Low membrane fluidity triggers lipid phase separation and protein segregation in living bacteria.

All living organisms adapt their membrane lipid composition in response to changes in their environment or diet. These conserved membrane-adaptive processes have been studied extensively. However, key concepts of membrane biology linked to regulation of lipid composition including homeoviscous adaptation maintaining stable levels of membrane fluidity, and gel-fluid phase separation resulting in domain formation, heavily rely upon in vitro studies with model membranes or lipid extracts. Using the bacterial model organisms Escherichia coli and Bacillus subtilis, we now show that inadequate in vivo membrane fluidity interferes with essential complex cellular processes including cytokinesis, envelope expansion, chromosome replication/segregation and maintenance of membrane potential. Furthermore, we demonstrate that very low membrane fluidity is indeed capable of triggering large-scale lipid phase separation and protein segregation in intact, protein-crowded membranes of living cells; a process that coincides with the minimal level of fluidity capable of supporting growth. Importantly, the in vivo lipid phase separation is not associated with a breakdown of the membrane diffusion barrier function, thus explaining why the phase separation process induced by low fluidity is biologically reversible.

Role of the extracellular matrix in regulating stem cell fate.

The field of stem cells and regenerative medicine offers considerable promise as a means of delivering new treatments for a wide range of diseases. In order to maximize the effectiveness of cell-based therapies - whether stimulating expansion of endogenous cells or transplanting cells into patients - it is essential to understand the environmental (niche) signals that regulate stem cell behaviour. One of those signals is from the extracellular matrix (ECM). New technologies have offered insights into how stem cells sense signals from the ECM and how they respond to these signals at the molecular level, which ultimately regulate their fate.

Phospholipid tail asymmetry allows cellular adaptation to anoxic environments.

Membrane biophysical properties are critical to cell fitness and depend on unsaturated phospholipid acyl tails. These can only be produced in aerobic environments since eukaryotic desaturases require molecular oxygen. This raises the question of how cells maintain bilayer properties in anoxic environments. Using advanced microscopy, molecular dynamics simulations, and lipidomics by mass spectrometry we demonstrated the existence of an alternative pathway to regulate membrane fluidity that exploits phospholipid acyl tail length asymmetry, replacing unsaturated species in the membrane lipidome. We show that the fission yeast, Schizosaccharomyces japonicus, which can grow in aerobic and anaerobic conditions, is capable of utilizing this strategy, whereas its sister species, the well-known model organism Schizosaccharomyces pombe, cannot. The incorporation of asymmetric-tailed phospholipids might be a general adaptation to hypoxic environmental niches.

AdipoR2 recruits protein interactors to promote fatty acid elongation and membrane fluidity.

The human AdipoR2 and its Caenorhabditis elegans homolog PAQR-2 are multipass plasma membrane proteins that protect cells against membrane rigidification. However, how AdipoR2 promotes membrane fluidity mechanistically is not clear. Using 13C-labeled fatty acids, we show that AdipoR2 can promote the elongation and incorporation of membrane-fluidizing polyunsaturated fatty acids into phospholipids. To elucidate the molecular basis of these activities, we performed immunoprecipitations of tagged AdipoR2 and PAQR-2 expressed in HEK293 cells or whole C. elegans, respectively, and identified coimmunoprecipitated proteins using mass spectrometry. We found that several of the evolutionarily conserved AdipoR2/PAQR-2 interactors are important for fatty acid elongation and incorporation into phospholipids. We experimentally verified some of these interactions, namely, with the dehydratase HACD3 that is essential for the third of four steps in long-chain fatty acid elongation and ACSL4 that is important for activation of unsaturated fatty acids and their channeling into phospholipids. We conclude that AdipoR2 and PAQR-2 can recruit protein interactors to promote the production and incorporation of unsaturated fatty acids into phospholipids.

Monomethyl branched-chain fatty acids are critical for Caenorhabitis elegans survival in elevated glucose conditions.

