Quantitative FRET Microscopy Reveals a Crucial Role of Cytoskeleton in Promoting PI(4,5)P2 Confinement

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) is an essential plasma membrane component involved in several cellular functions, including membrane trafficking and cytoskeleton organization. This function multiplicity is partially achieved through a dynamic spatiotemporal organization of PI(4,5)P₂ within the membrane. Here, we use a Förster resonance energy transfer (FRET) approach to quantitatively assess the extent of PI(4,5)P₂ confinement within the plasma membrane. This methodology relies on the rigorous evaluation of the dependence of absolute FRET efficiencies between pleckstrin homology domains (PH_PLCδ) fused with fluorescent proteins and their average fluorescence intensity at the membrane. PI(4,5)P₂ is found to be significantly compartmentalized at the plasma membrane of HeLa cells, and these clusters are not cholesterol-dependent, suggesting that membrane rafts are not involved in the formation of these nanodomains. On the other hand, upon inhibition of actin polymerization, compartmentalization of PI(4,5)P₂ is almost entirely eliminated, showing that the cytoskeleton network is the critical component responsible for the formation of nanoscale PI(4,5)P₂ domains in HeLa cells.     

Investigation of Shape Transformations of Vesicles,Induced by Their Adhesion to Flat Substrates Characterized by Different Adhesion Strength

The adhesion of lipid vesicles to a rigid flat surface is investigated. We examine the influence of the membrane spontaneous curvature, adhesion strength, and the reduced volume on the stability and shape transformations of adhered vesicles. The minimal strength of the adhesion necessary to stabilize the shapes of adhered vesicles belonging to different shape classes is determined. It is shown that the budding of an adhered vesicle may be induced by the change of the adhesion strength. The importance of the free vesicle shape for its susceptibility to adhesion is discussed.     

Observations of Membrane Domain Reorganization in Mechanically Compressed Artificial Cells

Giant unilamellar vesicles (GUVs) are considered to be the gold standard for assembling artificial cells from the bottom up. In this study, we investigated the behavior of such biomimetic vesicles as they were subjected to mechanical compression. A microfluidic device is presented that comprises a trap to capture GUVs and a microstamp that is deflected downwards to mechanically compress the trapped vesicle. After characterization of the device, we show that single-phase GUVs can be controllably compressed to a high degree of deformation (D=0.40) depending on the pressure applied to the microstamp. A permeation assay was implemented to show that vesicle bursting is prevented by water efflux. Next, we mechanically compressed GUVs with co-existing liquid-ordered and liquid-disordered membrane phases. Upon compression, we observed that the normally stable lipid domains reorganized themselves across the surface and fused into larger domains. This phenomenon, observed here in a model membrane system, not only gives us insights into how the multicomponent membranes of artificial cells behave, but might also have interesting consequences for the role of lipid rafts in biological cells that are subjected to compressive forces in a natural environment.     

Ezrin Regulates Ca2+ Ionophore-Induced Plasma Membrane Translocation of Aquaporin-5

Aquaporin-5 (AQP5) is selectively expressed in the apical membrane of exocrine glands, such as salivary, sweat, and submucosal airway glands, and plays important roles in maintaining their secretory functions. Because AQP5 is not regulated by gating, localization on the plasma membrane is important for its water-permeable function. Ezrin is an ezrin–radixin–moesin family protein that serves as a crosslinker between the plasma membrane and actin cytoskeleton network. It plays important roles in translocation of various membrane proteins to mediate vesicle trafficking to the plasma membrane. In this study, we examined the effects of ezrin inhibition on membrane trafficking of AQP5. Ezrin inhibition selectively suppressed an ionomycin-induced increase in AQP5 translocation to the plasma membrane of mouse lung epithelial cells (MLE-12) without affecting the steady-state level of plasma membrane AQP5. Taken together, our data suggest that AQP5 translocates to the plasma membrane through at least two pathways and that ezrin is selectively involved in a stimulation-dependent pathway.     

