Cell Membrane Interactions Research Articles

Pathways for external alkalinization in intact and in microwounded Chara cells are differentially sensitive to wortmannin

ABSTRACTProton flows across the plant cell membranes play a major role in electrogenesis and regulation of photosynthesis and ion balance. The profiles of external pH along the illuminated internodal cells of characean algae consist of alternating high- and low-pH zones that are spatially coordinated with the distribution of photosynthetic activity of chloroplasts underlying these zones. The results based on confocal laser scanning fluorescence microscopy, pH microsensors, and pulse-amplitude-modulated chlorophyll microfluorometry revealed that the coordination of H+ transport and photosynthesis is disrupted by the 2 different environmental cues (low light and wounding) and by a chemical, wortmannin interfering with the inositol phospholipid metabolism. On the one hand, the transition from moderate to low irradiance diminished the peaks in the profiles of photosystem II (PSII) quantum efficiency but did not remove the pH bands. On the other hand, the microwounding of the internode with a glass micropipette, impacting primarily the cell wall, resulted in a rapid local alkalinization of the external medium (by 2–2.5 pH units) near the cell surface, thus mimicking the appearance of natural pH bands. Despite their seeming similarity, the alkaline bands of intact cells were eliminated by wortmannin, whereas the wound-induced alkalinization was insensitive to this drug. Furthermore, the attenuation of natural pH bands in wortmannin-treated cells was accompanied by the enhancement in spatial heterogeneity of PSII efficiency and electron transport rates, which indicates the complexity of chloroplast–plasma membrane interactions. The results suggest that the light- and wound-induced alkaline areas on the cell surface are associated with different ion-transport systems.

Promysalin is a salicylate-containing antimicrobial with a cell-membrane-disrupting mechanism of action on Gram-positive bacteria

Promysalin was previously described as a narrow spectrum molecule with a unique species-specific activity against Pseudomonas aeruginosa. Here we demonstrate that promysalin is active against Gram-positive and Gram-negative bacteria using a microdilution assay. Promysalin acts on Gram-positive bacteria with a mechanism of action involving cell membrane damage with leakage of intracellular components. The evaluation of MICs and MBCs on 11 promysalin analogs, synthesized utilizing diverted total synthesis, allowed the identification of the structural moieties potentially involved in cell membrane interaction and damage. The mechanism of action of promysalin against Gram-negative bacteria is still not clarified, even if a synergistic effect with the bisguanidine chlorhexidine on cell membrane disruption has been observed.

Liquid-phase electron microscopy of molecular drug response in breast cancer cells reveals irresponsive cell subpopulations related to lack of HER2 homodimers.

The development of drug resistance in cancer poses a major clinical problem. An example is human epidermal growth factor receptor 2 (HER2) overexpressing breast cancer often treated with anti-HER2 antibody therapies, such as trastuzumab. Since drug resistance is rooted mainly in tumor cell heterogeneity, we examined the drug effect in different subpopulations of SKBR3 breast cancer cells, and compared the results with a drug resistant cell line, HCC1954. Correlative light microscopy and liquid-phase scanning transmission electron microscopy (STEM) were used to quantitatively analyze HER2 responses upon drug binding, whereby many tens of whole cells were imaged. Trastuzumab was found to selectively cross-link and down regulate HER2 homodimers from the plasma membranes of bulk cancer cells. In contrast, HER2 resided mainly as monomers in rare subpopulations of resting- and cancer stem cells (CSCs), and these monomers were not internalized after drug binding. The HER2 distribution was hardly influenced by trastuzumab for the HCC1954 cells. These findings show that resting cells and CSCs are irresponsive to the drug, and thus point towards a molecular explanation behind the origin of drug resistance. This analytical method is broadly applicable to study membrane protein interactions in the intact plasma membrane, while accounting for cell heterogeneity.

Size Effect on the Cytotoxicity of Layered Black Phosphorus and Underlying Mechanisms.

