Generalized Polarization Values Research Articles

Membrane fluidity properties of lipid-coated polylactic acid nanoparticles.

Lipid coating is considered a versatile strategy to equip nanoparticles (NPs) with a biomimetic surface coating, but the membrane properties of these nanoassemblies remain in many cases insufficiently understood. In this work, we apply C-Laurdan generalized polarization (GP) measurements to probe the temperature-dependent polarity of hybrid membranes consisting of a lipid monolayer adsorbed onto a polylactic acid (PLA) polymer core as function of lipid composition and compare the behavior of the lipid coated NPs (LNPs) with that of liposomes assembled from identical lipid mixtures. The LNPs were generated by nanoprecipitation of the polymer in aqueous solutions containing two types of lipid mixtures: (i) cholesterol, dipalmitoylphosphatidylcholine (DPPC), and the ganglioside GM3, as well as (ii) dioleoylphosphatidylcholine (DOPC), DPPC and GM3. LNPs were found to exhibit more distinct and narrower phase transitions than corresponding liposomes and to retain detectable phase transitions even for cholesterol or DOPC concentrations that yielded no detectable transitions in liposomes. These findings together with higher GP values in the case of the LNPs for temperatures above the phase transition temperature indicate a stabilization of the membrane through the polymer core. LNP binding studies to GM3-recognizing cells indicate that differences in the membrane fluidity affect binding avidity in the investigated model system.

Novel bioactive lipids enhanced HDL-mediated cholesterol efflux from macrophages through the ABCA1 receptor pathway

High-density lipoprotein (HDL) has traditionally been acknowledged as "good cholesterol" owing to its significant association with a decreased risk of atherosclerosis. This association is primarily attributed to HDL's direct involvement in cholesterol efflux capacity, which plays a pivotal role in reverse cholesterol transport. A novel active compound from Nannochloropsis microalgae termed lyso-DGTS, a lipid that contains EPA fatty acids, was previously isolated and found to increase paraoxonase 1 activity and enhance HDL-mediated cholesterol efflux and HDL-induced endothelial nitric oxide release. Here, the effect of different lyso-DGTS derivatives and analogs on HDL-mediated cholesterol efflux from macrophages was examined, and the mechanism was explored. Structure–activity relationships were established to characterize the essential lipid moieties responsible for HDL-mediated cholesterol efflux from macrophages. Lyso-DGTS, 1-carboxy-N-N-N-trimethyl-3-oleamidopropan-1-aminium, and lyso-platelet-activating factor increased HDL-mediated cholesterol efflux from macrophages dose-dependently, mainly via the ABCA1-mediated cholesterol efflux pathway. The effect of lyso-DGTS derivatives and analogs on the surface polarity of HDL was examined using the Laurdan generalized polarization (GP) assay. A reverse Pearson linear regression was obtained between Laurdan GP values and HDL-mediated cholesterol efflux. Because the incorporation of bioactive lipids into the surface phospholipid layer of HDL leads to a decrease in Laurdan GP, these bioactive lipids may induce lower phospholipid ordering and greater free space on the HDL particle surface, thereby enhancing apolipoprotein A1 binding to the ABCA1 receptor and improving ABCA1 cholesterol-mediated efflux. Our findings suggest a beneficial effect of lyso-DGTS and its bioactive lipid derivatives on increasing HDL-mediated cholesterol efflux activity from macrophages, which may impact atherosclerosis attenuation.

FRET-GP - A Local Measure of the Impact of Transmembrane Peptide on Lipids.

