Transbilayer Coupling of Lipids in Cells Investigated by Imaging Fluorescence Correlation Spectroscopy

Plasma membrane hosts numerous receptors, sensors, and ion channels involved in cellular signaling. Phase separation of the plasma membrane is emerging as a key biophysical regulator of signaling reactions in multiple physiological and pathological contexts. There is much evidence that plasma membrane composition supports the co-existence liquid-ordered (Lo) and liquid-disordered (Ld) phases or domains at physiological conditions. However, this phase/domain separation is nanoscopic and transient in live cells. It is recently proposed that transbilayer coupling between the inner and outer leaflets of the plasma membrane is driven by their asymmetric lipid distribution and by dynamic cytoskeleton-lipid composites that contribute to the formation and transience of Lo/Ld phase separation in live cells. In this Perspective, we highlight new approaches to investigate how transbilayer coupling may influence phase separation. For quantitative evaluation of the impact of these interactions, we introduce an experimental strategy centered around Imaging Fluorescence Correlation Spectroscopy (ImFCS), which measures membrane diffusion with very high precision. To demonstrate this strategy we choose two well-established model systems for transbilayer interactions: crosslinking by multivalent antigen of immunoglobulin E bound to receptor FcεRI, and crosslinking by cholera toxin B of GM1 gangliosides. We discuss emerging methods to systematically perturb membrane lipid composition, particularly exchange of outer leaflet lipids with exogenous lipids using methyl alpha cyclodextrin. These selective perturbations may be quantitatively evaluated with ImFCS and other high-resolution biophysical tools to discover novel principles of lipid-mediated phase separation in live cells in the context of their pathophysiological relevance.

Development of lung diffusion to adulthood following extremely preterm birth

BackgroundGas exchange in extremely preterm (EP) infants must take place in foetal lungs. Childhood lung diffusing capacity for carbon monoxide (DLCO) is reduced; however, longitudinal development has not been investigated. We describe growth of DLCO and its sub-components to adulthood in EP-born compared to term-born subjects.MethodsTwo area-based cohorts born at gestational age ≤28 weeks or birth weight ≤1000 grams in 1982–1985 (n=48) and 1991–1992 (n=35) were examined twice, at ages 18 and 25, and 10 and 18 years, respectively, and compared to matched term-born controls. Single-breath DLCO was measured at two oxygen pressures, with sub-components [membrane diffusion (DM) and pulmonary capillary blood volume (VC)] calculated using the Roughton–Forster equation.ResultsAge-, sex- and height-standardized transfer coefficients for carbon monoxide (KCO), and DLCO were reduced in EP-born compared to term-born and remained so during puberty and early adulthood (p-values for all time points and both cohorts ≤0.04), whereas alveolar volume was similar. Development occurred in parallel to term-born controls, with no signs of pubertal catch-up growth nor decline at 25 years (p-values for lack of parallelism within cohorts 0.99, 0.65, 0.71, 0.94, and 0.44 for z-DLCO, z-VA, z-KCO, DM, and VC, respectively). Split by membrane and blood volume components, findings were less clear; however, membrane diffusion seemed most affected.ConclusionPulmonary diffusing capacity was reduced in EP-born compared to term-born, and development from childhood to adulthood tracked in parallel to term-born, with no signs of catch-up growth nor decline at age 25.

Using kICS to Reveal Changed Membrane Diffusion of AQP-9 Treated with Drugs

The formation of nanodomains in the plasma membrane are thought to be part of membrane proteins regulation and signaling. Plasma membrane proteins are often investigated by analyzing the lateral mobility. k-space ICS (kICS) is a powerful image correlation spectroscopy (ICS) technique and a valuable supplement to fluorescence correlation spectroscopy (FCS). Here, we study the diffusion of aquaporin-9 (AQP9) in the plasma membrane, and the effect of different membrane and cytoskeleton affecting drugs, and therefore nanodomain perturbing, using kICS. We measured the diffusion coefficient of AQP9 after addition of these drugs using live cell Total Internal Reflection Fluorescence imaging on HEK-293 cells. The actin polymerization inhibitors Cytochalasin D and Latrunculin A do not affect the diffusion coefficient of AQP9. Methyl-β-Cyclodextrin decreases GFP-AQP9 diffusion coefficient in the plasma membrane. Human epidermal growth factor led to an increase in the diffusion coefficient of AQP9. These findings led to the conclusion that kICS can be used to measure diffusion AQP9, and suggests that the AQP9 is not part of nanodomains.

Protein Crystallization in a Microfluidic Contactor with Nafion®117 Membranes

Protein crystallization still remains mostly an empirical science, as the production of crystals with the required quality for X-ray analysis is dependent on the intensive screening of the best protein crystallization and crystal’s derivatization conditions. Herein, this demanding step was addressed by the development of a high-throughput and low-budget microfluidic platform consisting of an ion exchange membrane (117 Nafion® membrane) sandwiched between a channel layer (stripping phase compartment) and a wells layer (feed phase compartment) forming 75 independent micro-contactors. This microfluidic device allows for a simultaneous and independent screening of multiple protein crystallization and crystal derivatization conditions, using Hen Egg White Lysozyme (HEWL) as the model protein and Hg2+ as the derivatizing agent. This microdevice offers well-regulated crystallization and subsequent crystal derivatization processes based on the controlled transport of water and ions provided by the 117 Nafion® membrane. Diffusion coefficients of water and the derivatizing agent (Hg2+) were evaluated, showing the positive influence of the protein drop volume on the number of crystals and crystal size. This microfluidic system allowed for crystals with good structural stability and high X-ray diffraction quality and, thus, it is regarded as an efficient tool that may contribute to the enhancement of the proteins’ crystals structural resolution.