A systematic cytotoxicity study of layered black phosphorus (BP) is urgently needed before moving forward to its potential biomedical applications. Herein, bulk BP crystals are synthesized and exfoliated into layered BP with different lateral size and thickness. The cytotoxicity of as-exfoliated layered BP is evaluated by a label-free real-time cell analysis technique, displaying a concentration-, size-, and cell type-dependent response. The IC50 values can vary by 40 and 30 times among the BP sizes and cell types, respectively. BP-1 with the largest lateral size and thickness has the highest cytotoxicity; whereas the smallest BP-3 only shows moderate toxicity. The sensitivity of three tested cell lines follows the sequence of 293T > NIH 3T3 > HCoEpiC. Two possible mechanisms for BP to induce cytotoxicity are proposed and verified: (1) the generation of intracellular reactive oxygen species (ROS) is detected by a ROS sensitive probe using the inverted fluorescence microscopy and flow cytometry; (2) the interaction of layered BP and model cell membrane is examined by quartz crystal microbalance with dissipation, illustrating the disruption of cell membrane integrity especially by the largest BP-1. This systematic study of BP's cytotoxicity will shed light on its future biomedical and environmental applications.

Antimicrobial Peptides in the Cross Hairs of Computer Simulations

Antimicrobial peptides occur in a staggering variety throughout all organisms and range from just a few residues and no well-defined secondary structure to small proteins like human defensins (1,2). They are both of major biological interest in the chemical warfare between species and as possible new antibiotics, in their native forms or as a source of inspiration for the design of new compounds. Antimicrobial peptidelike peptides include cell-penetrating peptides, which may be useful in drug delivery and peptides targeted at other cell types, including cancer.

Graphene and the Immune System: A Romance of Many Dimensions.

Graphene-based materials (GBMs) are emerging as attractive materials for biomedical applications. Understanding how these materials are perceived by and interact with the immune system is of fundamental importance. Phagocytosis is a major mechanism deployed by the immune system to remove pathogens, particles, and cellular debris. Here, we discuss recent studies on the interactions of GBMs with different phagocytic cells, including macrophages, neutrophils, and dendritic cells. The importance of assessing GBMs for endotoxin contamination is discussed as this may skew results. We also explore the role of the bio-corona for interactions of GBMs with immune cells. Finally, we highlight recent evidence for direct plasma membrane interactions of GBMs.

Amphiphilic polymer layer – Model cell membrane interaction studied by QCM and AFM

The benefit gained by application of nanostructured drug delivery systems is highly influenced by their possible transport in the body. The affinity to cell membrane is a crucial parameter to be optimized by tuning the surface properties of nanoparticles (NPs). Poly(lactic/glycolic acid) (PLGA) NPs designed for drug delivery were stabilized with three different Pluronics (103, 105 and 108) as well as an amphiphilic monoalkyl hyperbranched polyglycerol (C18-HbPG). Their interaction with lipid bilayer was investigated in two types of arrangements using liposomes and polymeric adsorbed layer, while in the other model polymer stabilized PLGA NPs and supported lipid bilayer (SLB) were applied. Degree of adhesion of liposomes and PLGA NPs were characterized by quartz crystal microbalance (QCM) measurements in combination with the visual analysis of the surfaces by atomic force microscopy (AFM). The comparison of the various polymer coatings led to the conclusion that C18-HbPG resulted in the highest membrane affinity, followed by Pluronic105 with medium polarity within the Pluronic series. This finding further supports the results obtained previously regarding colloid stabilization efficacy (Kasza, 2017) that amphiphilic hyperbranched polyglycerol is an ideal alternative to Pluronics in the functional surface modification of drug carrier NPs.