Reconstitution of a transmembrane protein in model lipid systems allows studying its structure and dynamics in isolation from the complexity of the natural environment. This approach also provides a well-defined environment for studying the interactions of proteins with lipids. In this work, we describe the FRET-GP method, which utilizes Förster resonance energy transfer (FRET) to specifically probe the nanoenvironment of a transmembrane domain. The tryptophan residues flanking this domain act as efficient FRET donors, while Laurdan acts as acceptor. The fluorescence of this solvatochromic probe is quantified using generalized polarization (GP) to report on lipid mobility in the vicinity of the transmembrane domain. We applied FRET-GP to study the transmembrane peptide WALP incorporated in liposomes. We found that the direct excitation of Laurdan to its second singlet state strongly contributes to GP values measured in FRET conditions. Removal of this parasitic contribution was essential for proper determination of GPFRET - the local analogue of classical GP parameter. The presence of WALP significantly increased both parameters but the local effects were considerably stronger (GPFRET ≫ GP). We conclude that WALP restricts lipid movement in its vicinity, inducing lateral inhomogeneity in membrane fluidity. WALP was also found to influence lipid phase transition. Our findings demonstrated that FRET-GP simultaneously provides local and global results, thereby enhancing the depth of information obtained from the measurement. We highlight the simplicity and sensitivity of the method, but also discuss its potential and limitations in studying protein-lipid interactions.

Effect of Lipid Raft Disruptors on Cell Membrane Fluidity Studied by Fluorescence Spectroscopy.

Lipid rafts are specialized microdomains in cell membranes, rich in cholesterol and sphingolipids, and play an integrative role in several physiological and pathophysiological processes. The integrity of rafts can be disrupted via their cholesterol content-with methyl-β-cyclodextrin (MCD) or with our own carboxamido-steroid compound (C1)-or via their sphingolipid content-with sphingomyelinase (SMase) or with myriocin (Myr). We previously proved by the fluorescent spectroscopy method with LAURDAN that treatment with lipid raft disruptors led to a change in cell membrane polarity. In this study, we focused on the alteration of parameters describing membrane fluidity, such as generalized polarization (GP), characteristic time of the GP values change-Center of Gravity (τCoG)-and rotational mobility (τrot) of LAURDAN molecules. Myr caused a blue shift of the LAURDAN spectrum (higher GP value), while other agents lowered GP values (red shift). MCD decreased the CoG values, while other compounds increased it, so MCD lowered membrane stiffness. In the case of τrot, only Myr lowered the rotation of LAURDAN, while the other compounds increased the speed of τrot, which indicated a more disordered membrane structure. Overall, MCD appeared to increase the fluidity of the membranes, while treatment with the other compounds resulted in decreased fluidity and increased stiffness of the membranes.

Laurdan in live cell imaging: Effect of acquisition settings, cell culture conditions and data analysis on generalized polarization measurements.

Cell function is highly dependent on membrane structure, organization, and fluidity. Therefore, methods to probe the biophysical properties of biological membranes are required. Determination of generalized polarization (GP) values using Laurdan in fluorescence microscopy studies is one of the most widely-used methods to investigate changes in membrane fluidity in vitro and in vivo. In the last couple of decades, there has been a major increase in the number of studies using Laurdan GP, where several different methodological approaches are used. Such differences interfere with data interpretation inasmuch as it is difficult to validate if Laurdan GP variations actually reflect changes in membrane organization or arise from biased experimental approaches. To address this, we evaluated the influence of different methodological details of experimental data acquisition and analysis on Laurdan GP. Our results showed that absolute GP values are highly dependent on several of the parameters analyzed, showing that incorrect data can result from technical and methodological inconsistencies. Considering these differences, we further analyzed the impact of cell variability on GP determination, focusing on basic cell culture conditions, such as cell confluency, number of passages and media composition. Our results show that GP values can report alterations in the biophysical properties of cell membranes caused by cellular adaptation to the culture conditions. In summary, this study provides thorough analysis of the factors that can lead to Laurdan GP variability and suggests approaches to improve data quality, which would generate more precise interpretation and comparison within individual studies and among the literature on Laurdan GP.

