Isolated Plasma Membranes Research Articles

Development and Biological Characterization of Cancer Biomimetic Membrane Nanovesicles for Enhancing Therapy Efficacy in Human Glioblastoma Cells

As nanocarriers of a new generation, biomimetic nanovesicles are an emerging class of therapeutic tools whose surface is integrated or fabricated with biomaterials capable of mimicking the biological features and functions of native cells. Thanks to this, biomimetic nanovesicles, in particular, those made by plasma membrane moieties, possess greatly improved biocompatibility, high target specificity, a long retention time, and minimal undesired immune responses. For these reasons, a multitude of progenitor cells including cancer ones were employed as templates to generate biomimetic or membrane-camouflaged nanovesicles hosting different therapeutic compounds. In this contribution, different membrane-derived biomimetic vesicles (M-NVs) were generated by osmotic lysis or plasma membrane isolation approaches from normal and cancer cell lines and assayed against in vitro models of human glioblastoma. M-NVs were compared in their cellular internalization degrees of DNA and proteins, morphologically and molecularly characterized, expressing an extracellular membrane-associated marker. Then, Rose Bengal (RB), a photoactivable drug characterized by a relatively low cellular uptake, was incorporated into nascent glioblastoma-derived M-NVs and finally administered to homotypic receiving cells, showing an increased degree of internalization as well as induced cytotoxic effects, even in the absence of photodynamic direct stimulation. Similar results were also obtained assaying lyophilized M-NVs loaded with RB. In conclusion, M-NVs generated by cell membranes effectively deliver several cargoes, including therapeutic molecules, maintain functionality after lyophilization, and show significant internalization effects, making them a promising strategy for therapeutic applications against human glioblastoma cells.

Correlative Cryo-electron Tomography on Isolated Plasma Membranes

Correlative Cryo-electron Tomography on Isolated Plasma Membranes

Iron gallic acid biomimetic nanoparticles for targeted magnetic resonance imaging.

Developing T1-weighted magnetic resonance imaging (MRI) contrast agents with enhanced biocompatibility and targeting capabilities is crucial owing to concerns over current agents' potential toxicity and suboptimal performance. Drawing inspiration from "biomimetic camouflage," we isolated cell membranes (CMs) from human glioblastoma (T98G) cell lines via the extrusion method to facilitate homotypic glioma targeting. At an 8:1 mass ratio of ferric chloride hexahydrate to gallic acid (GA), the resulting iron (Fe)-GA nanoparticles (NPs) proved effective as a T1-weighted MRI contrast agent. T98G CM-coated Fe-GA NPs demonstrated improved homotypic glioma targeting, validated through Prussian blue staining and in vitro MRI. This biomimetic camouflage strategy holds promise for the development of targeted theranostic agents in a safe and effective manner.

Design, Synthesis, and Evaluation of a New Chemotype Fluorescent Ligand for the P2Y2 Receptor.

The P2Y2 receptor (P2Y2R) is a target for diseases including cancer, idiopathic pulmonary fibrosis, and atherosclerosis. However, there are insufficient P2Y2R antagonists available for validating P2Y2R function and future drug development. Evaluation of how (R)-5-(7-chloro-2-((2-ethoxyethyl)amino)-4H-benzo[5,6]cyclohepta[1,2-d]thiazol-4-yl)-1-methyl-4-thioxo-3,4-dihydropyrimidin-2(1H)-one, a previously published thiazole-based analogue of AR-C118925, binds in a P2Y2R homology model was used to design new P2Y2R antagonist scaffolds. One P2Y2R antagonist scaffold retained millimolar affinity for the P2Y2R and upon further functionalization with terminal carboxylic acid groups affinity was improved over 100-fold. This functionalized P2Y2R antagonist scaffold was employed to develop new chemotype P2Y2R fluorescent ligands, that were attainable in a convergent five-step synthesis. One of these fluorescent ligands demonstrated micromolar affinity (pK d = 6.02 ± 0.12, n = 5) for the P2Y2R in isolated cell membranes and distinct pharmacology from an existing P2Y2R fluorescent antagonist, suggesting it may occupy a different binding site on the P2Y2R.