Sperm-oocyte contact induces outside-in signaling via PYK2 activation

Fertilization is a multi-step process that begins with plasma membrane interactions that enable sperm – oocyte binding followed by fusion of the sperm and oocyte plasma membranes. Once membrane fusion has occurred, sperm incorporation involves actin remodeling events within the oocyte cortex that allow the sperm head to penetrate the cortical actin layer and gain access to the ooplasm. Despite the significance for reproduction, the control mechanisms involved in gamete binding, fusion, and sperm incorporation are poorly understood. While it is known that proline – rich tyrosine kinase 2 (PYK2 or PTK2b) kinase activity plays an important role in fertilization, its specific function has not been addressed. The present study made use of a zona-free mouse oocyte fertilization assay to investigate the relationship between PYK2 activity and sperm – oocyte binding and fusion, as well as localized changes in actin polymerization and sperm incorporation. In this assay, the majority of bound sperm had no apparent effect on the oocyte and only a few became incorporated into the ooplasm. However, a subset of bound sperm were associated with a localized response in which PYK2 was recruited to the oocyte cortex where it frequently co-localized with a ring or disk of f-actin. The frequency of sperm-oocyte binding sites that exhibited this actin response was reduced in pyk2-/- oocytes and the pyk2-/- oocytes proved less efficient at incorporating sperm, indicating that this protein kinase may have an important role in sperm incorporation. The response of PYK2 to sperm-oocyte interaction appeared unrelated to gamete fusion since PYK2 was recruited to sperm – binding sites under conditions where sperm – oocyte fusion was prevented and since PYK2 suppression or ablation did not prevent sperm – oocyte fusion. While a direct correlation between the PYK2 response in the oocyte and the successful incorporation of individual bound sperm remains to be established, these findings suggest a model in which the oocyte is not a passive participant in fertilization, but instead responds to sperm contact by localized PYK2 signaling that promotes actin remodeling events required to physically incorporate the sperm head into the ooplasm.

Dual functional small molecule fluorescent probes for image-guided estrogen receptor-specific targeting coupled potent antiproliferative potency for breast cancer therapy

A strategy by integrating biological imaging into early stages of the drug discovery process can improve our understanding of drug activity during preclinical and clinical study. In this article, we designed and synthesized coumarin-based nonsteroidal type fluorescence ligands for drug-target binding imaging. Among these synthesized compounds, 3e, 3f and 3h showed potent ER binding affinity and 3e (IC50=0.012μM) exhibited excellent ERα antagonistic activity, its antiproliferative potency in breast cancer MCF-7 cells is equipotent to the approved drug tamoxifen. The fluorescence of compounds 3e and 3f depended on the solvent properties and showed significant changes when mixed with ERα or ERβ in vitro. Furthermore, target molecule 3e could cross the cell membrane, localize and image drug-target interaction in real time without cell washing. Thus, the coumarin-based platform represents a promising new ER-targeted delivery vehicle with potential imaging and therapeutic properties.

Kinetic Off-Rates of Membrane Associated Proteins Measured in Unroofed Cells

Cellular function is orchestrated by the vast network of interacting biological molecules in the cell, and these interactions are governed by the kinetics of association and dissociation. The biochemist's in vitro toolkit provides a means to measure a small part of this network with high precision, unveiling a mechanism of action. It is in this regard that careful measurements of kinetics and binding equilibria of proteins and lipids reconstituted as mimetic plasma membrane has provided valuable insights into cell function (Knight and Falke, Biophys. J. 2009). From these studies in the in vitro setting it is worthwhile asking if the same measurements may be made in the cellular context to further refine our understanding of protein - plasma membrane interactions. In order to frame our study of protein - plasma membrane interaction within a cellular context we relied on cell “unroofing” for the nearly instantaneous production of a plasma membrane sheet exposed to a buffer solution. Upon unroofing, the rapid shift in equilibrium experienced by GFP-PLCδ-PH (a PIP2 binding protein, residing on the inner leaflet of the plasma membrane) led to an exponential loss of GFP fluorescence that was recorded and fit to a kinetic off-rate (1.1 ± 0.3 s−1; N=3). These results demonstrate a practical approach to measuring the dissociation rate constant for plasma membrane associated proteins in the cellular context and potentially open the door to studying binding interactions that have been intractable as in vitro experiments.