The maintenance of optimal membrane composition under basal and stress conditions is critical for the survival of an organism. High-glucose stress has been shown to perturb membrane properties by decreasing membrane fluidity, and the membrane sensor PAQR-2 is required to restore membrane integrity. However, the mechanisms required to respond to elevated dietary glucose are not fully established. In this study, we used a 13C stable isotope-enriched diet and mass spectrometry to better understand the impact of glucose on fatty acid dynamics in the membrane of Caenorhabditis elegans. We found a novel role for monomethyl branched-chain fatty acids (mmBCFAs) in mediating the ability of the nematodes to survive conditions of elevated dietary glucose. This requirement of mmBCFAs is unique to glucose stress and was not observed when the nematode was fed elevated dietary saturated fatty acid. In addition, when worms deficient in elo-5, the major biosynthesis enzyme of mmBCFAs, were fed Bacillus subtilis (a bacteria strain rich in mmBCFAs) in combination with high glucose, their survival rates were rescued to wild-type levels. Finally, the results suggest that mmBCFAs are part of the PAQR-2 signaling response during glucose stress. Taken together, we have identified a novel role for mmBCFAs in stress response in nematodes and have established these fatty acids as critical for adapting to elevated glucose.

Branched chain fatty acid synthesis drives tissue-specific innate immune response and infection dynamics of Staphylococcus aureus.

Fatty acid-derived acyl chains of phospholipids and lipoproteins are central to bacterial membrane fluidity and lipoprotein function. Though it can incorporate exogenous unsaturated fatty acids (UFA), Staphylococcus aureus synthesizes branched chain fatty acids (BCFA), not UFA, to modulate or increase membrane fluidity. However, both endogenous BCFA and exogenous UFA can be attached to bacterial lipoproteins. Furthermore, S. aureus membrane lipid content varies based upon the amount of exogenous lipid in the environment. Thus far, the relevance of acyl chain diversity within the S. aureus cell envelope is limited to the observation that attachment of UFA to lipoproteins enhances cytokine secretion by cell lines in a TLR2-dependent manner. Here, we leveraged a BCFA auxotroph of S. aureus and determined that driving UFA incorporation disrupted infection dynamics and increased cytokine production in the liver during systemic infection of mice. In contrast, infection of TLR2-deficient mice restored inflammatory cytokines and bacterial burden to wildtype levels, linking the shift in acyl chain composition toward UFA to detrimental immune activation in vivo. In in vitro studies, bacterial lipoproteins isolated from UFA-supplemented cultures were resistant to lipase-mediated ester hydrolysis and exhibited heightened TLR2-dependent innate cell activation, whereas lipoproteins with BCFA esters were completely inactivated after lipase treatment. These results suggest that de novo synthesis of BCFA reduces lipoprotein-mediated TLR2 activation and improves lipase-mediated hydrolysis making it an important determinant of innate immunity. Overall, this study highlights the potential relevance of cell envelope acyl chain repertoire in infection dynamics of bacterial pathogens.

Too rigid to fold: Carotenoid-dependent decrease in thylakoid fluidity hampers the formation of chloroplast grana.

In chloroplasts of land plants, the thylakoid network is organized into appressed regions called grana stacks and loosely arranged parallel stroma thylakoids. Many factors determining such intricate structural arrangements have been identified so far, including various thylakoid-embedded proteins, and polar lipids that build the thylakoid matrix. Although carotenoids are important components of proteins and the lipid phase of chloroplast membranes, their role in determining the thylakoid network structure remains elusive. We studied 2D and 3D thylakoid network organization in carotenoid-deficient mutants (ccr1-1, lut5-1, szl1-1, and szl1-1npq1-2) of Arabidopsis (Arabidopsis thaliana) to reveal the structural role of carotenoids in the formation and dynamics of the internal chloroplast membrane system. The most significant structural aberrations took place in chloroplasts of the szl1-1 and szl1-1npq1-2 plants. Increased lutein/carotene ratio in these mutants impaired the formation of grana, resulting in a significant decrease in the number of thylakoids used to build a particular stack. Further, combined biochemical and biophysical analyses revealed that hampered grana folding was related to decreased thylakoid membrane fluidity and significant changes in the amount, organization, and phosphorylation status of photosystem (PS) II (PSII) supercomplexes in the szl1-1 and szl1-1npq1-2 plants. Such changes resulted from a synergistic effect of lutein overaccumulation in the lipid matrix and a decreased level of carotenes bound with PS core complexes. Moreover, more rigid membrane in the lutein overaccumulating plants led to binding of Rubisco to the thylakoid surface, additionally providing steric hindrance for the dynamic changes in the level of membrane folding.