Film instability induced evolution of hierarchical structures in annealed ultrathin films of an asymmetric block copolymer on polar substrates

With an atomic-force microscope and a grazing-incidence small-angle X-ray scattering we studied ex situ the evolution of hierarchical structures in isothermally annealed ultrathin films of asymmetric polystyrene-block-poly(methyl methacrylate) P(S-b-MMA) that dewetted on polar substrates via a mechanism involving nucleation and growth. Film instability causes the surface to acquire an undulating thickness through incommensurability, producing not only the relief structures on a micrometer scale but also mesophase-separated domains on a nanometer scale. The dewetted morphologies strongly influence the ordering behavior of the nanoscale domains. The noncylindrical nanostructures become stable at the curved edges of the relief microstructures in the destabilized P(S-b-MMA) films, for which a preferential wetting of the PS block with the free surface is prohibited. Additionally, the shape of relief structures as result of film instability correlates with the formation of mesophase-separated nanodomains. At early stages of film instability, the formation of parallel-oriented PMMA cylindrical nanodomains increases the deformation energy and it further persists to force the shape of relief structures between irregular holes to have a facet-wedge shape. However, those relief structures are expected to be not at equilibrium. At high temperatures, the relief structures between irregular holes progressively developed to form hemispherical-cap drops accompanied by a transformation of cylindrical into noncylindrical nanodomains at curved surfaces.     

Pediatric Celiac Disease Patients Show Alterations of Dendritic Cell Shape and Actin Rearrangement

Celiac disease (CD) is a frequent intestinal inflammatory disease occurring in genetically susceptible individuals upon gluten ingestion. Recent studies point to a role in CD for genes involved in cell shape, adhesion and actin rearrangements, including a Rho family regulator, Rho GTPase-activating protein 31 (ARHGAP31). In this study, we investigated the morphology and actin cytoskeletons of peripheral monocyte-derived dendritic cells (DCs) from children with CD and controls when in contact with a physiological substrate, fibronectin. DCs were generated from peripheral blood monocytes of pediatric CD patients and controls. After adhesion on fibronectin, DCs showed a higher number of protrusions and a more elongated shape in CD patients compared with controls, as assessed by immunofluorescence actin staining, transmitted light staining and video time-lapse microscopy. These alterations did not depend on active intestinal inflammation associated with gluten consumption and were specific to CD, since they were not found in subjects affected by other intestinal inflammatory conditions. The elongated morphology was not a result of differences in DC activation or maturation status, and did not depend on the human leukocyte antigen (HLA)-DQ2 haplotype. Notably, we found that ARH-GAP31 mRNA levels were decreased while RhoA-GTP activity was increased in CD DCs, pointing to an impairment of the Rho pathway in CD cells. Accordingly, Rho inhibition was able to prevent the cytoskeleton rearrangements leading to the elongated morphology of celiac DCs upon adhesion on fibronectin, confirming the role of this pathway in the observed phenotype. In conclusion, adhesion on fibronectin discriminated CD from the controls’ DCs, revealing a gluten-independent CD-specific cellular phenotype related to DC shape and regulated by RhoA activity.     

Structure and Formation Mechanism of Antimicrobial Peptides Temporin B- and L-Induced Tubular Membrane Protrusion

Temporins are a family of antimicrobial peptides (AMPs) isolated from frog skin, which are very short, weakly charged, and highly hydrophobic. They execute bactericidal activities in different ways from many other AMPs. This work investigated morphological changes of planar bilayer membranes composed of mixed zwitterionic and anionic phospholipids induced by temporin B and L (TB and TL) using all-atom and coarse-grained molecular dynamics simulations. We found that TB and TL fold to α-helices at the membrane surface and penetrate shallowly into the bilayer. These short AMPs have low propensity to induce membrane pore formation but possess high ability to extract lipids out. At relatively high peptide concentrations, the strong hydrophobicity of TB and TL promotes them to aggregate into clusters on the membrane surface. These aggregates attract a large amount of lipids out of the membrane to release compression induced by other dispersed peptides binding to the membrane. The extruded lipids mix evenly with the peptides in the cluster and form tubule-like protrusions. Certain water molecules follow the movement of lipids, which not only fill the cavities of the protrusion but also assist in maintaining the tubular structures. In contrast, the peptide-free leaflet remains intact. The present results unravel distinctive antimicrobial mechanisms of temporins disturbing membranes.     