CaV1.3 channel clusters characterized by live-cell and isolated plasma membrane nanoscopy

A key player of excitable cells in the heart and brain is the L-type calcium channel CaV1.3. In the heart, it is required for voltage-dependent Ca2+-signaling, i.e., for controlling and modulating atrial cardiomyocyte excitation-contraction coupling. The clustering of CaV1.3 in functionally relevant channel multimers has not been addressed due to a lack of stoichiometric labeling combined with high-resolution imaging. Here, we developed a HaloTag-labeling strategy to visualize and quantify CaV1.3 clusters using STED nanoscopy to address the questions of cluster size and intra-cluster channel density. Channel clusters were identified in the plasma membrane of transfected live HEK293 cells as well as in giant plasma membrane vesicles derived from these cells that were spread on modified glass support to obtain supported plasma membrane bilayers (SPMBs). A small fraction of the channel clusters was colocalized with early and recycling endosomes at the membranes. STED nanoscopy in conjunction with live-cell and SPMB imaging enabled us to quantify CaV1.3 cluster sizes and their molecular density revealing significantly lower channel densities than expected for dense channel packing. CaV1.3 channel cluster size and molecular density were increased in SPMBs after treatment of the cells with the sympathomimetic compound isoprenaline, suggesting a regulated channel cluster condensation mechanism.

Rapid plasma membrane isolation via intracellular polymerization-mediated biomolecular confinement

Plasma membrane isolation is a foundational process in membrane proteomic research, cellular vesicle studies, and biomimetic nanocarrier development, yet separation processes for this outermost layer are cumbersome and susceptible to impurities and low yield. Herein, we demonstrate that cellular cytosol can be chemically polymerized for decoupling and isolation of plasma membrane within minutes. A rapid, non-disruptive in situ polymerization technique is developed with cell membrane-permeable polyethyleneglycol-diacrylate (PEG-DA) and a blue-light-sensitive photoinitiator, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). The photopolymerization chemistry allows for precise control of intracellular polymerization and tunable confinement of cytosolic molecules. Upon cytosol solidification, plasma membrane proteins and vesicles are rapidly derived and purified as nucleic acids and intracellular proteins as small as 15 kDa are stably entrapped for removal. The polymerization chemistry and membrane derivation technique are broadly applicable to primary and fragile cell types, enabling facile membrane vesicle extraction from shorted-lived neutrophils and human primary CD8 T cells. The study demonstrates tunable intracellular polymerization via optimized live cell chemistry, offers a robust membrane isolation methodology with broad biomedical utility, and reveals insights on molecular crowding and confinement in polymerized cells. Statement of significanceIsolating the minute fraction of plasma membrane proteins and vesicles requires extended density gradient ultracentrifugation processes, which are susceptible to low yield and impurities. The present work demonstrates that the membrane isolation process can be vastly accelerated via a rapid, non-disruptive intracellular polymerization approach that decouples cellular cytosols from the plasma membrane. Following intracellular polymerization, high-yield plasma membrane proteins and vesicles can be derived from lysis buffer and sonication treatment, respectively. And the intracellular content entrapped within the polymerized hydrogel is readily removed within minutes. The technique has broad utility in membrane proteomic research, cellular vesicle studies, and biomimetic materials development, and the work offers insights on intracellular hydrogel-mediated molecular confinement.

