Elongated membrane zones boost interactions of diffusing proteins.

Biological membranes are two dimensional, making the discovery of quasi-one-dimensional diffusion of membrane proteins puzzling. Jaqaman et al. (2011) now show that actomyosin and tubulin interact to establish long, thin diffusion corridors, thereby increasing the effective concentration of select membrane proteins to promote their interactions and modulate signaling.

Cetylpyridinium chloride (CPC) reduces zebrafish mortality from influenza infection: Super-resolution microscopy reveals CPC interference with multiple protein interactions with phosphatidylinositol 4,5-bisphosphate in immune function.

The COVID-19 pandemic raises significance for a potential influenza therapeutic compound, cetylpyridinium chloride (CPC), which has been extensively used in personal care products as a positively-charged quaternary ammonium antibacterial agent. CPC is currently in clinical trials to assess its effects on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) morbidity. Two published studies have provided mouse and human data indicating that CPC may alleviate influenza infection, and here we show that CPC (0.1 µM, 1 h) reduces zebrafish mortality and viral load following influenza infection. However, CPC mechanisms of action upon viral-host cell interaction are currently unknown. We have utilized super-resolution fluorescence photoactivation localization microscopy to probe the mode of CPC action. Reduction in density of influenza viral protein hemagglutinin (HA) clusters is known to reduce influenza infectivity: here, we show that CPC (at non-cytotoxic doses, 5-10 µM) reduces HA density and number of HA molecules per cluster within the plasma membrane of NIH-3T3 mouse fibroblasts. HA is known to colocalize with the negatively-charged mammalian lipid phosphatidylinositol 4,5-bisphosphate (PIP2); here, we show that nanoscale co-localization of HA with the PIP2-binding Pleckstrin homology (PH) reporter in the plasma membrane is diminished by CPC. CPC also dramatically displaces the PIP2-binding protein myristoylated alanine-rich C-kinase substrate (MARCKS) from the plasma membrane of rat RBL-2H3 mast cells; this disruption of PIP2 is correlated with inhibition of mast cell degranulation. Together, these findings offer a PIP2-focused mechanism underlying CPC disruption of influenza and suggest potential pharmacological use of this drug as an influenza therapeutic to reduce global deaths from viral disease.

Triclosan disrupts immune cell function by depressing Ca2+ influx following acidification of the cytoplasm.

Triclosan (TCS) is an antimicrobial agent that was effectively banned by the FDA from hand soaps in 2016, hospital soaps in 2017, and hand sanitizers in 2019; however, TCS can still be found in a few products. At consumer-relevant, non-cytotoxic doses, TCS inhibits the functions of both mitochondria and mast cells, a ubiquitous cell type. Via the store-operated Ca2+ entry mechanism utilized by many immune cells, mast cells undergo antigen-stimulated Ca2+ influx into the cytosol, for proper function. Previous work showed that TCS inhibits Ca2+ dynamics in mast cells, and here we show that TCS also inhibits Ca2+ mobilization in human Jurkat T cells. However, the biochemical mechanism behind the Ca2+ dampening has yet to be elucidated. Three-dimensional super-resolution microscopy reveals that TCS induces mitochondrial swelling, in line with and extending the previous finding of TCS inhibition of mitochondrial membrane potential via its proton ionophoric activity. Inhibition of plasma membrane potential (PMP) by the canonical depolarizer gramicidin can inhibit mast cell function. However, use of the genetically encoded voltage indicators (GEVIs) ArcLight (pH-sensitive) and ASAP2 (pH-insensitive), indicates that TCS does not disrupt PMP. In conjunction with data from a plasma membrane-localized, pH-sensitive reporter, these results indicate that TCS, instead, induces cytosolic acidification in mast cells and T cells. Acidification of the cytosol likely inhibits Ca2+ influx by uncoupling the STIM1/ORAI1 interaction that is required for opening of plasma membrane Ca2+ channels. These results provide a mechanistic explanation of TCS disruption of Ca2+ influx and, thus, of immune cell function.

Directing Stem Cell Commitment by Amorphous Calcium Phosphate Nanoparticles Incorporated in PLGA: Relevance of the Free Calcium Ion Concentration.