Optical tweezers study of membrane fluidity in small cell lung cancer cells.

The fluidity of the cell membrane is closely related to cancer metastasis/invasion. To test the relationship of membrane fluidity and invasiveness, we first demonstrated that transfection of small RNA miR-92b-3p can significantly increase invasiveness of the small cell lung cancer cell line SHP77. Then optical tweezers were used to measure membrane fluidity. This study employed continuous and step-like stretching methods to examine fluidity changes in SHP77 cell membranes before and after miR-92b-3p transfection. A newly developed physical model was used to derive the effective viscosity and static tension of the cell membrane from relaxation curves obtained via step-like stretching. Experiments showed that invasiveness and fluidity increased significantly after miR-92b-3p transfection. This study paved the way toward a better understanding of cancer cell invasion and membrane mechanical characteristics.

Erythrocyte membrane fluidity as a marker of diabetic retinopathy in type 1 diabetes mellitus.

BACKGROUND: A high level of glycosylated haemoglobin (HbA1c), which is a nonenzymatic glycosylation product, is correlated with an increased risk of developing microangiopathic complications in Diabetes Mellitus (DM). Erythrocyte membrane fluidity could provide a complementary index to monitor the development of complications since it is influenced by several hyperglycaemia-induced pathways and other independent risk factors. MATERIALS AND METHODS: 15 healthy controls and 33 patients with long-duration (≥20 years) type 1 Diabetes Mellitus (T1DM) were recruited. Diabetic subjects were classified into two groups: T1DM, constituted by 14 nonretinopathic patients, and T1DM + RD, constituted by 19 patients in any stage of diabetic retinopathy. Red blood cells (RBC) were incubated with the fluorescent Laurdan probe and median values of Generalized Polarization (GP), representative of membrane fluidity, were compared between the two groups. Baseline characteristics among groups have been compared with Student's t test or ANOVA. Values of P < .05 were considered statistically significant. RESULTS: All the participants were comparable for age, Body Mass Index (BMI), creatinine and lipid profile. The duration of diabetes was similar for T1DM (34.4 ± 7.8 years) and T1DM + RD (32.8 ± 7.5 years) subjects as well as values of HbA1c: (55.6 ± 8.1) mmol/mol for T1DM and (61.2 ± 11.0) mmol/mol for T1DM + RD, respectively. Erythrocyte plasmatic membranes of RD patients were found to be more fluid (GP: 0.40 ± 0.04) than non-RD patients (GP: 0.43 ± 0.03) with a statistically significant difference (P = .035). CONCLUSIONS: Altered erythrocyte membrane fluidity may therefore represent a marker of retinopathy in T1DM patients as a result of post-translational modifications of multifactorial aetiology (nonenzymatic glycosylation of proteins, generation of reactive oxygen species, lipid peroxidation).

Understanding the Link between Lipid Diversity and the Biophysical Properties of the Neuronal Plasma Membrane.

Cell membranes contain incredible diversity in the chemical structures of their individual lipid species and the ratios in which these lipids are combined to make membranes. Nevertheless, our current understanding of how each of these components affects the properties of the cell membrane remains elusive, in part due to the difficulties in studying the dynamics of membranes at high spatiotemporal resolution. In this work, we use coarse-grained molecular dynamics simulations to investigate how individual lipid species contribute to the biophysical properties of the neuronal plasma membrane. We progress through eight membranes of increasing chemical complexity, ranging from a simple POPC/CHOL membrane to a previously published neuronal plasma membrane [Ingólfsson, H. I., et al. (2017) Biophys. J. 113 (10), 2271-2280] containing 49 distinct lipid species. Our results show how subtle chemical changes can affect the properties of the membrane and highlight the lipid species that give the neuronal plasma membrane its unique biophysical properties. This work has potential far-reaching implications for furthering our understanding of cell membranes.