From Wastes To Functions: Preparation of Layered Double Hydroxides From Industrial Waste And Its Removal Performance Towards Phosphates

To control eutrophication and recover phosphate from wastewater, a calcium carbide slag and red mud composite material (CR-LDH) was prepared using industrial waste as raw material for phosphorus adsorption. The morphology and structure of synthesized CR-LDH was characterized by FT-IR, SEM, EDS and XRD measurements. The experimental data can be well described by pseudo-second-order kinetics and Langmuir isotherm models, suggesting that the adsorption process of CR-LDH with respect to phosphate is a chemical and monolayer process. The theoretical maximum adsorption capacity obtained by Langmuir isotherm model is 16.06 mg·g-1 at 25 oC. The intra-particle diffusion model fitting results indicated that the adsorption of phosphate by CR-LDH was controlled by both liquid membrane diffusion and intra-particle diffusion. Phosphate was bound to CR-LDH via synergistic effect of physical adsorption, ion exchange, anion intercalation and chemical precipitation as evidenced from a combination of microscopic analysis and adsorption mechanism study. The actual phosphate-containing wastewater investigation showed that CR-LDH not only exhibited good removal effect on phosphate, but also could greatly reduce turbidity, COD and ammonia nitrogen, which was suitable for disposal of practical wastewater. These results indicate that CR-LDH can be considered as potential adsorbent for the treatment of phosphate-containing wastewater, which will be helpful to achieve the goal of "treating waste with waste and turning waste into treasure".

Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions

Focal adhesions (FAs) initiate chemical and mechanical signals involved in cell polarity, migration, proliferation and differentiation. Super-resolution microscopy revealed that FAs are organized at the nanoscale into functional layers from the lower plasma membrane to the upper actin cytoskeleton. Yet, how FAs proteins are guided into specific nano-layers to promote interaction with given targets is unknown. Using single protein tracking, super-resolution microscopy and functional assays, we link the molecular behavior and 3D nanoscale localization of kindlin with its function in integrin activation inside FAs. We show that immobilization of integrins in FAs depends on interaction with kindlin. Unlike talin, kindlin displays free diffusion along the plasma membrane outside and inside FAs. We demonstrate that the kindlin Pleckstrin Homology domain promotes membrane diffusion and localization to the membrane-proximal integrin nano-layer, necessary for kindlin enrichment and function in FAs. Using kindlin-deficient cells, we show that kindlin membrane localization and diffusion are crucial for integrin activation, cell spreading and FAs formation. Thus, kindlin uses a different route than talin to reach and activate integrins, providing a possible molecular basis for their complementarity during integrin activation.

Adsorption Characteristics of Stone-Bentonite Mixtures towards Zn: Equilibrium and Kinetic Tests

In the treatment of industrial polluted sites and the construction of landfill sites, anti-pollution barriers are usually used to prevent the diffusion of pollutants. In this paper, the adsorption characteristics of Zn ions by the rock-bentonite anti-pollution barrier were observed by means of static equilibrium and dynamic adsorption tests. The experimental results showed that the adsorption of Zn by stone chips—bentonite was close to the nonlinear Freundlich and Langmuir models. When the concentration of Zn ion is constant, the adsorption capacity increases with the increase in temperature. At a certain temperature, the adsorption removal rate decreases with the increase in concentration. Further study found that the adsorption of Zn from mixed soil was mainly an ion exchange process, and the adsorption mode of Zn from mixed soil was controlled by both intra-particle diffusion and membrane diffusion. Zeta potential, X-ray diffraction (XRD) and The Fourier Transform Infrared spectroscopy (FTIR) showed that with the increase in concentration, the mixed soil adsorbed more metal ions, and the thickness of the double electric layer decreased. Moreover, the adsorption of Zn2+ by bentonite was mainly interlayer adsorption and ion exchange. As an anti-pollution barrier material, the mixed soil of stone chips -bentonite can prevent the diffusion of pollutants, which has certain reference significance for engineering construction.