Lipid-Protein Interactions in Fiber Cell Plasma Membrane Isolated From Human and Porcine Eye Lenses

The protein content in human lens fiber cell plasma membranes is extremely high, increases with age, and is higher in the nucleus than in cortex. This strongly affects the organization and physical properties of the lipid bilayer portion of intact fiber cell plasma membranes including the alkyl chain order, fluidity, hydrophobicity, and oxygen transport parameter. Investigated membranes were isolated from the eye lenses of pigs and human donors. They were studied using electron paramagnetic resonance spin-labeling method that provides an opportunity to discriminate coexisting lipid domains and to evaluate the relative amounts of phospholipids and cholesterol in them. Fiber-cell membranes were found to contain three distinct lipid environments: bulk, boundary and trapped domains that appear due to the presence of membrane proteins. In nuclear membranes the amount of boundary and trapped phospholipids as well as the amount of cholesterol in trapped lipid domains was greater than that in cortical membranes. Cholesterol was to a large degree excluded from trapped lipid domains in cortical membranes. The difference between the amounts of lipids in domains uniquely formed due to the presence of membrane proteins increased with the donors‘ age. The rigidity of nuclear membranes was greater than that of cortical membranes for all age groups. The nuclear fiber cell plasma membranes were less permeable to oxygen than cortical membranes and both of them become less permeable to oxygen with age. In nuclear porcine membranes the amounts of lipids in domains created due to the presence of membrane proteins were greater than those in cortical membranes and the differences were larger than the differences observed for human membranes. Lipids in porcine nuclear fiber cell membranes were more rigid and less permeable to oxygen than in human nuclear membranes.

Recent advances in occupational and environmental health hazards of workers exposed to gasoline compounds.

The impact of health and environmental hazards, associated with the constituents of gasoline, on occupationally exposed workers has been recorded over the past few decades. However, the scientific literature on their pathogenic potential remains incomplete, which could affect the current understanding of the associated health risks. This review provides current information based on recently improved research techniques to evaluate gasoline toxicity profiles for humans. Our current knowledge provides insight into the intricate mechanism of gasoline-induced adverse effects, including the formation of reactive metabolites via bio-activation and subsequent generation of reactive oxygen species (ROS) and oxidative stress, which are involved in multiple mechanisms that are central to the aetiology of gasoline-induced toxicity. These mechanisms include covalent binding to deoxyribonucleic acid (DNA), leading to oxidative damage, tumor-suppression gene activity, and activation of pro-oncogenes. Furthermore, it results in induction of autoimmunity and local inflammatory responses, disruption of multiple neurotransmitters and immune cell function, derangement of various enzyme activities (e.g., sodiumpotassium adenosine triphosphate (Na+/K+/ATPase) activity, cytochrome P450 (CYP450), nitric oxide synthase (NOS), antioxidant enzyme activities, etc.), conjugation of bile, and non-specific cell membrane interaction, leading to damage of the membrane lipid bilayer and proteins. Available data suggests that exposure to gasoline or gasoline constituents have the potential to cause different types of illnesses. The data highlights the need to maintain safety measures via suitable research, medical surveillance, regulatory control, life style modification, early detection, and intervention to minimize exposure and manage suspected cases. They also present novel opportunities to design and develop effective therapeutic strategies against gasoline-induced detrimental effects. Int J Occup Med Environ Health 2017;30(1):1-26.

Probing Induced Structural Changes in Biomimetic Bacterial Cell Membrane Interactions with Divalent Cations

Biological membranes, formed primarily by the self-assembly of complex mixtures of phospholipids, provide a structured scaffold for compartmentalization and structural processes in living cells. The specific physical properties of phospholipid species present in a given membrane play a key role in mediating these processes. Phosphatidylethanolamine (PE), a zwitterionic lipid present in bacterial, yeast, and mammalian cell membranes, is exceptional. In addition to undergoing the standard lipid polymorphic transition between the gel and liquid-crystalline phase, it can also assume an unusual polymorphic state, the inverse hexagonal phase (HII). Divalent cations are among the factors that drive the formation of the HII phase, wherein the lipid molecules form stacked tubular structures by burying the hydrophilic head groups and exposing the hydrophobic tails to the bulk solvent. Most biological membranes contain a lipid species capable of forming the HII state suggesting that such lipid polymorphic structural states play an important role in structural biological processes such as membrane fusion. In this study, the interactions between Mg2+ and biomimetic bacterial cell membranes composed of PE and phosphatidylglycerol (PG) were probed using differential scanning calorimetry (DSC), small-angle x-ray scattering (SAXS), and fluorescence spectroscopy. The lipid phase transitions were examined at varying ratios of PE to PG and upon exposure to physiologically relevant concentrations of Mg2+. An understanding of these basic interactions enhances our understanding of membrane dynamics and how membrane-mediated structural changes may occur in vivo.

