Membrane surfaces regulate assembly of a ribonucleoprotein condensate

Biomolecular condensates organize biochemistry in time and space, yet little is known about how cells control either the position or scale of these assemblies. In cells, condensates often appear as dispersed, relatively small assemblies that do not grow (coarsen) into a single droplet despite their propensity to coalesce. Here we report that ribonucleoprotein condensates of the Q-rich protein Whi3 interact with the endoplasmic reticulum, prompting us to hypothesize that membrane association controls the position and size of condensates. Reconstitution of Whi3 condensates on supported lipid bilayers reveals that association with a diffusive lipid surface promotes condensation at both physiological ionic strength and protein concentration. Notably, these assemblies rapidly arrest, matching size distributions seen in cells. The timing of the arrest is influenced by the ordering of protein-protein and protein-RNA interactions and controlled by the slow diffusion of complexes induced by the membrane. This slowed diffusion limits both transfer of small protein-RNA complexes between condensates and their coalescence, thus driving coarsening to arrest. Our experiments reveal a tradeoff between locally-enhanced protein concentration at membranes, which favors condensation, and an accompanying reduction in diffusion, which restricts coarsening. Thus, membranes can maintain a population of small condensates in the absence of active mechanisms. Given that many condensates are bound to endomembranes, we predict that the biophysical properties of lipid bilayers are key for controlling condensate sizes throughout the cell.One sentence summaryAssembly on a membrane surface positions and scales biomolecular condensates by controlling relative diffusion rates of proteins and nucleic acids.

Diagnostic and Therapeutic Applications of Exosome Nanovesicles in Lung Cancer: State-of-The-Art

Lung cancer is a malignant disease with a frequency of various morbidity, mortality, and poor prognosis in patients that the conventional therapeutic approaches are not efficient sufficiently. Recently, with the discovery of exosomes, researchers have examined new approaches in the development, diagnosis, treatment, and drug delivery of various cancer, such as lung cancer, and display various its potential. Investigation of exosome-derived lung cancer cells contents and preparation of their exhaustive profile by advanced technics such as labeling exosome with nanoparticle and types of mass spectroscopy methods will assist researchers for take advantage of the specific properties of exosomes. Moreover, scientists will present encouraging ways for the treatment of lung cancer with loaded of drugs, proteins, microRNA, and siRNA in specific antigen targeted exosomes. This manuscript will include brief details on the role of exosomes as a novel prognostic biomarker (by the content of lipid, surface and internal protein, miRNAs, and LnRNAs) and therapeutic agent (as vaccine and targeted drug delivery) in lung cancer.

Divalent cations bind to phosphoinositides to induce ion and isomer specific propensities for nano-cluster initiation in bilayer membranes

We report all-atom molecular dynamics simulations of asymmetric bilayers containing phosphoinositides in the presence of monovalent and divalent cations. We have characterized the molecular mechanism by which these divalent cations interact with phosphoinositides. Ca 2+ desolvates more readily, consistent with single-molecule calculations, and forms a network of ionic-like bonds that serve as a ‘molecular glue’ that allows a single ion to coordinate with up to three phosphatidylinositol-(4,5)-bisphosphate (PI(4, 5)P 2 ) lipids. The phosphatidylinositol-(3,5)-bisphosphate isomer shows no such effect and neither does PI(4, 5)P 2 in the presence of Mg 2+ . The resulting network of Ca 2+ -mediated lipid-lipid bonds grows to span the entire simulation space and therefore has implications for the lateral distribution of phosophoinositides in the bilayer. We observe context-specific differences in lipid diffusion rates, lipid surface densities and bilayer structure. The molecular-scale delineation of ion-lipid arrangements reported here provides insight into similar nanocluster formation induced by peripheral proteins to regulate the formation of functional signalling complexes on the membrane.