Acidic pH-induced changes in lipid nanoparticle membrane packing

To enable the release of the encapsulated nucleic acids into the cytosol of targeted cells, the interaction of lipid nanoparticles (LNPs) with endosomes is critical. We investigated changes in the physicochemical properties of LNPs containing ionizable cationic lipids that were induced by acidic pH, which reflects the conditions in the maturation of endosomes. We prepared a LNP containing an ionizable cationic lipid. The laurdan generalized polarization values, which are related to the hydration degree of the lipid membrane interface and are often used as an indicator of membrane packing, decreased with a decrease in pH value, showing that the membrane packing was decreased under acidic conditions. Furthermore, the pH-induced variation increased with an increasing percentage of ionizable cationic lipids in the LNPs. These results indicated that electrostatic repulsion between lipid molecules at acidic pH decreased the packing density of the lipids in the LNP membrane. Reducing the order of lipids could be a trigger to form a non-bilayer structure and allow fusion of the LNPs with the membrane of maturing endosomes in an acidic environment. The LNPs were used to incorporate and transport small interfering RNA (siRNA) into cells for knockdown of the expression of β-galactosidase. The knockdown efficiency of siRNA encapsulated in LNPs tended to increase with the ratio of KC2. These results, which demonstrate the underlying phenomena for the fusion of membranes, will help clarify the mechanism of the release of encapsulated nucleic acids.

Polyoxyethylene/polyoxypropylene dimethyl ether (EPDME) random copolymer improves lipid structural ordering in stratum corneum of an epidermal-equivalent model as seen by two-photon microscopy.

Topical application of polyoxyethylene/polyoxypropylene dimethyl ether (EPDME) random copolymer improves the barrier function of skin, whereas polyethylene glycol (PEG) and polypropylene glycol (PPG) are ineffective. The aim of this work was to examine the interaction between these polymers and lipid molecules in the stratum corneum in order to establish whether EPDME-specific changes in the structural ordering of lipids might account for the improvement of barrier function. We used two-photon microscopy to evaluate the effects of EPDME, PEG, and PPG on the structural ordering of lipids in an epidermal-equivalent model in terms of the fluorescence changes of Laurdan, a fluorescent dye that responds to changes of membrane fluidity. The generalized polarization (GP) value, a parameter that reflects lipid ordering, was measured at various depths from the surface of the stratum corneum. EPDME increased the GP value to a depth of about 3µm from the surface, indicating that lipid ordering was increased in this region, while PEG and PPG of the same molecular weight had no effect. Diffusion of Lucifer yellow into the epidermis was reduced after application of EPDME, indicating that the barrier function was improved. These results support the view that EPDME improves barrier function by increasing the ordering of lipid structures in the stratum corneum. The methodology described here could be useful for screening new compounds that would improve the structural ordering of lipids.

Sulfolobus acidocaldarius Microvesicles Exhibit Unusually Tight Packing Properties as Revealed by Optical Spectroscopy.