H2-driven reduction of CO2 to formate using bacterial plasma membranes

Bacterial membranes shield the intracellular compartment by selectively allowing unwanted substances to enter in, which in turn reduces overall catalytic efficiency. This report presents a model system using the isolated plasma membranes of Citrobacter sp. S-77 that harbor oxygen-stable [NiFe]hydrogenase and [Mo]formate dehydrogenase, which are integrated into a natural catalytic nanodevice through an electron transfer relay. This naturally occurring nanodevice exhibited selectivity and efficiency in catalyzing the H2-driven conversion of CO2 to formate with the rate of 817 mmol·L–1·gprotein–1·h−1 under mild conditions of 30 °C, pH 7.0, and 0.1 MPa. When the isolated plasma membranes of Citrobacter sp. S-77 was immobilized with multi-walled carbon nanotubes and encapsulated in hydrogel beads of gellan-gum cross-linked with calcium ions, the catalyst for formate production remained stable over 10 repeated uses. This paper reports the first case of efficient and selective formate production from H2 and CO2 using bacterial plasma membranes.

Unleashing the potential of cell biomimetic nanoparticles: Strategies and challenges in their design and fabrication for therapeutic applications.

In this contribution to the Orations - New Horizons of the Journal of Controlled Release, I present design strategies for the two main biomimetic nanoparticles (BNP) groups: BNP composed of isolated cell membrane proteins, and BNP composed of the native cell membrane. I furthermore describe BNP fabrication methods and analyze their advantages and challenges. Finally, I suggest future therapeutic applications of each BNP group and propose a new revolutionary concept for their use.

Surfactant protein A alters endosomal trafficking of influenza A virus in macrophages.

Influenza A virus infection (IAV) often leads to acute lung injury that impairs breathing and can lead to death, with disproportionate mortality in children and the elderly. Surfactant Protein A (SP-A) is a calcium-dependent opsonin that binds a variety of pathogens to help control pulmonary infections by alveolar macrophages. Alveolar macrophages play critical roles in host resistance and susceptibility to IAV infection. The effect of SP-A on IAV infection and antiviral response of macrophages, however, is not understood. Here, we report that SP-A attenuates IAV infection in a dose-dependent manner at the level of endosomal trafficking, resulting in infection delay in a model macrophage cell line. The ability of SP-A to suppress infection was independent of its glycosylation status. Binding of SP-A to hemagglutinin did not rely on the glycosylation status or sugar binding properties of either protein. Incubation of either macrophages or IAV with SP-A slowed endocytic uptake rate of IAV. SP-A interfered with binding to cell membrane and endosomal exit of the viral genome as indicated by experiments using isolated cell membranes, an antibody recognizing a pH-sensitive conformational epitope on hemagglutinin, and microscopy. Lack of SP-A in mice enhanced IFNβ expression, viral clearance and reduced mortality from IAV infection. These findings support the idea that IAV is an opportunistic pathogen that co-opts SP-A to evade host defense by alveolar macrophages. Our study highlights novel aspects of host-pathogen interactions that may lead to better understanding of the local mechanisms that shape activation of antiviral and inflammatory responses to viral infection in the lung.

Synthetic corticotropins and the GABA-receptor system: Direct and delayed effects.

The central effectors of the stress system are greatly interconnected and include, among others, a large group of peptides derived from proopiomelanocortin. In addition to natural corticotropins, a number of artificial molecules that contain some ACTH fragments in their structure are also referred to members of this family. Some of them possess a wide range of biological activity. The molecular mechanism underlying the biological activity of such peptides is partly based on allosteric modulation of various receptors. We analyzed the ability of some biologically active synthetic corticotropins (ACTH(4-7)PGP, ACTH(6-9)PGP, ACTH(7-10)PGP), and glyproline PGPL to affect the GABA-receptor system of rat brain. The effects of the peptides were studied in the isolated plasma membranes of brain cells, as well as after systemic peptide administration in the rat model of acute restraint stress. The delayed effect of stress or preadministration of each of the studied peptides on [3 H]GABA binding was different for its high- and low-affinity-specific sites. The studied peptides individually affected the binding of [3 H]GABA in their own way. Acute restraint stress caused a decrease in [3 H]GABA binding at its low-affine site and did not affected the high-affine site. Preliminary peptide administration did not influence this effect of stress.