X-Ray Crystallographic and Single-Molecule Fluorescence Studies of Beta-2 Glycoprotein I Reveal an Alternative Mechanism of Autoantibody Recognition

Background. Antiphospholipid antibodies (aPL) recognizing an epitope comprising residues R39-R43 in the N-terminal domain, Domain I (DI), of beta-2 glycoprotein I (b2GPI) are considered among the most pathogenic in patients with Antiphospholipid Syndrome (APS). How such autoantibodies engage b2GPI at the molecular level remains incompletely understood. Aim. To better understand how pathogenic anti-DI antibodies engage b2GPI at the molecular level. Results. Under physiological conditions, b2GPI is believed to adopt a closed conformation featuring an intramolecular interaction between DI and DV with amino acids R39 and R43 in DI being masked by DV. This conformation is therefore predicted to be immunologically inert, incapable of reacting against pathogenic anti-DI antibodies. Once bound to the membranes, however, b2GPI is believed to undergo a dramatic conformational change which liberates DI to the solvent. To get a better grasp of the molecular flexibility of b2GPI under conditions relevant to physiology, we expressed and purified fully-glycosylated human recombinant b2GPI (hr-b2GPI) from HEK293 cells at high yield and purity suitable for structural biology and biophysical studies. After native purification, we found that the recombinant protein bound to heparin and negatively charged phospholipids with affinities comparable to those obtained for b2GPI that was purified from plasma using the perchloric acid method (p-b2GPI); hr-b2GPI also displayed similar reactivity against anti-b2GPI immunoglobulin G antibodies that were isolated from 5 APS patients. Surprisingly, hr-b2GPI and p-b2GPI were structurally similar, too. The X-ray crystal structures of hr-b2GPI and p-b2GPI solved at 2.6 and 2.4 Å resolution were superimposable documenting a J-shaped elongated conformation of the molecule in which DI was located > 90 Å away from the C-terminal DV. Both structures were characterized by 22 oxidized cysteine residues forming 11 disulfide bonds, 4 N-glycosylations, and an intact yet flexible phospholipid-binding loop in DV. Since crystallization occurred at high salt concentrations, validation of the crystal structure of hr-b2GPI in solution was obtained by single-molecule Förster Resonance Energy Transfer (smFRET) and small-angle X-ray scattering (SAXS), while surface plasmon resonance (SPR) was used to probe the binding of a recently developed monoclonal anti-DI antibody, i.e., MBBS, to hr-b2GPI and p-b2GPI in solution. In keeping with the X-ray structural data, donor and acceptor fluorophores incorporated at positions 13/312 in DI and DV and 112/312 in DII and DV reported no measurable energy transfer whereas probes located at positions 13/112 in DI and DII displayed very high energy transfer. Likewise, the scattering profiles of the recombinant and plasma purified proteins returned similar hydrodynamic radii characteristic of elongated, flexible protein structures, and not circular. Notably, both hr-b2GPI and p-b2GPI in the elongated conformation were capable of interacting with MBBS without the need of phospholipids, even though addition of negatively charged phospholipids decreased the apparent dissociation affinity constant due to a reduction of the dissociation rate constant and a remarkable time-dependent accumulation of b2GPI onto the lipid surface, suggestive of a phospholipid-induced oligomerization mechanism. Conclusions. This study demonstrates that human b2GPI can adopt an elongated conformation in solution that is primed for phospholipid, heparin, and autoantibodies binding with DI constitutively exposed to the solvent. The fact that phospholipid-bound b2GPI is a better antigen for anti-DI autoantibody under physiological conditions as compared to the elongated form in solution can be explained by the relatively low affinity and bivalency of such autoantibodies that likely recognize a peptide motif pattern rather than a specific sequence of residues. Whether other context-dependent conformational changes occur after binding of the protein to the lipid surface, thus facilitating aPL binding, remain to be established. Since our studies failed to detect the closed form of b2GPI previously documented by electron and atomic force microscopy studies, it is possible that this conformation may arise from chemical and/or posttranslational modifications that occur in vivo while the protein circulates in the plasma. Disclosures No relevant conflicts of interest to declare.