In this study, we used optical spectroscopy to characterize the physical properties of microvesicles released from the thermoacidophilic archaeon Sulfolobus acidocaldarius (Sa-MVs). The most abundant proteins in Sa-MVs are the S-layer proteins, which self-assemble on the vesicle surface forming an array of crystalline structures. Lipids in Sa-MVs are exclusively bipolar tetraethers. We found that when excited at 275 nm, intrinsic protein fluorescence of Sa-MVs at 23 °C has an emission maximum at 303 nm (or 296 nm measured at 75 °C), which is unusually low for protein samples containing multiple tryptophans and tyrosines. In the presence of 10–11 mM of the surfactant n-tetradecyl-β-d-maltoside (TDM), Sa-MVs were disintegrated, the emission maximum of intrinsic protein fluorescence was shifted to 312 nm, and the excitation maximum was changed from 288 nm to 280.5 nm, in conjunction with a significant decrease (>2 times) in excitation band sharpness. These data suggest that most of the fluorescent amino acid residues in native Sa-MVs are in a tightly packed protein matrix and that the S-layer proteins may form J-aggregates. The membranes in Sa-MVs, as well as those of unilamellar vesicles (LUVs) made of the polar lipid fraction E (PLFE) tetraether lipids isolated from S. acidocaldarius (LUVPLFE), LUVs reconstituted from the tetraether lipids extracted from Sa-MVs (LUVMV) and LUVs made of the diester lipids, were investigated using the probe 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan). The generalized polarization (GP) values of Laurdan in tightly packed Sa-MVs, LUVMV, and LUVPLFE were found to be much lower than those obtained from less tightly packed DPPC gel state, which echoes the previous finding that the GP values from tetraether lipid membranes cannot be directly compared with the GP values from diester lipid membranes, due to differences in probe disposition. Laurdan’s GP and red-edge excitation shift (REES) values in Sa-MVs and LUVMV decrease with increasing temperature monotonically with no sign for lipid phase transition. Laurdan’s REES values are high (9.3–18.9 nm) in the tetraether lipid membrane systems (i.e., Sa-MVs, LUVMV and LUVPLFE) and low (0.4–5.0 nm) in diester liposomes. The high REES and low GP values suggest that Laurdan in tetraether lipid membranes, especially in the membrane of Sa-MVs, is in a very motionally restricted environment, bound water molecules and the polar moieties in the tetraether lipid headgroups strongly interact with Laurdan’s excited state dipole moment, and “solvent” reorientation around Laurdan’s chromophore in tetraether lipid membranes occurs very slowly compared to Laurdan’s lifetime.

Loosening of Lipid Packing by Cell-Surface Recruitment of Amphiphilic Peptides by Coiled-Coil Tethering.

Lipid packing has a strong influence on the formation and structural dynamics of cell membranes. Techniques to modulate lipid packing may thus enable modification of cellular functions and events. An 18-residue amphiphilic helical peptide derived from the N-terminal segment of epsin-1 (EpN18) is reported to induce positive membrane curvature and to loosen lipid packing in the cell membrane. In this study, it is shown that EpN18, crosslinked to a leucine-zipper peptide K4, is recruited to the cell surface by interacting with a cell-surface-expressed E3 leucine-zipper segment. Cell-surface tethering markedly enhanced loosening of lipid packing, which led to the promotion of membrane translocation of octaarginine. The loosening of lipid packing by EpN18 was also confirmed by analyzing the generalized polarization value with a membrane-environment-sensitive dye, 2-hydroxy-3-{2-[(2-hydroxyethyl)dimethylamino]ethyl}-4-{2-[6-(dibutylamino)-2-naphthyl]ethenyl}pyridiniumdibromide (di-4-ANEPPDHQ). This approach thus shows promise for the control of lipid packing and related cellular events.

Intragenic Antimicrobial Peptide Hs02 Hampers the Proliferation of Single- and Dual-Species Biofilms of P. aeruginosa and S. aureus: A Promising Agent for Mitigation of Biofilm-Associated Infections.

Pseudomonas aeruginosa and Staphylococcus aureus are two major pathogens involved in a large variety of infections. Their co-occurrence in the same site of infection has been frequently reported and is linked to enhanced virulence and difficulty of treatment. Herein, the antimicrobial and antibiofilm activities of an intragenic antimicrobial peptide (IAP), named Hs02, which was uncovered from the human unconventional myosin 1H protein, were investigated against several P. aeruginosa and S. aureus strains, including multidrug-resistant (MDR) isolates. The antibiofilm activity was evaluated on single- and dual-species biofilms of P. aeruginosa and S. aureus. Moreover, the effect of peptide Hs02 on the membrane fluidity of the strains was assessed through Laurdan generalized polarization (GP). Minimum inhibitory concentration (MIC) values of peptide Hs02 ranged from 2 to 16 μg/mL against all strains and MDR isolates. Though Hs02 was not able to hamper biofilm formation by some strains at sub-MIC values, it clearly affected 24 h preformed biofilms, especially by reducing the viability of the bacterial cells within the single- and dual-species biofilms, as shown by confocal laser scanning microscopy (CLSM) and atomic force microscopy (AFM) images. Laurdan GP values showed that Hs02 induces membrane rigidification in both P. aeruginosa and S. aureus. Peptide Hs02 can potentially be a lead for further improvement as an antibiofilm agent.