旋转管式膜分离技术的研究新进展

介绍了几种主要的膜分离技术的应用,叙述了近几年来旋转盘式膜和旋转管式膜在国内外的发展状况,重点讨论的是旋转管式膜与其中的流型,并指出了进一步研究它的重要性。     

Revising the Role of Cortical Cytoskeleton during Secretion: Actin and Myosin XI Function in Vesicle Tethering

In plants, secretion of cell wall components and membrane proteins plays a fundamental role in growth and development as well as survival in diverse environments. Exocytosis, as the last step of the secretory trafficking pathway, is a highly ordered and precisely controlled process involving tethering, docking, and fusion of vesicles at the plasma membrane (PM) for cargo delivery. Although the exocytic process and machinery are well characterized in yeast and animal models, the molecular players and specific molecular events that underpin late stages of exocytosis in plant cells remain largely unknown. Here, by using the delivery of functional, fluorescent-tagged cellulose synthase (CESA) complexes (CSCs) to the PM as a model system for secretion, as well as single-particle tracking in living cells, we describe a quantitative approach for measuring the frequency of vesicle tethering events. Genetic and pharmacological inhibition of cytoskeletal function, reveal that the initial vesicle tethering step of exocytosis is dependent on actin and myosin XI. In contrast, treatments with the microtubule inhibitor, oryzalin, did not significantly affect vesicle tethering or fusion during CSC exocytosis but caused a minor increase in transient or aborted tethering events. With data from this new quantitative approach and improved spatiotemporal resolution of single particle events during secretion, we generate a revised model for the role of the cortical cytoskeleton in CSC trafficking.     

Binding and Fusion of Extracellular Vesicles to the Plasma Membrane of Their Cell Targets

Exosomes and ectosomes, extracellular vesicles of two types generated by all cells at multivesicular bodies and the plasma membrane, respectively, play critical roles in physiology and pathology. A key mechanism of their function, analogous for both types of vesicles, is the fusion of their membrane to the plasma membrane of specific target cells, followed by discharge to the cytoplasm of their luminal cargo containing proteins, RNAs, and DNA. Here we summarize the present knowledge about the interactions, binding and fusions of vesicles with the cell plasma membrane. The sequence initiates with dynamic interactions, during which vesicles roll over the plasma membrane, followed by the binding of specific membrane proteins to their cell receptors. Membrane binding is then converted rapidly into fusion by mechanisms analogous to those of retroviruses. Specifically, proteins of the extracellular vesicle membranes are structurally rearranged, and their hydrophobic sequences insert into the target cell plasma membrane which undergoes lipid reorganization, protein restructuring and membrane dimpling. Single fusions are not the only process of vesicle/cell interactions. Upon intracellular reassembly of their luminal cargoes, vesicles can be regenerated, released and fused horizontally to other target cells. Fusions of extracellular vesicles are relevant also for specific therapy processes, now intensely investigated.     

AQP3 Increases Intercellular Cohesion in NSCLC A549 Cell Spheroids through Exploratory Cell Protrusions

Tumor cell aggregation is critical for cell survival following the loss of extracellular matrix attachment and dissemination. However, the underlying mechanotransduction of clustering solitary tumor cells is poorly understood, especially in non-small cell lung cancers (NSCLC). Here, we examined whether cell surface protrusions played an important role in facilitating the physical contact between floating cells detached from a substrate. We employed poly-2-hydroxyethyl methacrylate-based 3D culture methods to mimic in vivo tumor cell cluster formation. The suprastructural analysis of human NSCLC A549 cell spheroids showed that finger-like protrusions clung together via the actin cytoskeleton. Time-lapse holotomography demonstrated that the finger-like protrusions of free-floating cells in 3D culture displayed exploratory coalescence. Global gene expression analysis demonstrated that the genes in the organic hydroxyl transport were particularly enriched in the A549 cell spheroids. Particularly, the knockdown of the water channel aquaporin 3 gene (AQP3) impaired multicellular aggregate formation in 3D culture through the rearrangement of the actomyosin cytoskeleton. Moreover, the cells with reduced levels of AQP3 decreased their transmigration. Overall, these data indicate that cell detachment-upregulated AQP3 contributes to cell surface protrusions through actomyosin cytoskeleton remodeling, causing the aggressive aggregation of free-floating cells dependent on the property of the substratum and collective metastasis.     