Subtomogram averaging of protein structures in isolated HEK293 cell plasma membranes

The plasma membrane and its associated proteins together are key to the function and disfunction of many vital cell processes such as adhesion, sensing/signaling, endocytosis/exocytosis. To better understand their function and treat their disfunction with precision, we need high-resolution structures of membrane-associated molecular components in their native context. Cryo-electron tomography (cryoET) is a rapidly evolving structural determination technique capable of resolving proteins and protein complexes to ångström-level resolution. However, high-resolution reconstruction of membrane-associated proteins and organelles in situ has been met with obstacles including the necessity for ultrathin (<200 nm thick) sample preparation. Here, we isolate plasma membranes using a technique called “unroofing” immediately prior to sample vitrification. This produces 50-200-nm-thick vitreous samples containing the plasma membrane, the actin cortex, and the myriad of other protein complexes associated with the membrane. To verify this technique as a tool for membrane protein structure determination, we are performing a quantitative assessment of the preserved resolution using subtomogram averaging. We are currently working to utilize and modify existing subtomogram averaging tools to identify, segment, process, and refine objects of interest within reconstructed tomograms. We compare our reconstructions with known protein structures. This ongoing work is part of an effort aimed to develop a correlative pipeline for visualizing the proteome of the eukaryotic plasma membrane with efficiency and precision.

Comprehensive analysis of glycerolipid dynamics during tobacco pollen germination and pollen tube growth.

Pollen germination and subsequent pollen tube elongation are essential for successful land plant reproduction. These processes are achieved through well-documented activation of membrane trafficking and cell metabolism. Despite this, our knowledge of the dynamics of cellular phospholipids remains scarce. Here we present the turnover of the glycerolipid composition during the establishment of cell polarity and elongation processes in tobacco pollen and show the lipid composition of pollen plasma membrane-enriched fraction for the first time. To achieve this, we have combined several techniques, such as lipidomics, plasma membrane isolation, and live-cell microscopy, and performed a study with different time points during the pollen germination and pollen tube growth. Our results showed that tobacco pollen tubes undergo substantial changes in their whole-cell lipid composition during the pollen germination and growth, finding differences in most of the glycerolipids analyzed. Notably, while lysophospholipid levels decrease during germination and growth, phosphatidic acid increases significantly at cell polarity establishment and continues with similar abundance in cell elongation. We corroborated these findings by measuring several phospholipase activities in situ. We also observed that lysophospholipids and phosphatidic acid are more abundant in the plasma membrane-enriched fraction than that in the whole cell. Our results support the important role for the phosphatidic acid in the establishment and maintenance of cellular polarity in tobacco pollen tubes and indicate that plasma membrane lysophospholipids may be involved in pollen germination.

Surfaceome analyses uncover CD98hc as an antibody drug-conjugate target in triple negative breast cancer

BackgroundDespite the incorporation of novel therapeutics, advanced triple negative breast cancer (TNBC) still represents a relevant clinical problem. Considering this, as well as the clinical efficacy of antibody-drug conjugates (ADCs), we aimed at identifying novel ADC targets that could be used to treat TNBC.MethodsTranscriptomic analyses were performed on TNBC and normal samples from three different studies. Plasma membrane proteins of three cell lines representative of the TNBC subtype were identified by cell surface biotinylation or plasma membrane isolation, followed by analyses of cell surface proteins using the Surfaceome online tool. Immunofluorescence and western studies were used to characterize the action of a CD98hc-directed ADC, which was prepared by in house coupling of emtansine to an antibody that recognized the ectodomain of CD98hc. Xenografted TNBC cells were used to analyze the antitumoral properties of the anti-CD98hc ADC.ResultsComparative genomic studies between normal breast and TNBC tissues, together with proteomic and bioinformatic analyses resulted in the elaboration of a catalog of potential ADC targets. One of them, the CD98hc transmembrane protein, was validated as an ADC target. An antibody recognizing the ectodomain of CD98hc efficiently internalized and reached the lysosomal compartment. An emtansine-based ADC derived from such antibody was prepared and showed antitumoral properties in TNBC in vitro and in vivo models. Mechanistically, the anti-CD98hc ADC blocked cell cycle progression, that was followed by cell death caused by mitotic catastrophe.ConclusionsThis work describes a list of potential ADC targets in TNBC and validates one of them, the transmembrane protein CD98hc. The studies presented here also demonstrate the robustness of the multiomic approach herewith described to identify novel potential ADC targets.