Effect of Cholesterol on Membrane Fluidity and Association of Aβ Oligomers and Subsequent Neuronal Damage: A Double-Edged Sword.

Background: The beta-amyloid peptide (Aβ) involved in Alzheimer’s disease (AD) has been described to associate/aggregate on the cell surface disrupting the membrane through pore formation and breakage. However, molecular determinants involved for this interaction (e.g., some physicochemical properties of the cell membrane) are largely unknown. Since cholesterol is an important molecule for membrane structure and fluidity, we examined the effect of varying cholesterol content with the association and membrane perforation by Aβ in cultured hippocampal neurons.Methods: To decrease or increase the levels of cholesterol in the membrane we used methyl-β-cyclodextrin (MβCD) and MβCD/cholesterol, respectively. We analyzed if membrane fluidity was affected using generalized polarization (GP) imaging and the fluorescent dye di-4-ANEPPDHQ. Additionally membrane association and perforation was assessed using immunocytochemistry and electrophysiological techniques, respectively.Results: The results showed that cholesterol removal decreased the macroscopic association of Aβ to neuronal membranes (fluorescent-puncta/20 μm: control = 18 ± 2 vs. MβCD = 10 ± 1, p < 0.05) and induced a facilitation of the membrane perforation by Aβ with respect to control cells (half-time for maximal charge transferred: control = 7.2 vs. MβCD = 4.4). Under this condition, we found an increase in membrane fluidity (46 ± 3.3% decrease in GP value, p < 0.001). On the contrary, increasing cholesterol levels incremented membrane rigidity (38 ± 2.7% increase in GP value, p < 0.001) and enhanced the association and clustering of Aβ (fluorescent-puncta/20 μm: control = 18 ± 2 vs. MβCD = 10 ± 1, p < 0.01), but inhibited membrane disruption.Conclusion: Our results strongly support the significance of plasma membrane organization in the toxic effects of Aβ in hippocampal neurons, since fluidity can regulate distribution and insertion of the Aβ peptide in the neuronal membrane.

Atomic Force Microscopy Study on the Stiffness of Nanosized Liposomes Containing Charged Lipids

It has recently been recognized that the mechanical properties of lipid nanoparticles play an important role during in vitro and in vivo behaviors such as cellular uptake, blood circulation, and biodistribution. However, there have been no quantitative investigations of the effect of commonly used charged lipids on the stiffness of nanosized liposomes. In this study, by means of atomic force microscopy (AFM), we quantified the stiffness of nanosized liposomes composed of neutrally charged lipids combined with positively or negatively charged lipids while simultaneously imaging the liposomes in aqueous medium. Our results showed that charged lipids, whether negatively or positively charged, have the effect of reducing the stiffness of nanosized liposomes, independently of the saturation degree of the lipid acyl chains; the measured stiffness values of liposomes containing charged lipids are 30-60% lower than those of their neutral counterpart liposomes. In addition, we demonstrated that the Laurdan generalized polarization values, which are related to the hydration degree of the liposomal membrane interface and often used as a qualitative indicator of liposomal membrane stiffness, do not directly correlate with the physical stiffness values of the liposomes prepared in this study. However, our results indicate that direct quantitative AFM measurement is a valuable method to gain molecular-scale information about how the hydration degree of liposomal interfaces reflects (or does not reflect) liposome stiffness as a macroscopic property. Our AFM method will contribute to the quantitative characterization of the nano-bio interaction of nanoparticles and to the optimization of the lipid composition of liposomes for clinical use.