Deleting the DAG kinase Dgk1 augments yeast vacuole fusion through increased Ypt7 activity and altered membrane fluidity.

Diacylglycerol (DAG) is a fusogenic lipid that can be produced through phospholipase C activity on phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2 ], or through phosphatidic acid (PA) phosphatase activity. The fusion of Saccharomyces cerevisiae vacuoles requires DAG, PA and PI(4,5)P2 , and the production of these lipids is thought to provide temporally specific stoichiometries that are critical for each stage of fusion. Furthermore, DAG and PA can be interconverted by the DAG kinase Dgk1 and the PA phosphatase Pah1. Previously we found that pah1 Δ vacuoles were fragmented, blocked in SNARE priming and showed arrested endosomal maturation. In other pathways the effects of deleting PAH1 can be compensated for by additionally deleting DGK1 ; however, deleting both genes did not rescue the pah1 Δ vacuolar defects. Deleting DGK1 alone caused a marked increase in vacuole fusion that was attributed to elevated DAG levels. This was accompanied by a gain in resistance to the inhibitory effects of PA as well as inhibitors of Ypt7 activity. Together these data show that Dgk1 function can act as a negative regulator of vacuole fusion through the production of PA at the cost of depleting DAG and reducing Ypt7 activity.

Distinct cargo-specific response landscapes underpin the complex and nuanced role of galectin-glycan interactions in clathrin-independent endocytosis.

Clathrin-independent endocytosis (CIE) is a form of endocytosis that lacks a defined cytoplasmic machinery. Here, we asked whether glycan interactions, acting from the outside, could be a part of that endocytic machinery. We show that the perturbation of global cellular patterns of protein glycosylation by modulation of metabolic flux affects CIE. Interestingly, these changes in glycosylation had cargo-specific effects. For example, in HeLa cells, GlcNAc treatment, which increases glycan branching, increased major histocompatibility complex class I (MHCI) internalization but inhibited CIE of the glycoprotein CD59 molecule (CD59). The effects of knocking down the expression of galectin 3, a carbohydrate-binding protein and an important player in galectin-glycan interactions, were also cargo-specific and stimulated CD59 uptake. By contrast, inhibition of all galectin-glycan interactions by lactose inhibited CIE of both MHCI and CD59. None of these treatments affected clathrin-mediated endocytosis, implying that glycosylation changes specifically affect CIE. We also found that the galectin lattice tailors membrane fluidity and cell spreading. Furthermore, changes in membrane dynamics mediated by the galectin lattice affected macropinocytosis, an altered form of CIE, in HT1080 cells. Our results suggest that glycans play an important and nuanced role in CIE, with each cargo being affected uniquely by alterations in galectin and glycan profiles and their interactions. We conclude that galectin-driven effects exist on a continuum from stimulatory to inhibitory, with distinct CIE cargo proteins having unique response landscapes and with different cell types starting at different positions on these conceptual landscapes.

Studies on the interactions of neutral Galleria mellonella cecropin D with living bacterial cells.