The microenvironment of mesenchymal stem cells (MSCs) is responsible for the modulation in MSC commitment. Nanocomposites with an inorganic and an organic component have been investigated, and osteogenesis of MSCs has been attributed to inorganic phases such as calcium phosphate under several conditions. Here, electrospun meshes and two-dimensional films of poly(lactic-co-glycolic acid) (PLGA) or nanocomposites of PLGA and amorphous calcium phosphate nanoparticles (PLGA/aCaP) seeded with human adipose-derived stem cells (ASCs) were analyzed for the expression of selected marker genes. In a two-week in vitro experiment, osteogenic commitment was not found to be favored on PLGA/aCaP compared to pure PLGA. Analysis of the medium revealed a significant reduction of the Ca2+ concentration when incubated with PLGA/aCaP, caused by chemical precipitation of hydroxyapatite (HAp) on aCaP seeds of PLGA/aCaP. Upon offering a constant Ca2+ concentration, however, the previously observed anti-osteogenic effect was reversed: alkaline phosphatase, an early osteogenic marker gene, was upregulated on PLGA/aCaP compared to pristine PLGA. Hence, in addition to the cell-material interaction, the material-medium interaction was also important for the stem cell commitment here, affecting the cell-medium interaction. Complex in vitro models should therefore consider all factors, as coupled impacts might emerge.

Influenza Hemagglutinin Modulates Phosphatidylinositol 4,5-Bisphosphate Membrane Clustering.

The lipid phosphatidylinositol 4,5-bisphosphate (PIP2) forms nanoscopic clusters in cell plasma membranes; however, the processes determining PIP2 mobility and thus its spatial patterns are not fully understood. Using super-resolution imaging of living cells, we find that PIP2 is tightly colocalized with and modulated by overexpression of the influenza viral protein hemagglutinin (HA). Within and near clusters, HA and PIP2 follow a similar spatial dependence, which can be described by an HA-dependent potential gradient; PIP2 molecules move as if they are attracted to the center of clusters by a radial force of 0.079 ± 0.002 pN in HAb2 cells. The measured clustering and dynamics of PIP2 are inconsistent with the unmodified forms of the raft, tether, and fence models. Rather, we found that the spatial PIP2 distributions and how they change in time are explained via a novel, to our knowledge, dynamic mechanism: a radial gradient of PIP2 binding sites that are themselves mobile. This model may be useful for understanding other biological membrane domains whose distributions display gradients in density while maintaining their mobility.

Antimicrobial agent triclosan disrupts mitochondrial structure, revealed by super-resolution microscopy, and inhibits mast cell signaling via calcium modulation.

The antimicrobial agent triclosan (TCS) is used in products such as toothpaste and surgical soaps and is readily absorbed into oral mucosa and human skin. These and many other tissues contain mast cells, which are involved in numerous physiologies and diseases. Mast cells release chemical mediators through a process termed degranulation, which is inhibited by TCS. Investigation into the underlying mechanisms led to the finding that TCS is a mitochondrial uncoupler at non-cytotoxic, low-micromolar doses in several cell types and live zebrafish. Our aim was to determine the mechanisms underlying TCS disruption of mitochondrial function and of mast cell signaling. We combined super-resolution (fluorescence photoactivation localization) microscopy and multiple fluorescence-based assays to detail triclosan's effects in living mast cells, fibroblasts, and primary human keratinocytes. TCS disrupts mitochondrial nanostructure, causing mitochondria to undergo fission and to form a toroidal, "donut" shape. TCS increases reactive oxygen species production, decreases mitochondrial membrane potential, and disrupts ER and mitochondrial Ca2+ levels, processes that cause mitochondrial fission. TCS is 60 × more potent than the banned uncoupler 2,4-dinitrophenol. TCS inhibits mast cell degranulation by decreasing mitochondrial membrane potential, disrupting microtubule polymerization, and inhibiting mitochondrial translocation, which reduces Ca2+ influx into the cell. Our findings provide mechanisms for both triclosan's inhibition of mast cell signaling and its universal disruption of mitochondria. These mechanisms provide partial explanations for triclosan's adverse effects on human reproduction, immunology, and development. This study is the first to utilize super-resolution microscopy in the field of toxicology.

Selective Low-Energy Carbon Dioxide Adsorption Using Monodisperse Nitrogen-Rich Hollow Carbon Submicron Spheres.