Membrane phase separation drives organization and activation at bcr signaling complexes

Isolated plasma membranes separate into two coexisting liquid phases with distinct lipid and protein compositions but live cell plasma membranes do not macroscopically phase separate, leading to questions of whether and how the membrane phase transition contributes to functional heterogeneity in cells. Using quantitative super resolution microscopy we show that B cell receptor signaling platforms are nanoscale domains that quantitatively enrich membrane probes based on probe phase partitioning in isolated plasma membrane vesicles.

Exploring a role for the proximity to the plasma membrane miscibility phase transition in the gating of two temperature sensing channels

The Transient Receptor Potential (TRP) family channels TRPM8 and TRPV1 are well characterized temperature-gated ion channels whose functions are impacted by established ligands and a broad range of hydrophobic small molecules. For example, past work shows that ethanol lowers the gating temperature for both channels. Ethanol has also been shown to reduce the miscibility phase transition temperature in isolated plasma membrane vesicles isolated from mammalian membranes. This poster will present early investigations aimed at testing the hypothesis that the membrane phase transition influences the gating temperature of these channels, either directly by manipulating domain structure, or indirectly by impacting abundance and/or availability of allosteric regulators of channel function.

Characterization of Influenza Virus Binding to Receptors on Isolated Cell Membranes.

An interplay between receptor-binding properties of influenza viruses (IVs) and spectrum of sialic acid-containing receptors on target cells in birds and mammals determine viral host range, tissue tropism, and pathogenicity. Here, we describe method that allows to characterize binding of IVs to biologically relevant cellular receptors using a conventional solid-phase enzyme-linked assay. In this method, we isolate plasma membranes from respiratory and intestinal epithelial cells of animal origin (Subheading 3.2). We adsorb the membranes in the wells of 96-well ELISA plates, incubate the membrane-coated wells with serially diluted IVs, and determine amounts of IVs attached to the membranes using viral ability to bind peroxidase-labeled sialoglycoprotein fetuin. Based on the concentration dependence of IV binding to the membrane, we estimate binding avidity and number of binding sites. We describe two variants of the assay in Subheadings 3.6 and 3.7 and provide examples.

Sterol Extraction from Isolated Plant Plasma Membrane Vesicles Affects H+-ATPase Activity and H+-Transport.

Plasma membrane H+-ATPase is known to be detected in detergent-resistant sterol-enriched fractions, also called “raft” domains. Studies on H+-ATPase reconstituted in artificial or native membrane vesicles have shown both sterol-mediated stimulations and inhibitions of its activity. Here, using sealed isolated plasma membrane vesicles, we investigated the effects of sterol depletion in the presence of methyl-β-cyclodextrin (MβCD) on H+-ATPase activity. The rate of ATP-dependent ∆µH+ generation and the kinetic parameters of ATP hydrolysis were evaluated. We show that the relative sterols content in membrane vesicles decreased gradually after treatment with MβCD and reached approximately 40% of their initial level in 30 mM probe solution. However, changes in the hydrolytic and H+-transport activities of the enzyme were nonlinear. The extraction of up to 20% of the initial sterols was accompanied by strong stimulation of ATP-dependent H+-transport in comparison with the hydrolytic activity of enzymes. Further sterol depletion led to a significant inhibition of active proton transport with an increase in passive H+-leakage. The solubilization of control and sterol-depleted vesicles in the presence of dodecyl maltoside negated the differences in the kinetics parameters of ATP hydrolysis, and all samples demonstrated maximal hydrolytic activities. The mechanisms behind the sensitivity of ATP-dependent H+-transport to sterols in the lipid environment of plasma membrane H+-ATPase are discussed.