Quicker than Quick: Correlative Optical Tweezers-Fluorescence Microscopy (CTFM) for the Study of Short-Lived Metabolic Processes

Using optical tweezers in combination with confocal fluorescence microscopy in a controlled microfluidics environment, results in a robust and versatile methodology to study mechanisms occurring at all levels of the cellular metabolism, up to cell membrane interactions and beyond. Both the high temporal and spatial resolutions that correlative optical tweezers-fluorescence microscopy (CTFM) offers represent an asset to investigate processes with short lifetimes. Additionally, by being able to manipulate and exert tensions to selected molecules, we gain access to important structural features. To demonstrate the potential of this hybrid technique, we performed a series of experiments to study the folding/unfolding dynamics of an intracellular signaling protein and the packaging of DNA within a bacteriophage capsid. Both model systems present transient states with fast kinetics, which would be overlooked using other techniques. Our results show that the applications of this single‑molecule technology are not limited to the field of nucleic acids research and quickly advance to new venues.

Evolutionary plasticity of plasma membrane interaction in DREPP family proteins

The plant-specific DREPP protein family comprises proteins that were shown to regulate the actin and microtubular cytoskeleton in a calcium-dependent manner. Our phylogenetic analysis showed that DREPPs first appeared in ferns and that DREPPs have a rapid and plastic evolutionary history in plants. Arabidopsis DREPP paralogues called AtMDP25/PCaP1 and AtMAP18/PCaP2 are N-myristoylated, which has been reported as a key factor in plasma membrane localization. Here we show that N-myristoylation is neither conserved nor ancestral for the DREPP family. Instead, by using confocal microscopy and a new method for quantitative evaluation of protein membrane localization, we show that DREPPs rely on two mechanisms ensuring their plasma membrane localization. These include N-myristoylation and electrostatic interaction of a polybasic amino acid cluster. We propose that various plasma membrane association mechanisms resulting from the evolutionary plasticity of DREPPs are important for refining plasma membrane interaction of these signalling proteins under various conditions and in various cells.

Structural features of many circular and leaderless bacteriocins are similar to those in saposins and saposin-like peptides.

Bacteriocins are potent antimicrobial peptides that are ribosomally produced and exported by bacteria, presumably to aid elimination of competing microorganisms. Many circular and linear leaderless bacteriocins have a recuring three dimensional structural motif known as a saposin-like fold. Although these bacteriocin sizes and sequences are often quite different, and their mechanisms of action vary, this conserved motif of multiple helices appears critical for activity and may enable peptide-lipid and peptide-receptor interactions in target bacterial cell membranes. Comparisons between electrostatic surfaces and hydrophobic surface maps of different bacteriocins are discussed emphasizing similarities and differences in the context of proposed modes of action.

A low-cost, large field-of-view scanning ion conductance microscope for studying nanoparticle-cell membrane interactions.

Nanoparticles have the potential to become versatile tools in the medical and life sciences. One potential application is delivering drugs or other compounds to the cell cytoplasm, which requires the nanoparticles to bind to or cross the cell membrane. However, there are only a few tools available which allow studying the interaction of nanoparticles and the cell membrane of living cells in a physiological environment. Currently, the tool which least biases living cells is Scanning Ion Conductance Microscopy (SICM). Specialized SICMs allow imaging at high resolution, however, they are cost intensive, particularly when providing a large field-of-view. In contrast, less cost intensive SICMs which provide a large field-of-view do not allow imaging at high resolutions. We have developed a SICM setup consisting of a compact three-axis piezo system and an additional fast shear-force piezo actor. This combination allows imaging fields-of-view of up to 80 μm × 80 μm, recording sections of living cells with a temporal resolution in the range of minutes as well as imaging with a spatial resolution of below 70 nm. Using our SICM we found that the cell membrane of HeLa cells treated with carboxylated latex nanoparticles was significantly more convoluted compared to control cells. The SICM setup we introduce here combines high resolution imaging with a large field-of-view at low costs. Our setup only requires a mounting adapter to extend existing inverted light microscopes, thus it could be a valuable and cost effective tool for researchers in all fields of the medical and life sciences performing investigations at the nanometer scale.