Monodisperse, nitrogen-doped hollow carbon spheres of submicron size were synthesized using hexamethoxymethylmelamine as both a carbon and nitrogen source in a short (1 h) microwave-assisted synthesis. After carbonization at 550 °C, porous carbon spheres with a remarkably high nitrogen content of 37.1% were obtained, which consisting mainly of highly basic pyridinic moieties. The synthesized hollow spheres exhibited high selectivity for carbon dioxide (CO2) over nitrogen and oxygen gases, with a capture capacity up to 1.56 mmol CO2 g-1. The low adsorption enthalpy of the synthesized hollow carbon spheres permits good adsorbent regeneration. Evaluation of the feasibility of scaling up shows their potential for large-scale applications.

The Role of Probe Photophysics in Localization-Based Superresolution Microscopy.

Fluorescent proteins are used extensively for biological imaging applications; photoactivatable and photoconvertible fluorescent proteins (PAFPs) are used widely in superresolution localization microscopy methods such as fluorescence photoactivation localization microscopy and photoactivated localization microscopy. However, their optimal use depends on knowledge of not only their bulk fluorescence properties, but also their photophysical properties at the single molecule level. We have used fluorescence correlation spectroscopy and cross-correlation spectroscopy to quantify the diffusion, photobleaching, fluorescence intermittency, and photoconversion dynamics of Dendra2, a well-known PAFP used in localization microscopy. Numerous dark states of Dendra2 are observed both in inactive (green fluorescent) and active (orange fluorescent) forms; the interconversion rates are both light- and pH-dependent, as observed for other PAFPs. The dark states limit the detected count rate per molecule, which is a crucial parameter for localization microscopy. We then developed, to our knowledge, a new mathematical estimate for the resolution in localization microscopy as a function of the measured photophysical parameters of the probe such as photobleaching quantum yield, count rate per molecule, and intensity of saturation. The model was used to predict the dependence of resolution on acquisition parameters such as illumination intensity and time per frame, demonstrating an optimal set of acquisition parameters for a given probe for a variety of measures of resolution. The best possible resolution was then compared for Dendra2 and other widely used probes, including Alexa dyes and quantum dots. This work establishes a framework for determination of the best possible resolution using a localization microscope to image a particular fluorophore, and suggests that development of probes for use in superresolution localization microscopy must consider the count rate per molecule, the saturation intensity, the photobleaching yield, and, crucially, management of bright/dark state transitions, to optimize image resolution.

Precisely and accurately localizing single emitters in fluorescence microscopy.

Methods based on single-molecule localization and photophysics have brought nanoscale imaging with visible light into reach. This has enabled single-particle tracking applications for studying the dynamics of molecules and nanoparticles and contributed to the recent revolution in super-resolution localization microscopy techniques. Crucial to the optimization of such methods are the precision and accuracy with which single fluorophores and nanoparticles can be localized. We present a lucid synthesis of the developments on this localization precision and accuracy and their practical implications in order to guide the increasing number of researchers using single-particle tracking and super-resolution localization microscopy.

Response of human dental pulp cells to a silver-containing PLGA/TCP-nanofabric as a potential antibacterial regenerative pulp-capping material.

BACKGROUND: Damage or exposure of the dental pulp requires immediate therapeutic intervention. METHODS: This study assessed the biocompatibility of a silver-containing PLGA/TCP-nanofabric scaffold (PLGA/Ag-TCP) in two in vitro models, i.e. the material adapted on pre-cultured cells and cells directly cultured on the material, respectively. Collagen saffolds with and without hyaluronan acid (Coll-HA; Coll) using both cell culturing methods and cells growing on culture plates served as reference. Cell viability and proliferation were assessed after 24, 48, and 72 h based on formazan formation and BrdU incorporation. Scaffolds were harvested. Gene expression of interleukin(IL)-6, tumor necrosis factor (TNF)-alpha, and alkaline phosphatase (AP) was assessed 24 h after stimulation. RESULTS: In both models formazan formation and BrdU incorporation was reduced by PLGA/Ag-TCP on dental pulp cells, while no significant reduction was found in cells with Coll and Coll-HA. Cells with PLGA/Ag-TCP for 72 h showed similar relative BrdU incorporation than cells stimulated with Coll and Coll-HA. A prominent increase in the pro-inflammatory genes IL-6 and TNF-α was observed when cells were cultured with PLGA/Ag-TCP compared to the other groups. This increase was parallel with a slight increase in AP expression. Overall, no differences between the two culture methods were observed. CONCLUSIONS: PLGA/Ag-TCP decreased viability and proliferation rate of human dental pulp cells and increased the pro-inflammatory capacity and alkaline phosphatase expression. Whether these cellular responses observed in vitro translate into pulp regeneration in vivo will be assessed in further studies.