Nanoscale Imaging Reveals a Tetraspanin-CD9 Coordinated Elevation of Endothelial ICAM-1 Clusters

Endothelial barriers have a central role in inflammation as they allow or deny the passage of leukocytes from the vasculature into the tissue. To bind leukocytes, endothelial cells form adhesive clusters containing tetraspanins and ICAM-1, so-called endothelial adhesive platforms (EAPs). Upon leukocyte binding, EAPs evolve into docking structures that emanate from the endothelial surface while engulfing the leukocyte. Here, we show that TNF-α is sufficient to induce apical protrusions in the absence of leukocytes. Using advanced quantitation of atomic force microscopy (AFM) recordings, we found these structures to protrude by 160 ± 80 nm above endothelial surface level. Confocal immunofluorescence microscopy proved them positive for ICAM-1, JAM-A, tetraspanin CD9 and f-actin. Microvilli formation was inhibited in the absence of CD9. Our findings indicate that stimulation with TNF-α induces nanoscale changes in endothelial surface architecture and that—via a tetraspanin CD9 depending mechanism—the EAPs rise above the surface to facilitate leukocyte capture

Bacteria tracking by in vivo magnetic resonance imaging

Background: Different non-invasive real-time imaging techniques have been developed over the last decades to study bacterial pathogenic mechanisms in mouse models by following infections over a time course. In vivo investigations of bacterial infections previously relied mostly on bioluminescence imaging (BLI), which is able to localize metabolically active bacteria, but provides no data on the status of the involved organs in the infected host organism. In this study we established an in vivo imaging platform by magnetic resonance imaging (MRI) for tracking bacteria in mouse models of infection to study infection biology of clinically relevant bacteria. Results: We have developed a method to label Gram-positive and Gram-negative bacteria with iron oxide nano particles and detected and pursued these with MRI. The key step for successful labeling was to manipulate the bacterial surface charge by producing electro-competent cells enabling charge interactions between the iron particles and the cell wall. Different particle sizes and coatings were tested for their ability to attach to the cell wall and possible labeling mechanisms were elaborated by comparing Gram-positive and -negative bacterial characteristics. With 5-nm citrate-coated particles an iron load of 0.015 ± 0.002 pg Fe/bacterial cell was achieved for Staphylococcus aureus. In both a subcutaneous and a systemic infection model induced by iron-labeled S. aureus bacteria, high resolution MR images allowed for bacterial tracking and provided information on the morphology of organs and the inflammatory response. Conclusion: Labeled with iron oxide particles, in vivo detection of small S. aureus colonies in infection models is feasible by MRI and provides a versatile tool to follow bacterial infections in vivo. The established cell labeling strategy can easily be transferred to other bacterial species and thus provides a conceptual advance in the field of molecular MRI.<br

Biogenic Volatile Organic Compound and Respiratory CO2 Emissions after 13C-Labeling: Online Tracing of C Translocation Dynamics in Poplar Plants

Globally plants are the primary sink of atmospheric CO(2), but are also the major contributor of a large spectrum of atmospheric reactive hydrocarbons such as terpenes (e.g. isoprene) and other biogenic volatile organic compounds (BVOC). The prediction of plant carbon (C) uptake and atmospheric oxidation capacity are crucial to define the trajectory and consequences of global environmental changes. To achieve this, the biosynthesis of BVOC and the dynamics of C allocation and translocation in both plants and ecosystems are important.We combined tunable diode laser absorption spectrometry (TDLAS) and proton transfer reaction mass spectrometry (PTR-MS) for studying isoprene biosynthesis and following C fluxes within grey poplar (Populus x canescens) saplings. This was achieved by feeding either (13)CO(2) to leaves or (13)C-glucose to shoots via xylem uptake. The translocation of (13)CO(2) from the source to other plant parts could be traced by (13)C-labeled isoprene and respiratory (13)CO(2) emission.In intact plants, assimilated (13)CO(2) was rapidly translocated via the phloem to the roots within 1 hour, with an average phloem transport velocity of 20.3±2.5 cm h(-1). (13)C label was stored in the roots and partially reallocated to the plants' apical part one day after labeling, particularly in the absence of photosynthesis. The daily C loss as BVOC ranged between 1.6% in mature leaves and 7.0% in young leaves. Non-isoprene BVOC accounted under light conditions for half of the BVOC C loss in young leaves and one-third in mature leaves. The C loss as isoprene originated mainly (76-78%) from recently fixed CO(2), to a minor extent from xylem-transported sugars (7-11%) and from photosynthetic intermediates with slower turnover rates (8-11%).We quantified the plants' C loss as respiratory CO(2) and BVOC emissions, allowing in tandem with metabolic analysis to deepen our understanding of ecosystem C flux

Amyloid Precursor Protein Is Trafficked and Secreted via Synaptic Vesicles

A large body of evidence has implicated amyloid precursor protein (APP) and its proteolytic derivatives as key players in the physiological context of neuronal synaptogenesis and synapse maintenance, as well as in the pathology of Alzheimer's Disease (AD). Although APP processing and release are known to occur in response to neuronal stimulation, the exact mechanism by which APP reaches the neuronal surface is unclear. We now demonstrate that a small but relevant number of synaptic vesicles contain APP, which can be released during neuronal activity, and most likely represent the major exocytic pathway of APP. This novel finding leads us to propose a revised model of presynaptic APP trafficking that reconciles existing knowledge on APP with our present understanding of vesicular release and recycling

