Fluorescence labeling-induced structural rearrangement of a monoclonal IgG revealed by biophysical experiments and simulations.

Although it is known that labeling antibodies with fluorescent dyes impairs their function, the underlying mechanism remains unclear. In this study, we show that multiple antibody functions decline in a strikingly similar way as the degree of labeling increases, suggesting that labeling induces a global structural change affecting the entire IgG molecule. Fluorescence anisotropy decay experiments revealed faster nanosecond-scale dynamics in labeled antibodies.

Local ligand concentration gradients induced by the plasma membrane.

The plasma membrane is a dynamic structure surrounded by the extracellular matrix. Although stimulation of transmembrane proteins by soluble ligands takes place in this environment, its influence on receptor activation is usually overlooked. In the current manuscript, we quantitatively measured the concentration of epidermal growth factor (EGF) at the cell membrane and found that ligand distribution is non-homogeneous due to two concentration peaks causing significantly higher EGF concentrations at the membrane than in the bulk solution.

Epigenetic modulation via the C-terminal tail of H2A.Z.

H2A.Z-nucleosomes are present in both euchromatin and heterochromatin and it has proven difficult to interpret their disparate roles in the context of their stability features. Using an in situ assay of nucleosome stability and DT40 cells expressing engineered forms of the histone variant we show that native H2A.

Corrigendum: Human abdominal subcutaneous-derived active beige adipocytes carrying rs1421085 obesity-risk alleles exert lower thermogenic capacity.

[This corrects the article DOI: 10.3389/fcell.2023.

Human abdominal subcutaneous-derived active beige adipocytes carrying rs1421085 obesity-risk alleles exert lower thermogenic capacity.

White adipocytes store lipids, have a large lipid droplet and few mitochondria. Brown and beige adipocytes, which produce heat, are characterized by high expression of uncoupling protein (UCP) 1, multilocular lipid droplets, and large amounts of mitochondria. The rs1421085 T-to-C single-nucleotide polymorphism (SNP) of the human gene interrupts a conserved motif for ARID5B repressor, resulting in adipocyte type shift from beige to white.

Nucleotide binding is the critical regulator of ABCG2 conformational transitions.

ABCG2 is an exporter-type ABC protein that can expel numerous chemically unrelated xeno- and endobiotics from cells. When expressed in tumor cells or tumor stem cells, ABCG2 confers multidrug resistance, contributing to the failure of chemotherapy. Molecular details orchestrating substrate translocation and ATP hydrolysis remain elusive.

Palmitate Inhibits Mouse Macrophage Efferocytosis by Activating an mTORC1-Regulated Rho Kinase 1 Pathway: Therapeutic Implications for the Treatment of Obesity.

Every day, billions of our cells die and get cleared without inducing inflammation. When, clearance is improper, uncleared cells undergo secondary necrosis and trigger inflammation. In addition, proper efferocytosis would be required for inducing resolution of inflammation, thus clearance deficiencies in the long term lead to development of various chronic inflammatory diseases.

A Fluorescent Peptide Toxin for Selective Visualization of the Voltage-Gated Potassium Channel K1.3.

Upregulation of the voltage-gated potassium channel K1.3 is implicated in a range of autoimmune and neuroinflammatory diseases, including rheumatoid arthritis, psoriasis, multiple sclerosis, and type I diabetes. Understanding the expression, localization, and trafficking of K1.

Doxorubicin impacts chromatin binding of HMGB1, Histone H1 and retinoic acid receptor.

Doxorubicin (Dox), a widely used anticancer DNA-binding drug, affects chromatin in multiple ways, and these effects contribute to both its efficacy and its dose-limiting side effects, especially cardiotoxicity. Here, we studied the effects of Dox on the chromatin binding of the architectural proteins high mobility group B1 (HMGB1) and the linker histone H1, and the transcription factor retinoic acid receptor (RARα) by fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) in live cells. At lower doses, Dox increased the binding of HMGB1 to DNA while decreasing the binding of the linker histone H1.

Mitophagy Mediates the Beige to White Transition of Human Primary Subcutaneous Adipocytes Ex Vivo.

Brown and beige adipocytes have multilocular lipid droplets, express uncoupling protein (UCP) 1, and promote energy expenditure. In rodents, when the stimulus of browning subsides, parkin-dependent mitophagy is activated and dormant beige adipocytes persist. In humans, however, the molecular events during the beige to white transition have not been studied in detail.

Role of C-Terminal Domain and Membrane Potential in the Mobility of Kv1.3 Channels in Immune Synapse Forming T Cells.

Voltage-gated Kv1.3 potassium channels are essential for maintaining negative membrane potential during T-cell activation. They interact with membrane-associated guanylate kinases (MAGUK-s) via their C-terminus and with TCR/CD3, leading to enrichment at the immunological synapse (IS).

IL-15 -Presentation Is an Autonomous, Antigen-Independent Process.

IL-15 plays a pivotal role in the long-term survival of T cells and immunological memory. Its receptor consists of three subunits (IL-15Rα, IL-2/15Rβ, and γ). IL-15 functions mainly via -presentation (TP), during which an APC expressing IL-15 bound to IL-15Rα presents the ligand to the βγ receptor-heterodimer on a neighboring T/NK cell.

Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission.

Förster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the distance and orientation of the molecules as well as the extent of overlap between the donor emission and acceptor absorption spectra. FRET permits to study the interaction of proteins in the living cell over time and in different subcellular compartments. Different intensity-based algorithms to measure FRET using microscopy have been described in the literature.