MDGAs are fast-diffusing molecules that delay excitatory synapse development by altering neuroligin behavior

MDGAs are molecules that can bind neuroligins in cis and interfere with trans-synaptic neurexin-neuroligin interactions, thereby impairing synapse development. However, the sub-cellular localization and dynamics of MDGAs, as well as their specific mode of action in neurons are still unclear. Here, using both surface immunostaining of endogenous MDGAs and single molecule tracking of recombinant MDGAs in dissociated hippocampal neurons, we show that MDGA1 and MDGA2 molecules are homogeneously distributed and exhibit fast membrane diffusion, with a small reduction in mobility across neuronal maturation in culture Using shRNAs and CRISPR/Cas9 strategies to knock-down/out MDGA1 or MDGA2, we demonstrate an increase in the density of excitatory synapses accompanied by enhanced membrane immobilization and an increase in the phosphotyrosine level of neuroligins associated with excitatory post-synaptic differentiation. Finally, we show that decreasing MDGA expression level reduces the mobility of AMPA receptors and increases the frequency of AMPA receptor mediated mEPSCs. Overall, our results support a mechanism by which interactions between MDGAs and neuroligin-1 delays the assembly of functional excitatory synapses containing AMPA receptors.

The Release of Endogenous Nitrogen and Phosphorus in the Danjiangkou Reservoir: A Double-Membrane Diffusion Model Analysis

Endogenous contamination from the newly submerged sediment may have an impact on the water quality of the Danjiangkou Reservoir, the water source of the middle route of the South-to-North Water Diversion Project. In this study, in situ sediment samples were collected at different locations (Guojiashan (DJK1), Kongqueyuan (DJK2), Shijiagou (DJK3), Shiqiao Wharf (DJK4), and Songgang Wharf (DJK5) from the north to the south) of the Danjiangkou Reservoir and the upstream tributary using a columnar sediment sampler and statically cultured in laboratory, and the distribution and release of endogenous N and P from the sediment were investigated based on a double-membrane diffusion model. The results showed that the P contents in the main reservoir and the upstream tributary followed the order of inorganic phosphorus IP > organic phosphorus OP > calcium − phosphorus Ca − P > iron − phosphorus Fe − P > labile phosphorus LP . IP accounted for the largest proportion (47.10-59.70%) of total phosphorus (TP), while LP accounted for only a small proportion (0.10-0.90%) of TP. There is a significant difference in the spatial distribution of P at different sampling points, especially for the OP content (226.90 mg·kg-1) with a coefficient of variation of 26.90%. The contents of different forms of P and NH4+-N decreased from the upstream tributary to the main reservoir, which was mainly attributed to the land use type. In the vertical distribution, the contents of different forms of P in DJK4 and DJK5 decreased with the increase of sediment depth. The contents of NH4+-N and PO43--P in the sediment interstitial water of DJK4 and DJK5 were higher than those in the overlying water. In addition, the contents of NH4+-N and PO43--P were higher in DJK5 than that in DJK4. The static culture experiments showed that N and P were mainly released from the sediment-overlying water interface to the overlying water. The release rate of NH4+-N and PO43--P ranged from 13.08 mg·(m2·d)-1 to 21.39 mg·(m2·d)-1 and from 3.06 mg·(m2·d)-1 to 6.02 mg·(m2·d)-1, and the release amount calculated based on the double-membrane diffusion model was 1.17 × 10 3 t·a-1 and 0.53 × 10 3 t·a-1, respectively. Thus, endogenous contamination from the newly submerged land is an important factor affecting the water quality of the Danjiangkou Reservoir.

Genome-wide Characterization Deciphers Distinct Properties of Aquaporins in Six Phytophthora species

Background: Aquaporins, also known as major intrinsic proteins (MIPs), facilitate the membrane diffusion of water and some other small solutes. The role of MIPs in plant physiological processes is established and now their roles in plant-pathogen interactions are getting more attention. Objective: To investigate the evolution, diversity, and structural insights of Phytophthora MIPs (PhyMIPs) and to compare them to those in other domains of life. Methods: Bioinformatics approaches were used to identify and characterize the PhyMIPs. The phylogenetic analysis was done with MEGA7.0 using maximum likelihood method. The prediction of transmembrane α-helices was done by using SOSUI and TMpred servers, and that of subcellular localization was performed with WoLF PSORT and Cello prediction system. The structure of PhyMIP genes was predicted by GeneMark.hmm ES-3.0 program. The 3D homology models were generated using the Molecular Operating Environment software and the stereochemical quality of the templates and models was assessed by PROCHECK. The PoreWalker server was used to detect and characterize PhyMIP channels from their 3D structural models. Results: Herein, we identified 17, 24, 27, 19, 19, and 22 full-length MIPs, respectively, in the genomes of six Phytophthora species, P. infestans, P. parasitica, P. sojae, P. ramorum, P. capsici,and P. cinnamomi. Phylogenetic analysis showed that the PhyMIPs formed a completely distinct clade from their counterparts in other taxa and were clustered into nine subgroups. Sequence and structural properties indicated that the primary selectivity-related constrictions, including aromatic arginine (ar/R) selectivity filter and Froger's positions in PhyMIPs were distinct from those in other taxa. The substitutions in the conserved Asn-Pro-Ala motifs in loops B and E of many PhyMIPs were also divergent from those in other taxonomic domains. The group-specific consensus sequences/motifs deciphered in different loops and transmembrane α-helices of PhyMIPs were distinct from those in plants, animals, and other microbes. Conclusion: This study represents PhyMIPs with distinct evolutionary and structural properties, and the data collectively indicates that PhyMIPs might have novel functions.