AMPK and Endothelial Nitric Oxide Synthase Signaling Regulates K-Ras Plasma Membrane Interactions via Cyclic GMP-Dependent Protein Kinase 2.

K-Ras must localize to the plasma membrane and be arrayed in nanoclusters for biological activity. We show here that K-Ras is a substrate for cyclic GMP-dependent protein kinases (PKGs). In intact cells, activated PKG2 selectively colocalizes with K-Ras on the plasma membrane and phosphorylates K-Ras at Ser181 in the C-terminal polybasic domain. K-Ras phosphorylation by PKG2 is triggered by activation of AMP-activated protein kinase (AMPK) and requires endothelial nitric oxide synthase and soluble guanylyl cyclase. Phosphorylated K-Ras reorganizes into distinct nanoclusters that retune the signal output. Phosphorylation acutely enhances K-Ras plasma membrane affinity, but phosphorylated K-Ras is progressively lost from the plasma membrane via endocytic recycling. Concordantly, chronic pharmacological activation of AMPK → PKG2 signaling with mitochondrial inhibitors, nitric oxide, or sildenafil inhibits proliferation of K-Ras-positive non-small cell lung cancer cells. The study shows that K-Ras is a target of a metabolic stress-signaling pathway that can be leveraged to inhibit oncogenic K-Ras function.

Adsorption of Nanoceria by Phosphocholine Liposomes.

Nanoceria (CeO2 nanoparticle) possesses a number of enzyme-like activities. In particular, it scavenges reactive oxygen species based on in vitro and in vivo antioxidation studies. An important aspect of fundamental physical understanding is its interaction with lipid membranes that are the main components of the cell membrane. In this work, adsorption of nanoceria onto phosphocholine (PC) liposomes was performed. PC lipids are the main constituents of the cell outer membrane. Using a fluorescence quenching assay, a nanoceria adsorption isotherm was determined at various pH values and ionic strengths. A non-Langmuir isotherm occurred at pH 4 because of lateral electrostatic repulsion among the adsorbed cationic nanoceria. The phosphate group in the PC lipid is mainly responsible for the interaction, and the adsorbed nanoceria can be displaced by free inorganic phosphate. The tendency of the system to form large aggregates is a function of pH and the concentration of nanoceria, attributable to nanoceria being positively charged at pH 4 and neutral at physiological pH. Calcein leakage tests indicate that nanoceria induces liposome leakage because of transient lipid phase transition, and cryo-transmission electron microscopy indicates that the overall shape of the liposome is retained although deformation is still observed. This study provides fundamental biointerfacial information at a molecular level regarding the interaction of nanoceria and model cell membranes.

Rapid fluorometric bacteria detection assay and photothermal effect by fluorescent polymer of coated surfaces and aqueous state

A fluorescent dye and a photothermal agent were grafted onto a cationic polymer for rapid and simple bacteria detection in liquid and solid phase based fluorescence on/off. The integrated poly(vinylpyrrolidone) (PVP) backbone with catechol and bromoethane moieties possesses unique optical properties due to the presence of boron dipyrromethane (BODIPY) and near infared NIR-responsive IR825 (F-PVP). The cationic segments showed distinct fluorescence quenching patterns after interaction with gram-positive and gram-negative bacteria via polyion complex interactions. Fluorescence quenching depended on direct interaction of the bacterial cell membrane, as confirmed using SEM and confocal imaging. The detection limit was 1mg/mL for the liquid-phase assay and the minimal detectable concentration of bacteria using the solid-phase assay was 106CFU/mL. After bacterial detection in contaminated area, our system can directly kill bacteria via the photothermal conversion ability of the IR825 substituent using NIR exposure by polymer solution and limited in coated PP. Finally, the proposed biosensor is capable as potential material for detection of bacteria in simple liquid and solid phase assay.