Airborne Signals from a Wounded Leaf Facilitate Viral Spreading and Induce Antibacterial Resistance in Neighboring Plants

Many plants release airborne volatile compounds in response to wounding due to pathogenic assault. These compounds serve as plant defenses and are involved in plant signaling. Here, we study the effects of pectin methylesterase (PME)-generated methanol release from wounded plants (“emitters”) on the defensive reactions of neighboring “receiver” plants. Plant leaf wounding resulted in the synthesis of PME and a spike in methanol released into the air. Gaseous methanol or vapors from wounded PME-transgenic plants induced resistance to the bacterial pathogen Ralstonia solanacearum in the leaves of non-wounded neighboring “receiver” plants. In experiments with different volatile organic compounds, gaseous methanol was the only airborne factor that could induce antibacterial resistance in neighboring plants. In an effort to understand the mechanisms by which methanol stimulates the antibacterial resistance of “receiver” plants, we constructed forward and reverse suppression subtractive hybridization cDNA libraries from Nicotiana benthamiana plants exposed to methanol. We identified multiple methanol-inducible genes (MIGs), most of which are involved in defense or cell-to-cell trafficking. We then isolated the most affected genes for further analysis: β-1,3-glucanase (BG), a previously unidentified gene (MIG-21), and non-cell-autonomous pathway protein (NCAPP). Experiments with Tobacco mosaic virus (TMV) and a vector encoding two tandem copies of green fluorescent protein as a tracer of cell-to-cell movement showed the increased gating capacity of plasmodesmata in the presence of BG, MIG-21, and NCAPP. The increased gating capacity is accompanied by enhanced TMV reproduction in the “receivers”. Overall, our data indicate that methanol emitted by a wounded plant acts as a signal that enhances antibacterial resistance and facilitates viral spread in neighboring plants

Oxygen and light accelerate recovery from SO2-induced inhibition of leaf photosynthesis and from cytoplasmic acidification

Leaves of several plant species were illuminated either in air or in a gas atmosphere with reduced oxygen content, and photosynthesis and transpiration were recorded after brief exposure to high concentrations of SO2. Inhibition of photosynthesis by SO2 was similar in air and at reduced oxygen concentrations or, in some experiments, even larger under low oxygen. Recovery from inhibition was always faster in high than in low oxygen. In leaves of Pelargonium zonale, stomata remained open or closed only slowly after fumigation, whereas in spinach and potato leaves stomata closed rapidly under the influence of high SO2. After cessation of fumigation, stomata always reopened. pH-indicating fluorescent dyes were employed to monitor SO2-dependent pH changes in the apoplast and the cytosol of leaf tissue. In the cytosol, acidification was weaker in the dark than in the light. In the light, it was stronger in 21 % oxygen than in 1 % oxygen. Cytosolic acidification could not fully be attributed to the hydration and oxidation of SO2 but was also caused by a transient breakdown of transmembrane proton gradients that resulted in the influx of acid from acidic leaf compartments into the cytosol. This was concluded from opposite pH changes in the cytosol and the apoplast under the influence of SO2. Even chloroplasts were transiently acidified by SO2 as shown by the kinetics of 505 nm absorption, which reflect the pH-sensitive interconversion of violaxanthin to zeaxanthin. Differences in the kinetics of SO2-dependent inhibition of photosynthesis and of cytosolic acidification in high and low oxygen suggest combined nucleophilic attack of sulfite, and of radicals generated in the light, on sensitive cellular constituents. However, the reversibility of photosynthesis inhibition and of acidification, particularly in the presence of air levels of oxygen, demonstrates fast repair. The observations suggest that detrimental effects on plants caused by long-term exposure to ambient concentrations of SO2, which are several hundredfold lower than used in the present investigation, can neither be explained by radical damage nor by immediate effects of cellular acidification. Both radical detoxification and pH regulation are highly effective in healthy plants

Simultaneous growth and emission measurements demonstrate an interactive control of methanol release by leaf expansion and stomata

Emission from plants is a major source of atmospheric methanol. Growing tissues contribute most to plant-generated methanol in the atmosphere, but there is still controversy over biological and physico-chemical controls of methanol emission. Methanol as a water-soluble compound is thought to be strongly controlled by gas-phase diffusion (stomatal conductance), but growth rate can follow a different diurnal rhythm from that of stomatal conductance, and the extent to which the emission control is shared between diffusion and growth is unclear. Growth and methanol emissions from Gossypium hirsutum, Populus deltoides, and Fagus sylvatica were measured simultaneously. Methanol emission from growing leaves was several-fold higher than that from adult leaves. A pronounced diurnal rhythm of methanol emission was observed; however, this diurnal rhythm was not predominantly determined by the diurnal rhythm of leaf growth. Large methanol emission peaks in the morning when the stomata opened were observed in all species and were explained by release of methanol that had accumulated in the intercellular air space and leaf liquid pool at night in leaves with closed stomata. Cumulative daily methanol emissions were strongly correlated with the total daily leaf growth, but the diurnal rhythm of methanol emission was modified by growth rate and stomatal conductance in a complex manner. While in G. hirsutum and in F. sylvatica maxima in methanol emission and growth coincided, maximum growth rates of P. deltoides were observed at night, while maximum methanol emissions occurred in the morning. This interspecific variation was explained by differences in the share of emission control by growth processes, by stomatal conductance, and methanol solubilization in tissue water