Evaluation of membrane fluidity of multidrug-resistant isolates of Escherichia coli and Staphylococcus aureus in presence and absence of antibiotics

In the face of the serious problem of antimicrobial resistance and the global dissemination of multidrug-resistant (MDR) bacteria, it is relevant to deeply study such bacteria both genetically and phenotypically. It is well known that bacteria have the ability to modify the biophysical properties of their cytoplasmic membranes, namely fluidity, in order to survive and thrive in hostile environments. The aim of this study was to assess and compare the membrane fluidity among multidrug-resistant (MDR) isolates of Escherichia coli and Staphylococcus aureus in absence and in presence of antibiotics (ceftazidime or ciprofloxacin). The membrane fluidity was monitored at 24-h intervals up to three days and at the sixth day, by measuring the anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and the generalized polarization (GP) of Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene). The anisotropy values as well as the Laurdan excitation GP (GPexc) values obtained from all three E. coli strains (two MDR isolates and one susceptible reference strain) were quite similar and indicative of a very alike membrane phospholipid composition, regardless harboring or not resistance to multiple antimicrobials. Nonetheless, in the case of S. aureus, the anisotropy values were more increased in methicillin-resistant S. aureus (MRSA) isolates in comparison to the reference strain, meaning they have a less fluid membrane. Equally, GPexc values were statistically different among the three S. aureus strains and showed that the two MRSA isolates had more rigid membranes than the susceptible strain. The exposition of MDR isolates of S. aureus to subinhibitory concentrations of ciprofloxacin did not affect neither the anisotropy values nor the GPexc values, therefore, not altering membrane fluidity. Membrane integrity, assessed by the Live/Dead staining, of all strains and conditions studied was maintained over the six days. Thus, these preliminary studies on membrane properties of MDR isolates demonstrate that i) MRSA seem to have a more rigid membrane that susceptible S. aureus and ii) the presence of subinhibitory concentrations of antibiotics does not significantly alter the membrane fluidity of S. aureus, regardless being MDR or susceptible, but slightly affect the membrane fluidity of E. coli.

Use of quantitative optical imaging to examine the role of cholesterol-rich lipid raft microdomains in the migration of breast cancer cells

Lipid rafts have been extensively studied and shown to be involved in many cancers, including breast cancer. However, the exact role of lipid rafts in the migration of breast cancer cells remains unclear. This study was designed to examine lipid rafts (cholesterol) in the plasma membrane of breast cancer cells (MDA-MB-231 and MCF-7) and normal breast epithelial cells (MCF-10A) through generalized polarization values, and further investigate the role of cholesterol-rich lipid rafts in the migration of breast cancer cells. The results showed that the plasma membrane in breast cancer cells was more orderly than that in normal epithelial cells; this was correlated with expression changes of matrix metallopeptidase 9 (MMP-9) and urokinase-type plasminogen activator receptor (uPAR), the markers of cancer cell migration. Moreover, the breast cancer cells were more sensitive to the reagent that induced cholesterol depletion than the normal breast epithelial cells, while the capacity of cancer cells to migrate decreased significantly according to changes in MMP-9 and uPAR expression. To our best knowledge, this is the first demonstration of the relationship between cholesterol-rich lipid rafts and the migration of breast cancer cells; it could be useful for the prevention of breast cancer and early treatment through reduction of the level of cholesterol in the plasma membrane of the cells.

Interaction of membrane palmitoylated protein-1 with model lipid membranes.