Genomic and Targeted Approaches Unveil the Cell Membrane as a Major Target of the Antifungal Cytotoxin Amantelide A

Amantelide A, a polyhydroxylated macrolide isolated from a marine cyanobacterium, displays broad-spectrum activity against mammalian cells, bacterial pathogens, and marine fungi. We conducted comprehensive mechanistic studies to identify the molecular targets and pathways affected by amantelide A. Our investigations relied on chemical structure similarities with compounds of known mechanisms, yeast knockout mutants, yeast chemogenomic profiling, and direct biochemical and biophysical methods. We established that amantelide A exerts its antifungal action by binding to ergosterol-containing membranes followed by pore formation and cell death, a mechanism partially shared with polyene antifungals. Binding assays demonstrated that amantelide A also binds to membranes containing epicholesterol or mammalian cholesterol, thus suggesting that the cytotoxicity to mammalian cells might be due to its affinity to cholesterol-containing membranes. However, membrane interactions were not completely dependent on sterols. Yeast chemogenomic profiling suggested additional direct or indirect effects on actin. Accordingly, we performed actin polymerization assays, which suggested that amantelide A also promotes actin polymerization in cell-free systems. However, the C-33 acetoxy derivative amantelide B showed a similar effect on actin dynamics in vitro but no significant activity against yeast. Overall, these studies suggest that the membrane effects are the most functionally relevant for amantelide A mechanism of action.     

Lipid Self-Assemblies under the Atomic Force Microscope

Lipid model membranes are important tools in the study of biophysical processes such as lipid self-assembly and lipid–lipid interactions in cell membranes. The use of model systems to adequate and modulate complexity helps in the understanding of many events that occur in cellular membranes, that exhibit a wide variety of components, including lipids of different subfamilies (e.g., phospholipids, sphingolipids, sterols…), in addition to proteins and sugars. The capacity of lipids to segregate by themselves into different phases at the nanoscale (nanodomains) is an intriguing feature that is yet to be fully characterized in vivo due to the proposed transient nature of these domains in living systems. Model lipid membranes, instead, have the advantage of (usually) greater phase stability, together with the possibility of fully controlling the system lipid composition. Atomic force microscopy (AFM) is a powerful tool to detect the presence of meso- and nanodomains in a lipid membrane. It also allows the direct quantification of nanomechanical resistance in each phase present. In this review, we explore the main kinds of lipid assemblies used as model membranes and describe AFM experiments on model membranes. In addition, we discuss how these assemblies have extended our knowledge of membrane biophysics over the last two decades, particularly in issues related to the variability of different model membranes and the impact of supports/cytoskeleton on lipid behavior, such as segregated domain size or bilayer leaflet uncoupling.     

Growth mechanisms of WC in WC-5.75 wt% Co

The grain growth mechanisms of WC in WC-5.75 wt% Co are studied in this work. The two well known growth modes of WC in cemented carbides (coalescence process and solution/reprecipitation process) are both observed and show temperature dependence. At low temperatures (1100 and 1200 °C), the growth of WC grains is mainly through coalescence. At intermediate temperatures (1300 °C), coarsening of most WC grains takes place via isotropic growth, accompanied by the shape relaxation leading to the faceting of round grains. At high temperatures (1400 °C), layer-by-layer structures, resulting from anisotropic growth, are observed. Both isotropic growth at 1300 °C and anisotropic growth at 1400 °C are assisted by the solution/reprecipitation process. This is the first time that layer-by-layer structures are observed for WC crystals in WC-Co. The formation mechanism of layer-by-layer structures has been discussed based on the 2D nucleation and growth mechanism and the diffusion rates of W and C atoms within the Co binder at different temperatures.     

Loose Morphology and High Dynamism of OSER Structures Induced by the Membrane Domain of HMG-CoA Reductase

The membrane domain of eukaryotic HMG-CoA reductase (HMGR) has the conserved capacity to induce endoplasmic reticulum (ER) proliferation and membrane association into Organized Smooth Endoplasmic Reticulum (OSER) structures. These formations develop in response to overexpression of particular proteins, but also occur naturally in cells of the three eukaryotic kingdoms. Here, we characterize OSER structures induced by the membrane domain of Arabidopsis HMGR (1S domain). Immunochemical confocal and electron microscopy studies demonstrate that the 1S:GFP chimera co-localizes with high levels of endogenous HMGR in several ER compartments, such as the ER network, the nuclear envelope, the outer and internal membranes of HMGR vesicles and the OSER structures, which we name ER-HMGR domains. After high-pressure freezing, ER-HMGR domains show typical crystalloid, whorled and lamellar ultrastructural patterns, but with wide heterogeneous luminal spaces, indicating that the native OSER is looser and more flexible than previously reported. The formation of ER-HMGR domains is reversible. OSER structures grow by incorporation of ER membranes on their periphery and progressive compaction to the inside. The ER-HMGR domains are highly dynamic in their formation versus their disassembly, their variable spherical-ovoid shape, their fluctuating borders and their rapid intracellular movement, indicating that they are not mere ER membrane aggregates, but active components of the eukaryotic cell.     