Farmakoterapia skóry. Część 3. Nawilżanie skóry i naturalne środki nawilżające

Moisturizing of the skin depends on both the penetration of water present in dermis, and the skin’s ability to retain water on its surface. The movement of water from the dermis to the surface encounters barriers: a natural moisturizing factor (NMF), as well as a water-lipid surface covering the skin. An important role in the evaporation of water through the skin is also played by external factors, such as air temperature, surfactants and disease states. To reduce water loss by the skin, many natural moisturizers are used, including hydrophilic water-binding on the epidermis (hyaluronic acid, chitosan, collagen, sorbitol), penetrating into the epidermis (glycerol, urea, biotin), hydrophilic occlusive substances (beeswax, cetyl alcohol, soybean lecithin), and modifying the epidermal barrier (ceramides, cholesterol, NUFA).

Known and Unknown Features of Hair Cuticle Structure: A Brief Review

The cuticle is the outermost layer of overlapping flattened cells of hair and has been subjected to many years of study to understand its structure and how it develops in the follicle. The essential function of the cuticle with its tough inelastic protein content is to protect the inner cortex that provides the elastic properties of hair. Progress in our knowledge of hair came from studies with the electron microscope, initially transmission electron microscopy (TEM) for internal structure and later the scanning electron microscope (SEM) for cuticle surface shape and for investigating changes caused by various environmental influences such as cosmetic treatments and industrial processing of wool. Other physical techniques have been successfully applied in conjunction with proteomics. The outstanding internal features of the cuticle cells are the internal layers consisting of keratin filament proteins and the keratin-associated proteins. The stability and physical toughness of the cuticle cell is partly accounted for by the high content of disulphide crosslinking. The material between the cells that holds them tightly together, the cell membrane complex, consists of a layer of lipid on both sides of a central protein layer. The lipid contains 18-methyleicosanoic acid that is part of the hydrophobic lipid surface of hair. For the past decade there have been aspects that remained unanswered because they are difficult to study. Some of these are discussed in this brief review with suggestions for experimental approaches to shed more light.

Escherichia coliZipA Organizes FtsZ Polymers into Dynamic Ring-Like Protofilament Structures

ABSTRACTZipA is an essential cell division protein inEscherichia coli. Together with FtsA, ZipA tethers dynamic polymers of FtsZ to the cytoplasmic membrane, and these polymers are required to guide synthesis of the cell division septum. This dynamic behavior of FtsZ has been reconstituted on planar lipid surfacesin vitro, visible as GTP-dependent chiral vortices several hundred nanometers in diameter, when anchored by FtsA or when fused to an artificial membrane binding domain. However, these dynamics largely vanish when ZipA is used to tether FtsZ polymers to lipids at high surface densities. This, along with somein vitrostudies in solution, has led to the prevailing notion that ZipA reduces FtsZ dynamics by enhancing bundling of FtsZ filaments. Here, we show that this is not the case. When lower, more physiological levels of the soluble, cytoplasmic domain of ZipA (sZipA) were attached to lipids, FtsZ assembled into highly dynamic vortices similar to those assembled with FtsA or other membrane anchors. Notably, at either high or low surface densities, ZipA did not stimulate lateral interactions between FtsZ protofilaments. We also usedE. colimutants that are either deficient or proficient in FtsZ bundling to provide evidence that ZipA does not directly promote bundling of FtsZ filamentsin vivo. Together, our results suggest that ZipA does not dampen FtsZ dynamics as previously thought, and instead may act as a passive membrane attachment for FtsZ filaments as they treadmill.IMPORTANCEBacterial cells use a membrane-attached ring of proteins to mark and guide formation of a division septum at midcell that forms a wall separating the two daughter cells and allows cells to divide. The key protein in this ring is FtsZ, a homolog of tubulin that forms dynamic polymers. Here, we use electron microscopy and confocal fluorescence imaging to show that one of the proteins required to attach FtsZ polymers to the membrane duringE. colicell division, ZipA, can promote dynamic swirls of FtsZ on a lipid surfacein vitro. Importantly, these swirls are observed only when ZipA is present at low, physiologically relevant surface densities. Although ZipA has been thought to enhance bundling of FtsZ polymers, we find little evidence for bundlingin vitro. In addition, we present several lines ofin vivoevidence indicating that ZipA does not act to directly bundle FtsZ polymers.