Cecropins constitute an important family of insect antimicrobial peptides involved in humoral innate immune response. In comparison with the highly basic cecropins A and B, cecropins D are less cationic and more hydrophobic. Interestingly, cecropins D were described only in lepidopteran insects, e.g., the greater wax moth Galleria mellonella. In the present study, interactions of neutral cecropin D (pI 6.47) purified from hemolymph of G. mellonella with living Escherichia coli cells were investigated. Fluorescence lifetime imaging microscopy using fluorescein isothiocyanate-labeled cecropin D revealed very fast binding of the peptide to E. coli cells. Fourier transform infrared spectroscopy analyses showed that G. mellonella cecropin D interacted especially with E. coli LPS and probably other lipid components of the bacterial cell envelope and exhibited an ordering effect with regard to lipid chains. This effect is consistent with the peptide binding mechanism based upon its incorporation into the lipid phase of the cell membrane. The interaction resulted in permeabilization of the bacterial cell membrane. Upon cecropin D binding, the cells lost characteristic surface topography, which was accompanied by altered nanomechanical properties, as revealed by atomic force microscopy. The interaction of the peptide with the bacterial cells also led to intracellular damage, i.e., loss of the cell envelope multilayer structure, formation of membrane vesicles, and enlargement of periplasmic space, which eventually caused death of the bacteria. In summary, it can be concluded that amphipathic character of α-helices, exposure of small positively charged patches on their polar surfaces and hydrophobic interactions are important physicochemical characteristics related to effective binding to E. coli cells and antibacterial activity of neutral G. mellonella cecropin D.

Two-dimensional Finite Element Model of Breast Cancer Cell Motion Through a Microfluidic Channel.

A two-dimensional model for red blood cell motion is adapted to consider the dynamics of breast cancer cells in a microfluidic channel. Adjusting parameters to make the membrane stiffer, as is the case with breast cancer cells compared with red blood cells, allows the model to produce reasonable estimates of breast cancer cell trajectories through the channel. In addition, the model produces estimates of quantities not as easily obtained from experiment such as velocity and stress field information throughout the fluid and on the cell membrane. This includes locations of maximum stress along the membrane wall. A sensitivity analysis shows that the model is capable of producing useful insights into various systems involving breast cancer cells. Current results suggest that dynamics taking place when cells are near other objects are most sensitive to membrane and cytoplasm elasticity, dynamics taking place when cells are not near other objects are most sensitive to cytoplasm viscosity, and dynamics are significantly affected by low membrane bending elasticity. These results suggest that continued calibration and application of this model can yield useful predictions in other similar systems.

Factors influencing the membrane fluidity and the impact on production of lactic acid bacteria starters.

Production of lactic acid bacteria starters for manufacturing food, probiotic, and chemical products requires the application of successive steps: fermentation, concentration, stabilization, and storage. Despite process optimization, losses of bacterial viability and functional activities are observed after stabilization and storage steps due to cell exposure to environmental stresses (thermal, osmotic, mechanical, and oxidative). Bacterial membrane is the primary target for injury and its damage is highly dependent on its physical properties and lipid organization. Membrane fluidity is a key property for maintaining cell functionality, and depends on lipid composition and cell environment. Extensive evidence has been reported on changes in membrane fatty acyl chains when modifying fermentation conditions. However, a deep characterization of membrane physical properties and their evolution following production processes is scarcely reported. Therefore, the aims of this mini-review are (i) to define the membrane fluidity and the methods used to assess it and (ii) to summarize the effect of environmental conditions on membrane fluidity and the resulting impact on the resistance of lactic acid bacteria to the stabilization processes. This will make it possible to highlight existing gaps of knowledge and opens up novel approaches for future investigations.

Time-resolved Fluorescence and Generalized Polarization: Innovative tools to assess bull sperm membrane dynamics during slow freezing.

During slow freezing, spermatozoa undergo membrane alterations that compromise their ability of fertilizing. These alterations are cause either by cold shock or by the use of cryoprotectants known to be cytotoxic. However, little is known about the membrane changes that occurred during freezing. Here, we combined Generalized Polarization (GP), Time-resolved Fluorescence and laurdan fluorescence properties to investigate the changes in membrane fluidity and dynamics during slow freezing of bull sperm. We successfully demonstrated that laurdan may be distributed in three different local environments that correspond to different membrane lipid composition. These environments wont behave the same way when the cells will be subjected to either a chemical treatment (adding the cryoprotectants) or a physical treatment (freezing).

Caenorhabditis elegans PAQR-2 and IGLR-2 Protect against Glucose Toxicity by Modulating Membrane Lipid Composition.