Membrane palmitoylated protein-1 (MPP1) plays an important role in the formation of raft domains in erythroid membranes. We have shown recently that MPP1 interacts with membrane lipids composed of dioleoylphosphatidylcholine (DOPC), sphingomyelin (SM) and cholesterol. Here we further extend this investigation. Our results obtained from FRET assays revealed that MPP1 binds liposomes with high affinity (KD ~ 135 ± 15 nM). Preincubation of MPP1 with cholesterol before its addition to the Langmuir subphase resultedin a dramatic reduction in the membrane insertion/binding of MPP1, indicating the role of direct MPP1/cholesterol complexes in the interaction of MPP1 with membrane lipids. The generalized polarization values of liposomes as well as the constant surface area experiments on monolayers composed of DOPC/SM/Chol indicated a change in the lipid mono- and bilayer properties upon the addition of MPP1. Furthermore, the presence of flotillins did not affect the binding of MPP1 to membrane lipids. Also, MPP1 containing palmitoylation-mimicking mutation (C242F) bound DOPC/SM/Chol mono- and bilayer with an affinity very similar to that obtained for wild-type MPP1. In conclusion, our results suggest that the direct binding of MPP1 with membrane lipids could be involved in the mechanism of membrane association of MPP1 in erythroid cells.

The dynamics of intracellular water constrains glycolytic oscillations in Saccharomyces cerevisiae

We explored the dynamic coupling of intracellular water with metabolism in yeast cells. Using the polarity-sensitive probe 6-acetyl-2-dimethylaminonaphthalene (ACDAN), we show that glycolytic oscillations in the yeast S. cerevisiae BY4743 wild-type strain are coupled to the generalized polarization (GP) function of ACDAN, which measures the physical state of intracellular water. We analysed the oscillatory dynamics in wild type and 24 mutant strains with mutations in many different enzymes and proteins. Using fluorescence spectroscopy, we measured the amplitude and frequency of the metabolic oscillations and ACDAN GP in the resting state of all 25 strains. The results showed that there is a lower and an upper threshold of ACDAN GP, beyond which oscillations do not occur. This critical GP range is also phenomenologically linked to the occurrence of oscillations when cells are grown at different temperatures. Furthermore, the link between glycolytic oscillations and the ACDAN GP value also holds when ATP synthesis or the integrity of the cell cytoskeleton is perturbed. Our results represent the first demonstration that the dynamic behaviour of a metabolic process can be regulated by a cell-wide physical property: the dynamic state of intracellular water, which represents an emergent property.

Spectral imaging toolbox: segmentation, hyperstack reconstruction, and batch processing of spectral images for the determination of cell and model membrane lipid order

BackgroundSpectral imaging with polarity-sensitive fluorescent probes enables the quantification of cell and model membrane physical properties, including local hydration, fluidity, and lateral lipid packing, usually characterized by the generalized polarization (GP) parameter. With the development of commercial microscopes equipped with spectral detectors, spectral imaging has become a convenient and powerful technique for measuring GP and other membrane properties. The existing tools for spectral image processing, however, are insufficient for processing the large data sets afforded by this technological advancement, and are unsuitable for processing images acquired with rapidly internalized fluorescent probes.ResultsHere we present a MATLAB spectral imaging toolbox with the aim of overcoming these limitations. In addition to common operations, such as the calculation of distributions of GP values, generation of pseudo-colored GP maps, and spectral analysis, a key highlight of this tool is reliable membrane segmentation for probes that are rapidly internalized. Furthermore, handling for hyperstacks, 3D reconstruction and batch processing facilitates analysis of data sets generated by time series, z-stack, and area scan microscope operations. Finally, the object size distribution is determined, which can provide insight into the mechanisms underlying changes in membrane properties and is desirable for e.g. studies involving model membranes and surfactant coated particles. Analysis is demonstrated for cell membranes, cell-derived vesicles, model membranes, and microbubbles with environmentally-sensitive probes Laurdan, carboxyl-modified Laurdan (C-Laurdan), Di-4-ANEPPDHQ, and Di-4-AN(F)EPPTEA (FE), for quantification of the local lateral density of lipids or lipid packing.ConclusionsThe Spectral Imaging Toolbox is a powerful tool for the segmentation and processing of large spectral imaging datasets with a reliable method for membrane segmentation and no ability in programming required. The Spectral Imaging Toolbox can be downloaded from https://uk.mathworks.com/matlabcentral/fileexchange/62617-spectral-imaging-toolbox.