Thermogenic Activation Downregulates High Mitophagy Rate in Human Masked and Mature Beige Adipocytes.

Thermogenic brown and beige adipocytes oxidize metabolic substrates producing heat, mainly by the mitochondrial uncoupling protein UCP1, and can thus counteract obesity. Masked beige adipocytes possess white adipocyte-like morphology, but can be made thermogenic by adrenergic stimuli. We investigated the regulation of mitophagy upon thermogenic activation of human masked and mature beige adipocytes.

The voltage-gated proton channel hHv1 is functionally expressed in human chorion-derived mesenchymal stem cells.

The voltage-gated proton channel Hv1 is widely expressed, among others, in immune and cancer cells, it provides an efficient cytosolic Hextrusion mechanism and regulates vital functions such as oxidative burst, migration and proliferation. Here we demonstrate the presence of human Hv1 (hHv1) in the placenta/chorion-derived mesenchymal stem cells (cMSCs) using RT-PCR. The voltage- and pH-dependent gating of the current is similar to that of hHv1 expressed in cell lines and that the current is blocked by 5-chloro-2-guanidinobenzimidazole (ClGBI) and activated by arachidonic acid (AA).

Simultaneous Mapping of Molecular Proximity and Comobility Reveals Agonist-Enhanced Dimerization and DNA Binding of Nuclear Receptors.

Single Plane Illumination Microscopy (SPIM) revolutionized time lapse imaging of live cells and organisms due to its high speed and reduced photodamage. Quantitative mapping of molecular (co)mobility by fluorescence (cross-)correlation spectroscopy (F(C)CS) in a SPIM has been introduced to reveal molecular diffusion and binding. A complementary aspect of interactions is proximity, which can be studied by Förster resonance energy transfer (FRET).

IL-2 receptors preassemble and signal in the ER/Golgi causing resistance to antiproliferative anti-IL-2Rα therapies.

Interleukin-2 (IL-2) and IL-15 play pivotal roles in T cell activation, apoptosis, and survival, and are implicated in leukemias and autoimmune diseases. Their heterotrimeric receptors share their β- and γ-chains, but have distinct α-chains. Anti-IL-2Rα (daclizumab) therapy targeting cell surface-expressed receptor subunits to inhibit T cell proliferation has only brought limited success in adult T cell leukemia/lymphoma (ATL) and in multiple sclerosis.

Minimum degree of overlap between IL-9R and IL-2R on human T lymphoma cells: A quantitative CLSM and FRET analysis.

The heterodimeric receptor complex of IL-9 consists of the cytokine-specific α-subunit and the common γ -chain shared with other cytokines, including IL-2, a central regulator of T cell function. We have shown previously the bipartite spatial relationship of IL-9 and IL-2 receptors at the surface of human T lymphoma cells: in addition to common clusters, expression of the two receptor kinds could also be observed in segregated membrane areas. Here we analyzed further the mutual cell surface organization of IL-9 and IL-2 receptors.

Membrane Potential Distinctly Modulates Mobility and Signaling of IL-2 and IL-15 Receptors in T Cells.

The high electric field across the plasma membrane might influence the conformation and behavior of transmembrane proteins that have uneven charge distributions in or near their transmembrane regions. Membrane depolarization of T cells occurs in the tumor microenvironment and in inflamed tissues because of K release from necrotic cells and hypoxia affecting the expression of K channels. However, little attention has been given to the effect of membrane potential (MP) changes on membrane receptor function.

MHC I Expression Regulates Co-clustering and Mobility of Interleukin-2 and -15 Receptors in T Cells.

MHC glycoproteins form supramolecular clusters with interleukin-2 and -15 receptors in lipid rafts of T cells. The role of highly expressed MHC I in maintaining these clusters is unknown. We knocked down MHC I in FT7.

FPGA implementation of a 32x32 autocorrelator array for analysis of fast image series.

With the evolving technology in CMOS integration, new classes of 2D-imaging detectors have recently become available. In particular, single photon avalanche diode (SPAD) arrays allow detection of single photons at high acquisition rates (≥ 100 kfps), which is about two orders of magnitude higher than with currently available cameras. Here we demonstrate the use of a SPAD array for imaging fluorescence correlation spectroscopy (imFCS), a tool to create 2D maps of the dynamics of fluorescent molecules inside living cells.

New cholesterol-specific antibodies remodel HIV-1 target cells' surface and inhibit their in vitro virus production.

The importance of membrane rafts in HIV-1 infection is still in the focus of interest. Here, we report that new monoclonal anticholesterol IgG antibodies (ACHAs), recognizing clustered membrane cholesterol (e.g.

Dual role of poly(ADP-ribose) glycohydrolase in the regulation of cell death in oxidatively stressed A549 cells.

Activation of poly(ADP-ribose) polymerase-1 (PARP1) has been shown to mediate cell death induced by genotoxic stimuli. The role of poly(ADP-ribose) glycohydrolase (PARG), the enzyme responsible for polymer degradation, has been largely unexplored in the regulation of cell death. Using lentiviral gene silencing we generated A549 lung adenocarcinoma cell lines with stably suppressed PARG and PARP1 expression (shPARG and shPARP1 cell lines, respectively) and determined parameters of apoptotic and necrotic cell death following hydrogen peroxide exposure.

A biophysical approach to IL-2 and IL-15 receptor function: localization, conformation and interactions.

Interleukin-2 and interleukin-15 (IL-2, IL-15) are key participants in T and NK cell activation and function. Sharing the beta and gamma receptor subunits results in several common functions: e.g.