In spite of the worldwide impact of diabetes on human health, the mechanisms behind glucose toxicity remain elusive. Here we show that C. elegans mutants lacking paqr-2, the worm homolog of the adiponectin receptors AdipoR1/2, or its newly identified functional partner iglr-2, are glucose intolerant and die in the presence of as little as 20 mM glucose. Using FRAP (Fluorescence Recovery After Photobleaching) on living worms, we found that cultivation in the presence of glucose causes a decrease in membrane fluidity in paqr-2 and iglr-2 mutants and that genetic suppressors of this sensitivity act to restore membrane fluidity by promoting fatty acid desaturation. The essential roles of paqr-2 and iglr-2 in the presence of glucose are completely independent from daf-2 and daf-16, the C. elegans homologs of the insulin receptor and its downstream target FoxO, respectively. Using bimolecular fluorescence complementation, we also show that PAQR-2 and IGLR-2 interact on plasma membranes and thus may act together as a fluidity sensor that controls membrane lipid composition.

Modulating membrane fluidity corrects Batten disease phenotypes in vitro and in vivo.

The neuronal ceroid lipofuscinoses are a class of inherited neurodegenerative diseases characterized by the accumulation of autofluorescent storage material. The most common neuronal ceroid lipofuscinosis has juvenile onset with rapid onset blindness and progressive degeneration of cognitive processes. The juvenile form is caused by mutations in the CLN3 gene, which encodes the protein CLN3. While mouse models of Cln3 deficiency show mild disease phenotypes, it is apparent from patient tissue- and cell-based studies that its loss impacts many cellular processes. Using Cln3 deficient mice, we previously described defects in mouse brain endothelial cells and blood-brain barrier (BBB) permeability. Here we expand on this to other components of the BBB and show that Cln3 deficient mice have increased astrocyte endfeet area. Interestingly, this phenotype is corrected by treatment with a commonly used GAP junction inhibitor, carbenoxolone (CBX). In addition to its action on GAP junctions, CBX has also been proposed to alter lipid microdomains. In this work, we show that CBX modifies lipid microdomains and corrects membrane fluidity alterations in Cln3 deficient endothelial cells, which in turn improves defects in endocytosis, caveolin-1 distribution at the plasma membrane, and Cdc42 activity. In further work using the NIH Library of Integrated Network-based Cellular Signatures (LINCS), we discovered other small molecules whose impact was similar to CBX in that they improved Cln3-deficient cell phenotypes. Moreover, Cln3 deficient mice treated orally with CBX exhibited recovery of impaired BBB responses and reduced autofluorescence. CBX and the compounds identified by LINCS, many of which have been used in humans or approved for other indications, may find therapeutic benefit in children suffering from CLN3 deficiency through mechanisms independent of their original intended use.

Effect of 25-hydroxycholesterol in viral membrane fusion: Insights on HIV inhibition.

Recently, it was demonstrated that 25-hydroxycholesterol (25HC), an oxidized cholesterol derivative, inhibits human immunodeficiency virus type 1 (HIV) entry into its target cells. However, the mechanisms involved in this action have not yet been established. The aim of this work was to study the effects of 25HC in biomembrane model systems and at the level of HIV fusion peptide (HIV-FP). Integration of different biophysical approaches was made in the context of HIV fusion process, to clarify the changes at membrane level due to the presence of 25HC that result in the suppressing of viral infection. Lipid vesicles mimicking mammalian and HIV membranes were used on spectroscopy assays and lipid monolayers in surface pressure studies. Peptide-induced lipid mixing assays were performed by Förster resonance energy transfer to calculate fusion efficiency. Liposome fusion is reduced by 50% in the presence of 25HC, comparatively to cholesterol. HIV-FP conformation was assessed by infrared assays and it relies on sterol nature. Anisotropy, surface pressure and dipole potential assays indicate that the conversion of cholesterol in 25HC leads to a loss of the cholesterol modulating effect on the membrane. With different biophysical techniques, we show that 25HC affects the membrane fusion process through the modification of lipid membrane properties, and by direct alterations on HIV-FP structure. The present data support a broad antiviral activity for 25HC.