cytoskeleton by pharmacological and genetic approaches), (ii) simulation of deformation (calcium channel activation, stretching in silicon chambers) and (iii) simulation of shape restoration after deformation (stimulation of calcium efflux). Indeed, high-curvature cholesterol-enriched domains gather under deformation while low-curvature domains become enriched in sphingomyelin and lose some of their ganglioside GM1 enrichment. We hypothesize that cholesterol-enriched domains can help to stabilize high curvature membranes during deformation while polar lipid-enriched domains, especially those enriched in sphingomyelin, are involved into calcium balance regulation during or after deformation. This hypothesis is under investigation.