Quality and freshness of human bone marrow-derived mesenchymal stem cells decrease over time after trypsinization and storage in phosphate-buffered saline

Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) have been studied for their therapeutic potential. However, evaluating the quality of hBM-MSCs before transplantation remains a challenge. We addressed this issue in the present study by investigating deformation, the expression of genes related to reactive oxygen species (ROS) generation, changes in amino acid profiles, and membrane fluidity in hBM-MSCs. Deformability and cell size were decreased after storage for 6 and 12 h, respectively, in phosphate-buffered saline. Intracellular ROS levels also increased over time, which was associated with altered expression of genes related to ROS generation and amino acid metabolism. Membrane fluidity measurements revealed higher Laurdan generalized polarization values at 6 and 12 h; however, this effect was reversed by N-acetyl-l-cysteine-treatment. These findings indicate that the quality and freshness of hBM-MSCs is lost over time after dissociation from the culture dish for transplantation, highlighting the importance of using freshly trypsinized cells in clinical applications.

Enzymatic studies on planar supported membranes using a widefield fluorescence LAURDAN Generalized Polarization imaging approach

We introduce a custom-built instrument designed to perform fast LAURDAN Generalized Polarization (GP) imaging on planar supported membranes. It is mounted on a widefield fluorescence microscope and allows kinetic analysis of the GP function in the millisecond time scale, largely improving the temporal resolution previously achieved using laser scanning based microscopes. A dedicated protocol to calibrate LAURDAN GP data obtained with charge-coupled device (CCD) cameras as detectors is also presented, enabling reliable assignment of GP values in the field of view. Using this methodology we studied structural and dynamical transformations induced by Sphingomyelinase D (SM-D) on planar supported membranes composed of N-lauroyl sphingomyelin (C12SM). GP data show the evolution of an initially compositionally homogeneous symmetric bilayer existing in a single liquid disordered phase, to an intermediate configuration showing coexistence of liquid disordered and solid ordered domains, which are not always in-register across the axial plane of the bilayer. This intermediate state, caused by the transformation of C12SM to C12-ceramide-1-phosphate in the distal leaflet of the bilayer, evolved to a single solid ordered phase at longer time scales. Additionally, we comparatively studied this system using the membrane fluorophore DiIC18. The advantages and limitations of both fluorescent dyes are discussed, emphasizing the adequacy of LAURDAN GP imaging to explore this type of membrane phenomena.

Assessment of Membrane Fluidity Fluctuations during Cellular Development Reveals Time and Cell Type Specificity.

Cell membrane is made up of a complex structure of lipids and proteins that diffuse laterally giving rise to what we call membrane fluidity. During cellular development, such as differentiation cell membranes undergo dramatic fluidity changes induced by proteins such as ARC and Cofilin among others. In this study we used the generalized polarization (GP) property of fluorescent probe Laurdan using two-photon microscopy to determine membrane fluidity as a function of time and for various cell lines. A low GP value corresponds to a higher fluidity and a higher GP value is associated with a more rigid membrane. Four different cell lines were monitored such as hN2, NIH3T3, HEK293 and L6 cells. Membrane fluidity was measured at 12h, 72h and 92 h. Our results show significant changes in membrane fluidity among all cell types at different time points. GP values tend to increase significantly within 92 h in hN2 cells and 72 h in NIH3T3 cells and only at 92 h in HEK293 cells. L6 showed a marked decrease in membrane fluidity at 72 h and starts to increase at 92 h. As expected, NIH3T3 cells have more rigid membrane at earlier time points. On the other hand, neurons tend to have the highest membrane fluidity at early time points emphasizing its correlation with plasticity and the need for this malleability during differentiation. This study sheds light on the involvement of membrane fluidity during neuronal differentiation and development of other cell lines.