Structural Basis of Membrane Trafficking by Rab Family Small G Protein

The Ras-superfamily of small G proteins is a family of GTP hydrolases that is regulated by GTP/GDP binding states. One member of the Ras-superfamily, Rab, is involved in the regulation of vesicle trafficking, which is critical to endocytosis, biosynthesis, secretion, cell differentiation and cell growth. The active form of the Rab proteins, which contains GTP, can recruit specific binding partners, such as sorting adaptors, tethering factors, kinases, phosphatases and motor proteins, thereby influencing vesicle formation, transport, and tethering. Many Rab proteins share the same interacting partners and perform unique roles in specific locations. Because functional loss of the Rab pathways has been implicated in a variety of diseases, the Rab GTPase family has been extensively investigated. In this review, we summarize Rab GTPase- mediated membrane trafficking while focusing on the structures of Rab protein and Rab-effector complexes. This review provides detailed information that helps explain how the Rab GTPase family is involved in membrane trafficking.     

Exosomes and Microvesicles: Identification and Targeting By Particle Size and Lipid Chemical Probes

Exosomes and microvesicles are two classes of submicroscopic vesicle released by cells into the extracellular space. Collectively referred to as extracellular vesicles, these membrane containers facilitate important cell–cell communication by carrying a diverse array of signaling molecules, including nucleic acids, proteins, and lipids. Recently, the role of extracellular vesicle signaling in cancer progression has become a topic of significant interest. Methods to detect and target exosomes and microvesicles are needed to realize applications of extracellular vesicles as biomarkers and, perhaps, therapeutic targets. Detection of exosomes and microvesicles is a complex problem as they are both submicroscopic and of heterogeneous cellular origins. In this Minireview, we highlight the basic biology of extracellular vesicles, and address available biochemical and biophysical detection methods. Detectible characteristics described here include lipid and protein composition, and physical properties such as the vesicle membrane shape and diffusion coefficient. In particular, we propose that detection of exosome and microvesicle membrane curvature with lipid chemical probes that sense membrane shape is a distinctly promising method for identifying and targeting these vesicles.     

Study on the Flow Field inside the Microfiltration Separator with Rotary Tubular Membrane

Abstract

By using a new type of laser surveying instrument named particle image velocimetry (PIV), the flow field inside a rotary tubular membrane separator with a rotating inner tubular microfiltration membrane and a transparent outer cylinder was measured experimentally, and from which some new observations were resulted. Pairs of stable Taylor vortices with similar dimensions and opposite flowing directions were directly visualized by the measured streamlines and vorticity of flow field in the annular gap of the membrane separator. No matter how the axial Reynolds number, radial Reynolds number and Taylor number changed, the dimensions of the Taylor vortices and the distances between the centers of adjacent Taylor vortices were almost the same, but the shapes of the Taylor vortices at lower Taylor numbers were more regular than those at higher Taylor numbers. The Taylor vortices disappeared because of the turbulence when the Taylor number was too high. The maximum axial velocity near the membrane surface was about 20 times larger than the mean velocity of axial flow inside the annular gap, and the maximum outward radial velocity near the membrane surface was even about 3000 times larger than the average velocity of the radial permeating flow through the tubular microfiltration membrane. The large velocities near the membrane surface, which were due to the Taylor vortices, could prevent solid fine particles from depositing onto the membrane surface and/or entering into the membrane pores and therefore result in reduced concentration polarization and reduced membrane fouling. The results in this study provided some valuable guidelines on the hydrodynamic way to reduce membrane fouling.     

UHMWPE管式微滤膜的过滤性能研究

膜分离是一项高效的分离技术,超高分子量聚乙烯(UHMWPE)是应用于其中的主要膜材料之一。在本研究中,采用UHMWPE管式微滤膜过滤含高岭土颗粒的水样,通过对比过滤前后水样中高岭土颗粒的含量,辅之以UHMWPE管式微滤膜的电镜照片分析,对微滤膜的过滤性能进行了研究,并对其过滤机理进行了分析和讨论。研究表明:UHMWPE管式微滤膜内部存在着网络状通孔,微滤管的过滤精度可达0.20μm;从过滤机理来看,可分为物理截流、吸附、“堵塞截流”等三种方式。