Cell Membrane-Coated Mimics: A Methodological Approach for Fabrication, Characterization for Therapeutic Applications, and Challenges for Clinical Translation.

Cell membrane-coated (CMC) mimics are micro/nanosystems that combine an isolated cell membrane and a template of choice to mimic the functions of a cell. The design exploits its physicochemical and biological properties for therapeutic applications. The mimics demonstrate excellent biological compatibility, enhanced biointerfacing capabilities, physical, chemical, and biological tunability, ability to retain cellular properties, immune escape, prolonged circulation time, and protect the encapsulated drug from degradation and active targeting. These properties and the ease of adapting them for personalized clinical medicine have generated a significant research interest over the past decade. This review presents a detailed overview of the recent advances in the development of cell membrane-coated (CMC) mimics. The primary focus is to collate and discuss components, fabrication methodologies, and the significance of physiochemical and biological characterization techniques for validating a CMC mimic. We present a critical analysis of the two main components of CMC mimics: the template and the cell membrane and mapped their use in therapeutic scenarios. In addition, we have emphasized on the challenges associated with CMC mimics in their clinical translation. Overall, this review is an up to date toolbox that researchers can benefit from while designing and characterizing CMC mimics.

Quantitation of Plasma Membrane Drug Transporters in Kidney Tissue and Cell Lines Using a Novel Proteomic Approach Enabled a Prospective Prediction of Metformin Disposition.

The successful prospective incorporation of in vitro transporter kinetics in physiologically based pharmacokinetic (PBPK) models to describe drug disposition remains challenging. Although determination of scaling factors to extrapolate in vitro to in vivo transporter kinetics has been facilitated by quantitative proteomics, no robust assessment comparing membrane recoveries between different cells/tissues has been made. HEK293 cells overexpressing OCT2, MATE1, and MATE2K or human kidney cortex were homogenized and centrifuged to obtain the total membrane fractions, which were subsequently subjected to liquid-liquid extraction followed by centrifugation and precipitation to isolate plasma membrane fractions. Plasma membrane recoveries determined by quantitation of the marker Na+/K+-ATPase in lysate and plasma membrane fractions were ≤20% but within 3-fold across different cells and tissues. A separate study demonstrated that recoveries are comparable between basolateral and apical membranes of renal proximal tubules, as measured by Na+/K+-ATPase and γ-glutamyl transpeptidase 1, respectively. The plasma membrane expression of OCT2, MATE1, and MATE2K was quantified and relative expression factors (REFs) were determined as the ratio between the tissue and cell concentrations. Corrections using plasma membrane recovery had minimal impact on REF values (<2-fold). In vitro transporter kinetics of metformin were extrapolated to in vivo using the corresponding REFs in a PBPK model. The simulated metformin exposures were within 2-fold of clinical exposure. These results demonstrate that transporter REFs based on plasma membrane expression enable a prediction of transporter-mediated drug disposition. Such REFs may be estimated without the correction of plasma membrane recovery when the same procedure is applied between different matrices. SIGNIFICANCE STATEMENT: Transporter REFs based on plasma membrane expression enable in vitro-in vivo extrapolation of transporter kinetics. Plasma membrane recoveries as determined by the quantification of sodium-potassium adenosine triphosphatase were comparable between the in vitro and in vivo systems used in the present study, and therefore had minimal impact on the transporter REF values.

Small-Scale Plasma Membrane Preparation for the Analysis of &lt;em&gt;Candida albicans&lt;/em&gt; Cdr1-mGFPHis

This article presents a small-scale plasma membrane isolation protocol for the characterization of Candida albicans ABC (ATP-binding cassette) protein Cdr1, overexpressed in Saccharomyces cerevisiae. A protease-cleavable C-terminal mGFPHis double tag with a 16-residue linker between Cdr1 and the tag was designed to facilitate the purification and detergent-screening of Cdr1.